Biological activities of extracts and isolated compounds from Bauhinia

Biological activities of extracts and isolated compounds from Bauhinia
Biological activities of extracts and isolated compounds from Bauhinia
galpinii (Fabaceae) and Combretum vendae (Combretaceae) as potential
antidiarrhoeal agents
Ahmed Aroke Shahid
BSc (Chemistry) (ABU), MSc (Chemistry) (UNILAG)
A dissertation submitted in fulfilment of the requirements for the degree of Doctor of
Philosophy (PhD)
in the
Phytomedicine Programme, Department of Paraclinical Sciences,
Faculty of Veterinary Science
Promoter: Prof. Jacobus N. Eloff
Co-promoters: Dr. Nivan Moodley (CSIR)
Prof. Vinny Naidoo
Dr. Lyndy McGaw
January 2012
i
© University of Pretoria
Declaration
The research work described in the thesis was conducted in the Phytomedicine Programme in the Department of
Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria under the supervision of Professor
JN. Eloff, Dr. N. Moodley, Prof. V. Naidoo and Dr. LJ. McGaw
The results presented herewith were generated from my own experiments, except where the work of others are
quoted and referenced. There is no part of this work that has been submitted to any other University.
_______________________________
Aroke Shahid, Ahmed
ii
Dedication
This work is dedicated to the memory of the following: My Father (Late Mr. Ahmed Aninya Aroke), my brothers
(Late Salihu Aroke and Late Ibrahim Onimisi Ahmed), Late Olukemi Ore Udom (A friend and colleague who
started her PhD, but could not finish the programme before death) and my dear sister (Late Mrs. Husseinatu
Ohunene Abubakar).
iii
Acknowledgements
This Ph.D. study would not have been possible without the support and encouragement of many people. I will
thank my mum Mrs Omeneke Aminatu Ahmed who has been the pillar of my life. The love of my wife Mrs
Rabiatu Isoyiza Ahmed and my children Enehu Mazidah, Onize Nusrah, Ometere Shamsiyah, Eneze Azeemah
and Ava’ami AbdulAleem have been essential to my success. I thank you all for your understanding and
endurance during my absence. I also wish to thank my siblings Mr. Aroke Haruna, Mr. Umar Omeiza Aroke and
his wife Rukkayat, Mrs. Zulaiha Mubarak, Dr. Halidu Aroke Ahmed, Mrs. Khaltum Khamilu and Aisha Ahmed for
their enormous support. I am also deeply indebted to my friends who stuck by me through the years: Dr.
Muhammed Awwalu Usman, Engr. Yakubu Adajah, Mr. Yahaya Ohida Yusuf, Mr. Tijani Muhammed Isah,
Abdulmumuni Enesi Umar, Dr. Muhammed Onujagbe Onoda, Mrs. Ukachi Ezenwa Igbo, Dr. Caroline
Anyakorah, Dr. Oluwatoyin Taiwo and Dr. Chima Cartney Igwe among others. Financial support was a crucial
element of my ability to devote so much time to this research. Major support that keeps me and my family afloat
during the study was provided by a study leave with pay provide by Federal Institute of Industrial Research
Oshodi (FIIRO), Lagos, Nigeria. The University of Pretoria has provided support through University bursary,
National Research Foundation (NRF) of South Africa provide fund for the research, Faculty of Veterinary Science
also provide research fund through the research committee (RESCOM), and Department of Paraclinical Science
also provide research fund.
I am extremely grateful to my supervisor, Prof. J. N. Eloff (overall supervisor) and my co-supervisors, Dr. Nivan
Moodley (characterization of isolated compounds), Prof. Vinny Naidoo (isolated organ studies), and Dr. Lyndy J.
McGaw (cellular toxicology) for allowing me to tap from the wealth of their knowledge and also giving me much of
their time and energy through the duration of this study. I have a great respect and appreciated the instruction
and assistance I received from each and every one of you. I sincere thank the Secretary to Phytochemical
Programme, Tharien de Winnaar for all her assistance in coordinating the purchase of materials and other
important aspects of this project.
I feel honoured to have opportunity to do some of the work at Bioscience, CSIR South Africa and lucky to meet
with Dr. Vinesh Maharaj, Dr. Jacqueline Ndlebe, and Ms. Teresa Faleschini who are friends as well as crucial
resources of information, techniques and instructions. I express my gratitude to CSIR for allowing me to use her
NMR spectroscopic facilities.
At the UPBRC where I got my isolated organ part, the assistance of Dr. Tamsyn Pulleer, Mrs. Stephanie Keulder
and Mrs. Ilse Janse van Rensburg were highly appreciated. The ability to collect plant material was essential to
this project, and for that I want to recognize the help and outstanding collaboration of Ms Magda Nel of the Manie
van der Schyff Botanical Garden and Ms Elsa van Wyk (Curator of the HGW Schweickert Herbarium of the
University of Pretoria) for assistance in collection, identification and authentication of the plant samples. My
appreciation also goes to Ms Lita Pauw of the Phytomedicine Programme for allowing me access her stored
iv
plant samples. I also appreciated working in harmony with all other students of the Phytomedicine Programme,
most especially Mr. Thanyani Ramandwa, Ms Bellonah Sakong, Ms Salaelo Raphatlelo, Ms Imelda Ledwaba and
Mrs Edwina Muleya. I thank you all for you co-operation and immeasurable assistance.
v
Table of Contents
Page number
Abstract
xiv
List of abbreviations
xvi
List of figures
xix
List of tables
xxi
List of appendix
xxii
CHAPTER ONE
Gastrointestinal disorders in diarrhoea diseases mechanisms and medicinal plants
potentiality as therapeutic agents
1.0. Introduction
1
1.1. Plant metabolites as potential therapeutic agent
2
1.2. Aims
3
1.3. Specific objectives
4
1.4. Hypothesis
4
CHAPTER TWO
2.0 Literature review
2.1. Diarrhoea as a disease
5
2.2. Pathophysiology of Diarrhoea
6
2.3. Detailed pathophysiology of diarrhoea
8
2.3.1. Inflammation in diarrhoea
8
2.3.2. Oxidative damage in diarrhoea
11
2.3.3. Enteric nervous system in diarrhoea
15
2.3.4. Cystic fibrosis transmembrane conductance regulator (CFTR) regulation
16
2.4. Specific Agents of Diarrhoea
16
2.4.1. Bacterial causes of diarrhoea
16
2.4.1.1. Escherichia coli
16
2.4.1.2. Staphylococcus aureus
17
2.4.1.3. Campylobacter jejuni
18
2.4.1.4. Shigella spp
18
2.4.1.5. Vibrio cholerae
18
2.4.1.6. Bacillus cereus
19
2.4.1.7. Yersinia enterocolitica
19
2.4.1.8. Listeria monocytogenes
20
vi
2.4.1.9. Clostridium spp
20
2.4.1.10. Salmonella typhimurium
20
2.4.1.11. Enterococcus faecalis
21
2.5. Fungal induced diarrhoea symptoms
21
2.5.1. Candida albicans
21
2.6. Viral induced diarrhoea
21
2.6.1. Rotavirus
21
2.6.2. Norovirus
22
2.6.3. Hepatitis A virus
22
2.6.4. Human immunodeficiency virus (HIV)
22
2.7. Protozoa induced diarrhoea
22
2.7.1. Giardia intestinalis
22
2.7.2. Entamoeba histolytica
23
2.7.3. Cryptosporidium parvum
23
2.7.4. Cyclospora cayetanensis
23
2.8. Parasitic induced diarrhoea
23
2.8.1. Trichinella spiralis
23
2.9. Immune disordered induced diarrhoea
24
2.9.1. Compromised immune system
24
2.9.2. Hyperactive immune system
24
2.10. Antibiotic therapy induced diarrhoea
24
2.10.1. Antibiotic toxicity
24
2.10.2. Alteration of digestive functionality
25
2.10.3. Overgrowth of pathogenic microorganisms
25
2.11. Diabetic complications induced diarrhoea
25
2.12. Food allergy induced diarrhoea
26
2.13. Potential mechanisms in the control of diarrhoea
26
2.13.1. Oxidative damage and antioxidants in diarrhoeal management
26
2.13.2. Inflammation and anti-inflammatory agents in diarrhoea management
26
2.13.3. Enteric nervous system in diarrhoea symptoms and treatment
26
2.14. Plants as potential source of therapeutic agents in alleviating diarrhoeal symptoms
28
2.14.1. Anti-infectious mechanisms of plant secondary metabolites against diarrhoeal pathogens 28
2.14.2. Antioxidative mechanisms of plant phytochemical as potential antidiarrhoeal agents
29
2.14.3. Anti-inflammatory mechanisms of plant phytochemical in diarrhoea management
29
2.14.4. Antidiarrhoeal mechanisms of plant phytochemical
30
2.15. Classification of phytochemicals with antidiarrhoea potential
30
2.15.1. Terpenoids
30
vii
2.15.2. Alkaloids
33
2.15.3. Phenolic
35
2.16. Ethnobotany and scientific investigation of plant species used traditionally in treating
diarrhoea in South Africa
38
2.17.Conclusion
38
CHAPTER THREE
Plant selection, collection, extraction and analysis of selected species
3.1. Introduction
39
3.2. Solid-liquid extraction
40
3.3. Liquid-liquid fractionation
41
3.4. Thin layer chromatography (TLC)
41
3.4.1. Phytochemical fingerprints
41
3.5. Materials and Methods
42
3.5.1. Selection of South Africa medicinal plants for antidiarrhoeal screening
42
3.5.2. Collection of plant materials
42
3.5.3. Preparation of plant material and optimization of phenolic-enriched extraction process
42
3.5.4. Phytochemical profiling
44
3.6. Quantification of the phenolic constituents of the extracts
45
3.6.1. Determination of total phenolic constituents
45
3.6.2. Determination of total tannin
45
3.6.3. Determination of proanthocyanidin
45
3.6.4. Determination of condensed tannin
46
3.6.5. Determination of hydrolysable tannin (gallotannin)
46
3.6.6. Determination of total flavonoids and flavonol
46
3.6.7. Determination of anthocyanin
47
3.7.
47
Results
3.7.1. Yield of extractions and fractionations processes
47
3.7.2. Phytochemical screening (fingerprints)
48
3.7.3. Phenolic composition of the crude extracts
52
3.8. Discussion
57
3.8.1. Yield
57
3.8.2. Thin layer chromatogram
57
3.8.3. Phenolic constituents of the crude extract
58
3.9.
60
Conclusion
CHAPTER FOUR
Antimicrobial activities of the plant extracts against potential diarrhoeal pathogens
61
4.0. Introduction
viii
4.1. Qualitative antimicrobial (Bioautography) assay
62
4.2. Quantitative antimicrobial activity (Minimum inhibitory concentration (MIC)) assay
63
4.3.Selection of microorganisms used in the study
63
4.4. Material and Methods
64
4.4.1. Microorganism strains
64
4.4.2. Culturing of the Bacteria
64
4.4.3. Bioautography against some pathogenic microorganisms
64
4.4.4. Determination of Minimum Inhibitory Concentration (MIC) against the bacteria pathogens
64
4.4.5. Determination of Minimum Inhibitory Concentration (MIC) against the fungal pathogens
65
4.5. Results
65
4.5.1. Microbial bioautography
65
4.5.2. Minimum inhibitory concentration against bacteria
70
4.5.3. Minimum inhibitory concentration (MIC)
73
4.6. Discussion
75
4.6.1. Antimicrobial bioautography
75
4.6.2. Minimum inhibitory concentration (MIC)
75
4.7. Conclusion
77
CHAPTRER FIVE
Free radical scavenging and antioxidant activities of the extracts and fractions as
antidiarrhoeal mechanism
5.1. Introduction
79
5.1.1. Superoxide ion
81
5.1.2. Hydrogen peroxide
81
5.1.3. Hydroxyl radical
81
5.1.4. Peroxyl radical
82
5.1.5. Hypochlorous acid
82
5.1.6. Nitric oxide
82
5.2. Antioxidant assays
83
5.2.1. Antioxidant bioautography
83
5.2.2. The chemistry of some common antioxidant assays
83
5.2.2.1. Hydroxyl radical
83
5.2.2.2. Hydrogen peroxide scavenging
84
5.2.2.3. Superoxide scavenging capacity
84
5.2.2.4. DPPH
84
5.2.2.5. ABTS
85
5.2.2.6. Ferric reducing antioxidant power (FRAP)
85
5.3.
85
Materials and Methods
ix
5.3.1.
Antioxidative profile of the crude extracts and fractions using DPPH radical solution
86
5.3.2. Antioxidative assays
86
5.3.2.1. DPPH free radical-scavenging method
86
5.3.2.2. ABTS free radical-scavenging method
86
5.3.2.3. Ferric reducing antioxidant power (FRAP)
87
5.3.2.4. Hydroxyl radical scavenging assay
87
5.3.2.5. Lipid peroxidation inhibition assay
87
5.4. Result
87
5.4.1. TLC-DPPH analyses
87
5.4.2. DPPH effective concentration (EC50)
90
5.4.3. ABTS effective concentration (EC50)
92
5.4.4. FRAP gradient
93
5.4.5. Hydroxyl radical effective concentration (EC50)
94
5.4.6. Lipid peroxidation inhibition effective concentration (EC50)
95
5.5. Discussion
96
5.5.1. Qualitative antioxidant analyses (DPPH-TLC bioautography)
96
5.6.
98
Conclusion
CHAPTER SIX
Anti-inflammatory activities of the crude extracts as antidiarrhoeal mechanisms
6.0. Introduction
100
6.1. Effect of cyclooxygenases (COX) on GIT
101
6.2. Effects of lipoxygenase (LOX) on GIT
101
6.3. Effects of cytokines on GIT
102
6.4. Oxidative species as inflammatory mediator
102
6.5. Allopathic anti-inflammatory therapies and adverse effects on GIT
103
6.6. Plant phytochemicals as anti-inflammatory agents
105
6.7. Mechanisms of anti-inflammatory assay models
105
6.8. Materials and Methods
106
6.8.1. COX assay
106
6.8.2. LOX assay
106
6.9. Results
107
6.9.1. COX
107
6.9.2. LOX
108
6.10. Discussion
109
6.10.1. COX
109
6.10.2. LOX
109
6.11. Conclusion
110
x
CHAPTER SEVEN
Cytotoxicity evaluation of the crude extracts against Vero African green monkey kidney cell lines
7.0. Introduction
111
7.1. Materials and Methods
112
7.1.1. Preparation of plant extracts
112
7.1.2. Cytotoxicity assay against Vero cell
112
7.2. Results
113
7.3. Discussion
114
7.4. Conclusion
115
CHAPTER EIGHT
Motility modulation potential of Bauhinia galpinii and Combretum vendae phenolic-enriched
leaf extracts on isolated rat ileum
8.0. Introduction
116
8.1. Drugs and reagents
117
8.2. Animal care
117
8.2.1. Isolated ileum preparation
118
8.3. Contractility test
118
8.3.1. Spasmogen assay
118
8.3.2. Spasmolytic assays
118
8.3.2.1. Effects on acetylcholine-induced contractility
118
8.3.2.2. Effects on serotonin-induced contractility
118
8.3.2.3. Effects on KCl-induced contractility
119
8.4. Data analysis
119
8.5.
119
Results
8.5.1. Effect of B. galpinii crude extract on isolated rat ileum
119
8.5.2. Effect of C. vendae crude extract on isolated rat ileum
122
8.6. Discussion
123
8.7. Conclusion
126
CHAPTER NINE
Isolation and characterization of antimicrobial and antioxidant compounds from
Bauhinia galpinii and Combretum vendae
9.0. Introduction
127
9.1.1. Column chromatography
128
9.1.2. Mass spectrometry
128
9.2. Materials and Methods
128
9.2.1. Preparation of plant extracts
128
9.2.2. Bioautography
128
xi
9.2.3. Isolation of bioactive triterpenoids from C. vendae
129
9.2.4. Isolation of phenolic compounds from C. vendae
130
9.3.
130
Isolation of compounds from B. galpinii
9.3.1. Isolation of bioactive triterpenoids from B. galpinii
130
9.3.2. Isolation of phenolic compounds from B. galpinii
130
9.4. Characterization of the isolated compounds
131
9.4.1. NMR spectroscopy
132
9.4.2 .Mass spectrometry
132
9.4.3. Ultra-violet spectroscopy
132
9.5. Results
132
9.5.1. Identification of the chemical structures of isolated compounds from C. vendae
132
9.5.2. Antimicrobial activity of isolated compounds from C. vendae
135
9.5.3. Identification of the chemical structures of isolated compounds from B. galpinii
136
9.5.4. Antimicrobial activity of isolated compounds from B. galpinii
140
9.6. Discussion
140
9.6.1. Bioactive compounds from C. vendae
140
9.6.2. Bioactive compounds from B. galpinii
141
9.7. Conclusion
143
CHAPTER 10
General conclusion and future prospects
144
10. Introduction
10.1. Identification of diarrhoeal pathogenesis and medicinal plants used as therapeutic
Agents
145
10.2. Antimicrobial evaluation of the extracts against infectious pathogens
145
10.3. Antioxidant evaluation of the extracts
145
10.4. Anti-inflammatory potential of the extracts
146
10.5. Toxicity risk of the extracts
146
10.6 Motility modulatory effects of Bauhinia galpinii and Combretum vendae
147
10.7. Isolation and characterisation of bioactive compounds
147
CHAPTER 11
149
References
xii
Abstract
Diarrhoea is one of the killer diseases resulting from the dehydration and loss of electrolytes through profuse and
excessive excretion of loose stool. The pathoaetiologies include infections, intestinal inflammation, imbalanced
intestinal oxidative homeostasis and altered motility. Treatment with oral rehydration therapy (ORT) is a key
intervention especially in secretory diarrhoea as supportive therapy. Symptomatic and non-symptomatic
therapies directed at treating the intestinal tissues are available. However, these conventional treatments are still
not sufficient in curing diarrhoea due to their associated hazards such as the development and spread of drugresistant pathogens, changes in normal intestinal bacteria flora and potential chronic toxicity. Therapies targeted
at intestinal tissue include antimotility and antisecretory agents have adverse effects such as addictiveness,
constipation and fatal ischaemic colitis. Many ethnopharmacological and ethnobotanical therapies for treating
diarrhoea exist among different cultures. The aims of this study were to evaluate the biological activities of plant
extracts against some diarrhoeal pathophysiologies.
A literature search in English of published articles and books that discussed ethnobotanical uses of
medicinal plants in southern Africa was conducted. A list of 230 medicinal plants used in South African traditional
medicines for treating diarrhoea and associated complications was created. The list included family, genus,
species, biological activities and bioactive isolates as well as the remedies for diarrhoea. Twenty seven species
were selected to evaluate for antimicrobial, antioxidant and anti-inflammatory activities. Safety of the plants was
determined by determining the cytotoxicity of the crude extracts against Vero African green monkey kidney cell
lines using a standard method. Motility effects of Bauhinia galpinii (BGE) and Combretum vendae (CVE) were
determined by modulation of the contractility process of the isolated rat ileum induced by spasmogens.
Phenolic compositions of the crude extract were determined using various standard methods and finally
bioactivity guided isolation of antimicrobial and antioxidant compounds from BGE and CVE were carried out
using open column chromatography. Identification and characterization of the isolated compounds was achieved
by NMR, EI-MS and UV spectroscopy.
The non-polar fractions had good antimicrobial activities with MIC ranged between 19 – 1250 µg/ml
while the polar fraction had moderate antimicrobial activities with MIC ranged between 39 - >2500 µg/ml. In
general the non-polar fractions had a higher antimicrobial activity.
The crude extracts contained wide range phenolic compounds with a total phenolic (74.91±1.26 to
467.04±15.82 mg GAE/g plant material), and total flavonoids (11.27±3.37 to 176±5.96 mg EQ/g plant material).
The antioxidant activities were concentrated and potentiated in the polar fractions. The non-polar fractions had
poor antioxidant activities with EC50 values ranging from 0.21±0.03 to 303.65±3.84 µg/ml for DPPH radical
scavenging and 0.43±0.03 to 1709±91.44 µg/ml for ABTS radical scavenging.
The crude extracts had selective COX-1 inhibitory activities ranging between 41.70 to 84.61% and had
no COX-2 inhibitory activity. All the extracts tested had 15-LOX inhibitory capacity with LC50 values ranging
between 0.86±0.27 and 111.44±37.28 µg/ml. The cytotoxicity results indicated a wide variation in toxic potential
of the crude extracts with LC50 values ranging from 3.51 to 741.90µg/ml.
xiii
The BGE extracts had dual activities as spasmolytic by stimulating the spontaneous contractility and
also agonised contractions induced by spasmogens but it inhibited K+ induced contraction. CVE had spasmodic
activities through a multiple mechanisms inhibiting contractions induced by spasmogens and K+ in a dosedependent manner.
Several bioactive xompoundswere isolated from the Combretum vendae leaves,
There were
triterpenoids (ursol-12-en-28-oic acid, mixtures of corosolic acid and maslinic acid, and asiatic acid and arjunolic
acid) as well as bibenzyls combretastatin B5-O-2’-β-D-glucopyranoside, combretastatin B1-O-2’-β-Dglucopyranoside and a flavonoid (apigenin)..
From Bauhinia galpinii the following bioactive compounds were isolated and characterized: β-3 ethoxy
sitosterol, one new flavone (5, 7, 4’ 5’ tetrahydroxy-2’-methoxyflavone (isoetin 2’-methyl ether) or 5, 7, 2’ 5’
tetrahydroxy-4’-methoxyflavone (isoetin 4’-methyl ether)), 3, 5, 7, 3’, 4’-pentahydroxyflavone and 3, 5, 7, 3’, 4’, 5’hexahydroxyflavone, quercetin-3-O-β-galactopyranoside and myricetin-3-O-β-galactopyranoside
The extraction protocol used in this work potentiated the antimicrobial activities in the non-polar
fractions while antioxidant activities were potentiated in the polar fractions. This indicated that using polar
solvents as extractant for treating infectious diarrhoea may not be quite effective unless some other
antidiarrhoeal mechanisms are involved. Therefore, mixture of organic solvent (ethanol) and water can be
recommended for broad-based activity.
Bauhinia galpinii extracts had a dual- mechanism of action (prokinetic and relaxant) on gastro-intestinal
motility, depending on the prevalent patho-physiological condition and Combretum vendae mediated spasmolytic
effects on isolated rat ileum through multiple inhibitions of a wide range of contractile stimuli. Hence, the
presence of multiple acting spasmolytic activities in the plant extract might be contributing towards its
effectiveness in treating diarrhoea and abdominal spasm. The uses of these plants in traditional medicine need
to be monitored closely because of the selective inhibition of COX-1 and its associated GIT injury, and the high
toxicity potential of some of the extracts.
Further work evaluating the antidiarrhoea mechanisms, identification and isolation of bioactive
compounds, sub-acute and acute toxicity of the plant extracts is recommended.
Key words: Antimicrobial, antioxidant, anti-inflammatory, diarrhoeal, antispasmolytic, enteric nervous system,
cytotoxicity.
xiv
List of Abbreviations
A
ABTS=2.2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid
AMP=Antimicrobial peptides
B
BAB= Bauhinia bowkeri
BAG= Bauhinia galpinii
BAP= Bauhinia petersiana
BAV= Bauhinia variegata
BGE= Bauhinia galpinii extract
C
Ca2+= Calcium ion
Cl-= chloride ions
CNF-1= Cytotoxic necrotising factor 1
CNS= Central nervous system
COB= Combretum bracteosum
COP= Combretum padoides
COV= Combretum vendae
COX= Cyclooxygenase
COW= Combretum woodii
CVE= Combretum vendae extract
D
DAEC= diffusively adherent Escherichia coli
DNA
DPPH=2, 2-diphenyl-1-picrylhydrazyl
E
EAEC= Enteroaggregative Escherichia coli
EHEC= Enterohaemorrhagic Escherichia coli
EIEC= Enteroinvasive Escherichia coli
ENS= Enteric nervous system
EPEC= Enteropathogenic Escherichia coli
ETEC= Enterotoxigenic Escherichia coli
EUC=Euclea crispa
EUN= Euclea natalensis
xv
F
FIC= Ficus cratestoma
FIG=Ficus glumosa
FRAP= Ferric reducing antioxidant capacity
G
GIT= Gastrointestinal tract
H
HIV/AIDS= Human immune deficiency virus/Acquired immune deficiency syndrome
HOCl= hypochlorite
HUB= Haemolytic uremic syndrome
I
IBS= Irritable bowel syndrome
IL= Interleukin
INC= Indigofera cylindrica
iNOS= inducible nitric oxide synthase
INT= p-iodonitrotetrazolium
L
LT= Heat labile enterotoxin
LTB= Leukotriene B
M
MDA= Malondialdehyde
MCP-1= Monocyte chemoattractant protein
MIC= Minimum inhibitory concentration
MPD= Maytenus peduncularis
MPR= Maytenus procumbens
MSE= Maytenus senegalensis
MUN= Maytenus undata
N
Na+= sodium ions
NAME= nitro
NH2Cl= Ammonium chloride
NO= Nitric oxide
O
OH¯= Hydroxyl radical
ORT=Oral rehydration therapy
OZM= Ozoroa mucronata
OZP= Ozoroa paniculosa
xvi
P
PG= Prostaglandin
R
ROS= Reactive oxygen species
RNS= Reactive nitrogen species
S
SCB=Schotia brachypetala
SLE= Searsia leptodictya
SPD= Searsia pendulina
SPT= Searsia pentheri
ST= Heat stable enterotoxins
SYP= Syzygium paniculatum
T
TLC=Thin layer chromatography
TNF-α= Tumour necrosis factor-α
Trolox= 6-hydroxy-2, 5, 7, 8-tetrahydroxyl-chroman-2-carboxylic acid
U
UNICEF=United Nation Children Fund
W
WHO= World Health Organization
xvii
List of Figures
Chapter 2
Fig. 2.1. Classification of the diarrhoea and the stimulants
6
Fig. 2.2. Cytokines production network in the tissues
8
Fig. 2.3. Biosynthetic pathways for the eicosanoids
9
Fig. 2.4. Intestinal epithelial TJs as a physical barrier
10
Fig.2.5. The integrative pathophysiology and mechanism of diarrhoeal disease
13
Fig. 2.6. Lipid peroxidation chain reactions
14
Fig. 2.7. Chemical structures of the lipid peroxidation intermediates
14
Fig. 2.8. Mechanisms of antibiotic-induced diarrhoea
25
Fig. 2.9. Chemical structures of bioactive terpenoids against diarrhoeal mechanisms
31
Fig. 2.10. Chemical structures of bioactive alkaloids against diarrhoeal mechanisms
33
Fig. 2.11. Sub-classes of biologically important phenolic compounds
34
Fig. 2.12. Chemical structures of bioactive phenolics against diarrhoeal mechanisms
36
Chapter 3
Fig.3.1. Flow chart for the extraction, phytochemical analysis and fractionation of the crude extracts
43
Fig.3.2. TLC phytochemical profile of the crude extracts
48
Fig.3.3. TLC phytochemical profile of the hexane fractions
49
Fig.3.4. TLC phytochemical profile of the dichloromethane fraction
50
Fig.3.5. TLC phytochemical profile of the ethyl acetate fraction
51
Fig.3.6. Total phenolic and non-tannin constituents of the crude extract
52
Fig.3.7. Total tannin and condensed tannin constituents of the crude extracts
53
Fig.3.8. Proanthocyanidin and gallotannin constituents of the crude extract
54
Fig.3.9. Total flavonoid and flavonol constituents of the crude extract
56
Chapter 4
Fig.4.1. The classification of microbiological methods for biological detection
63
Fig.4.2. Bioautography of the hexane fractions against S. aureus
65
Fig.4.3. Bioautography of the dichloromethane fractions against S. aureus
66
Fig.4.4. Bioautography of hexane fractions of different plant species against E. faecalis
66
Fig.4.5. Bioautography of dichloromethane fractions of different plant species against E. coli
67
Fig.4.6. Bioautography of dichloromethane fractions of different plant species against E. faecalis
67
Fig.4.7. Bioautography of hexane of different plant speicies against C. neoformans
68
Fig.4.8. Bioautography of dichloromethane fractions against C. neoformans
68
Fig.4.9. Bioautography of hexane fractions against A. fumigatus
69
Fig.4.10. Bioautography of dichloromethane fractions against A. fumigatus
69
Fig.4.11. Bioautography of hexane fractions against C. albicans
70
xviii
Fig.4.12. Bioautography of dichloromethane fractions against C. albicans
70
Chapter 5
Fig.5.1. Some deleterious reactions from the production of reactive free radicals in biological systems
80
Fig.5.2. TLC-DPPH profiles of the crude extracts of extracts of different plants
88
Fig.5.3. TLC-DPPH profile of the hexane fractions of different plants
89
Fig.5.4. TLC-DPPH profiles of the dichloromethane fractions of different plants
89
Fig.5.5. TLC-DPPH profiles of the ethyl acetate fractions of different plants
90
Chapter 6
Fig.6.1: Roles of COX in the pathogenesis mechanism of NSAID-induced intestinal damage
103
Fig.6.2: Factors involved in the pathogenesis of indomethacin-induced small intestinal lesions
104
Fig.6.3: COX-1 inhibitory activity of some selected phenolic-enriched crude extracts
106
Chapter 8
Fig.8.1.Schematic presentation of the contractility assay using isolated rat ileum
118
Fig.8.2.Stimulatory effects B. galpinii on spontaneous contraction of isolated rat ileum
119
Fig.8.3.Effects of 70% acetone crude leaf extract of B. galpinii on the acetylcholine cumulative concentration
dependent-induced contraction in the absence and presence of atropine
119
Fig.8.4.Agonized effects of B. galpinii on serotonin-induced contraction of the isolated rat ileum
120
Fig.8.5.Relaxant effects of B. galpinii on KCl-induced contraction of the isolated rat ileum
120
Fig.8.6.Spasmolytic effects of 70% acetone crude leaf extract of C. vendae on acetylcholine-induced contraction
of the isolated rat ileum
121
Fig.8.7.Spasmolytic effects of 70% acetone crude leaf extract of C. vendae on serotonin-induced contraction of
the isolated rat ileum
121
Fig.8.8.Spasmolytic effect of the C. vendae on the depolarised KCl-induced isolated rat ileum contractions
122
Chapter 9
Fig.9.1. Extraction, fractionation and isolation of bioactive compounds from the leaf extract of Combretum
vendae
128
Fig.9.2. Extraction, fractionation and isolation of bioactive compounds from the leaf extract of Bauhinia galpinii
130
Fig.
9.3 Chemical structures isolated bioactive compounds from the leaf extract of C. vendae
134
Fig.
9.4 Chemical structures isolated bioactive compounds from the leaf extract of B. galpinii
138
xix
List of Tables
Chapter 2
Table 2.1. The mechanism of action and symptoms of enteric pathogenic E.coli
17
Table 2.2. Neurotransmitters of ENS causing intestinal secretion in diarrhoea
27
Chapter 3
Table 3.1. Medicinal plants selected for the antidiarrhoeal investigations in this study
42
Table 3.2. The percentage yield of the crude extract and fractions (g/g dried plant material)
47
Chapter 4
Table 4.1. The minimum inhibitory concentration (MIC) of the crude extracts and fractions against bacterial
strains tested
72
Table 4.2 The minimum inhibitory concentration (MIC) of the crude extracts and fractions against fungal strains
tested
74
Chapter 5
Table 5.1 DPPH radical scavenging potential of the crude extracts and fractions expressed as EC50 (µg/ml)
91
Table 5.2. ABTS radical scavenging potential of the crude extracts and fractions expressed as EC50 (µg/ml)
92
Table 5.3. FRAP
94
Table 5.4. Hydroxyl radical scavenging potential of the crude extracts and fractions expressed as EC50 (µg/ml)
94
Table 5.5. Linoleic acid peroxidation inhibition expressed as LC50 (µg/ml)
95
Chapter 6
Table 6.1. Lipoxygenase inhibitory activity of the crude extracts
107
Chapter 7
Table 7.1. The LD50 of the cytotoxicity assay of some medicinal plants used in South African traditional
medicine to treat diarrhoea and related ailments
112
Chapter 9
Table 9.1: NMR experiments commonly applied for natural product structural elucidation
126
Table 9.2: Minimum inhibitory concentration (µg/ml) of the isolated compounds from the leaf extract of C. vendae
135
Table 9.2: Minimum inhibitory concentration (µg/ml) of the isolated compounds from the leaf extract of B. galpinii
139
xx
List of Appendix
Page number
Appendix 2.1. Ethnobotanical and literature information of medicinal plant species used traditionally for treating
diarrhoea in South Africa
172
Appendix 9.1. 1D and 2D NMR spectra data of Ursolic acid
197
Appendix 9.2. 1D and 2D NMR spectra data of mixture of corosolic acid and maslinic acid
198
Appendix 9.3. 1D and 2D NMR spectra data of mixture of asiatic aicd and arjunolic acid
199
Appendix 9.4. 1D and 2D NMR spectra data of combretastatin B5-2’-O- glucopyranoside
200
Appendix 9.5. 1D and 2D NMR spectra data of combretastatin B1-2’-O- glucopyranoside
200
Appendix 9.6. 1D and 2D NMR spectra data of 3β-ethoxy sitosterol
201
Appendix 9.7. 1D and 2D NMR spectra data of quercetin
201
Appendix 9.8. 1D and 2D NMR spectra data of myricetin
202
Appendix 9.9. 1D and 2D NMR spectra data of isoetin 2’ methyl ether/ isoetin 4’ methyl ether
202
Appendix 9.10. 1D and 2D NMR spectra data of quercetin-3-O-β-galactopyranoside
203
Appendix 9.11. 1D and 2D NMR spectra data of myricetin-3-O-β-galactopyranoside
203
xxi
CHAPTER ONE
Gastrointestinal disorders in diarrhoea diseases mechanisms and medicinal plants potentiality as
therapeutic agents.
1. Introduction
The gastrointestinal tract (GIT) is dedicated to processing and absorbing nutrients and fluids essential for the
maintenance of good health (Martinez-Augustin et al., 2009). For the GIT to function optimally, a balance is
maintained between intestinal motility and intestinal fluid volume. The latter process is finely regulated through
the control of fluid absorption via intestinal villous epithelial cells and secretion across the intestine via intestinal
crypt cells (Martinez-Augustin et al., 2009). Net fluid absorption driven by osmotic gradients controlling the
movement of electrolytes (sodium ions [Na+] and chloride ions [Cl-]), sugars and amino acids across the epithelial
lining of the lumen, predominate in these opposing processes (Pash et al, 2009). In contrast, motility is controlled
by the activation of enteric nervous system (ENS) by either neurotransmitters, inflammatory mediators or
epithelium membrane lipid peroxidation by-products (Wood, 2004). Any upset of this delicate intestinal fluid
balance (decrease fluid absorption and increase fluid secretion), and/or changes in GIT motility usually causes
intestinal disorders clinically evident as diarrhoea (Vitali et al., 2006).
Diarrhoea is loosely defined as an alteration in the normal bowel movement characterized by an increase in the
volume, frequency and water content of stool (Baldi et al., 2009). The pathophysiology of diarrhoea include
microbial and parasitic infections (Hodges and Gill, 2010), stress (oxidative or physical) (Soderholm and Perdue,
2006), dysfunctional immunity (Schulzke et al., 2009), disrupt GIT integrity and neurohumoral mechanisms (Vitali
et al., 2006; Spiller, 2004). Diarrhoea can also be a symptom of other diseases such as cholera, irritable bowel
syndrome (IBS), gastroenteritis (intestinal inflammation and ulcerative colitis) (Schiller, L. R., 1999; Baldi et al.,
2009), malaria (Gale et al., 2007) and diabetes mellitus (Forgacs and Patel, 2011).
The mechanism causing diarrhoea can be secretory (resulting from osmotic load within the intestine), hyper
motility (resulting from rapid intestinal transitions) or hypo motility (resulting in decreased intestinal fluid reabsorption) or combination of these mechanisms (Vitali et al., 2006). The symptoms are either caused by an
increase in fluid and electrolyte secretion predominantly in the small intestine or a decrease in absorption which
can involve both small and large intestine (Pash et al, 2009; Spiller and Garsed, 2009). Physiologically, diarrhoea
is considered beneficial to the GIT as it provides an important mechanism of flushing away harmful luminal
substances (Valeur et al., 2009). However, diarrhoea becomes pathological when the loss of fluids and
electrolytes exceeds the body’s ability to replace the losses.
As a disease, diarrhoea is considered one of the most dangerous GIT disorders as death can result in severe
cases due to dehydration and loss of electrolytes (WHO and UNICEF, 2004). According to the World Health
Organization (WHO)/United Nation Children Fund (UNICEF) report, more than 1 billion diarrhoeal episodes
occurred in human across the world yearly, with about 5 million deaths especially in infants (Thapar and
1
Sanderson, 2004). In addition to causing acute disease and mortality, diarrhoea associated malnutrition could
result in stunted growth, non-optimal immune functionality and increase susceptibility to infections. Diarrhoea
therefore poses a major health challenge to human, as it could lead to premature mortality, disability and/or
increase health-care costs (Guerrant et al., 2005).
In animal production, diarrhoea is presumed to impose heavy productivity losses on affected farms, although true
effects in monetary terms cannot be easily appreciated. The apparent on-farm losses are reduction in
productivity (milk, wool, egg, meat and meat quality), increased mortality and morbidity, weight loss and abortion
(Chi et al., 2002). Episodes of diarrhoeal diseases can also affect the export market and hurt consumer’s
confidence in the products (Yarnell, 2007).
The most common modern method of managing diarrhoea is the replacement of lost fluids and electrolytes with
either oral or intravenous electrolyte preparation (Thapar and Sanderson, 2004). While fluid replacement is
usually effective, severe fluid losses requires additional pharmacological treatment to mitigate the on-going fluid
loss. For this, drugs with antispasmodic, antimotility, antioxidative, anti-secretory/pro-absorptive and/or antiinflammatory properties (depending on the causative agents) may be used to treat diarrhoeal (Wynn and
Fougere, 2007). The issue of antimicrobial therapy for self-limiting and non-infectious diarrhoea is usually not
encouraged to avoid development of drug resistance microbes. However, in cases of established infectious
diarrhoea with known pathogenic agents, specific therapeutic intervention using antimicrobial drugs targeting the
causative microbes may be applied. At present, the current standard therapeutic options are insufficient because
of limited available modalities with broad based activities against the large number of diarrhoeal disease
mechanisms and apparent side effects. The problems associated with some of the standard therapies include
antimicrobial resistance, drug toxicity, constipation and addiction.
As a result there is an urgent need for new therapeutic drugs with lower cost, high efficacy, little or no side
effects and wider availability especially in rural areas where diarrhoea causes large scale infant mortality. Plants,
which servs as dietary source to animals and people, may also provide a good source of new therapeutic drugs.
1.2. Plant metabolites as potential therapeutic agent
Plant serves as dietary source to animals and humans providing sufficient nutrients to meet metabolic
requirements for their well being, growth and productivity. However, it can also contribute to achieving optimal
health and development as well as serving an essential role in reducing the risk or delaying the onset of diseases
and disorders (Kosar et al, 2006; Halliwell, 1997). Medicinal plants have therapeutic properties due to
biosynthesis of various complex phytochemical substances grouped broadly as phenolics, alkaloids and
terpenoids. Synergistic interaction among the multiple phytochemicals may be responsible for the overall
bioactivity of a given medicinal plant. Pharmacological and clinical studies of phytochemical in plants have shown
that they exhibit various medicinal uses and serve as the major backbone of traditional medicine (Van Wyk and
Wink, 2004). Medicinal plants have played some key roles in the health care needs of rural and urban
2
settlements for human, livestock and animals. Plant extracts, formulations, or pure natural compounds are used
in controlling diverse diseases ranging from coughs, inflammation, and diarrhoea to parasitic infection in human
and veterinary medicine. A large number of these medicinal plants have been screened and validated for their
ethnopharmacological use as antidiarrhoeal agents of varied mechanisms (Gutierrez et al., 2007). However, the
literatures available on the pharmacological evaluation of medicinal plants used traditionally in treating diarrhoea
in South Africa are mainly on antimicrobial screening models. Little literature information is available on other
antidiarrhoeal mechanisms and in vivo study
For this study, 27 South African medicinal plants used as diarrhoeal remedies with ethnopharmacological
background identified as requiring further biological evaluation. Thereafter, Bauhinia galpinii and Combretum
vendae were choosing for further investigation based on the results from preliminary screening. Bauhinia galpinii
was previously investigated for its antioxidant activities and three compounds (two active and one inactive) were
isolated from the acetone leave extract (Aderogba et al., 2007). Methanol and dichloromethane leaf extracts of B.
galpinii are reported to have antimutagenic property (Reid et al., 2006). The acetone root extract of B. galpinii
has also been found to be highly cytotoxic (LD50 2.70 µg/ml) against Vero cell lines (Samie et al., 2009).
Antimicrobial activity of Combretum vendae against four bacterial pathogens (Ahmed et al., 2009) and apigenin
has been isolated from the acetone leaf extract (Eloff et al., 2008).
In many previous studies relatively non-polar extractants were used despite the fact that traditionally aqueous
extracts are used. This is probably due to difficulties in analyzing complex molecules extracted by polar
extractants, because phenolics may play an important role in managing diarrhoea the focus of this study will be
on more polar extractant.
1.3. Aims
To investigate the biological activities of the phenolic-enriched extracts and fractions of 27 medicinal plants
against some diarrhoea pathoaetiologies and evaluating the antidiarrhoeal mechanisms of Bauhinia galpinii and
Combretum vendae extracts using in vitro isolated organ methods, as means of validating their
ethnopharmacological used in South African traditional medicine to treat diarrhoea.
1.4. Specific objectives
To evaluate the effect of the extracts, fractions and isolated compound(s) against pathogenic microbes
that are known to induce diarrhoea.
To determine the antioxidative properties of the extracts, fractions and isolated compound(s) using the
DPPH radical scavenging, the ABTS radical scavenging, the hydroxyl radical scavenging, the linoleic
acid peroxidation inhibition and the ferric reducing antioxidant power (FRAP).
To determine the effects of the most promising extracts on the contractility process of the isolated rat
ileum induced by spasmogens, receptor agonists, antagonists and ion channels activators.
3
To fractionate the extracts and elucidate the component(s) that exhibit antimicrobial and antioxidant
properties.
To evaluate the safety, efficacy and toxicity of the crude extracts and the pure active component(s).
1.5. Hypothesis
The phytochemical constituents of medicinal plants used in traditional medicine have antioxidant, antiinflammatory, antimicrobial and /or anti-spasmodic activities that could help in alleviating diarrhoeal diseases in
human and animals.
4
CHAPTER TWO
2.0. Literature review
2.1. Diarrhoea as a disease
Diarrhoea is a common clinical sign following on altered bowel movement, decreased intestinal absorption of
fluids and increased intestinal electrolyte secretion resulting in loose and watery stool (Baldi et al., 2009). The
mechanisms of diarrhoea diseases can be secretory due to impaired electrolyte absorption and osmotic load
within the intestine, hyper motility resulting from rapid intestinal transitions of material or hypo motility resulting in
decreased intestinal fluid re-absorption or combination of these mechanisms (Vitali et al., 2006). The symptoms
are either caused by an increase in fluid and electrolyte secretion predominantly in the small intestine or a
decrease in absorption which can involve both small and large intestine (Pash et al, 2009; Spiller and Garsed,
2009).
Diarrhoeal disease can be either infectious or non-infectious in nature with infection pathogenesis responsible for
the major total episode worldwide. In infectious diarrhoea, the potential causative pathogens include bacterial
agents (Mathabe et al., 2006), rarely fungal (Robert et al., 2001), viral and parasite pathogens (Brijesh et al.,
2006). Non-infectious diarrhoea can be caused by adverse reactions to drugs, toxins, allergy to food, poisons
and acute inflammation which promote the release of secretagogues and some enteric nervous system (ENS)
receptors (prostaglandin, serotonin, substance P, vasoactive intestinal peptides, and hormone) in the GIT (Wynn
and Fougere, 2007). Diarrhoea is usually classified according to the duration of the symptoms:
•
Acute diarrhoea: mostly caused by enteric pathogenic infections, intoxicants or food allergy. This type of
diarrhoea is self-limiting without pharmacological intervention and usually resolves within two week from
onset or,
•
Persistent diarrhoea: mostly result from a secondary cause such as enteric infections or malnutrition,
and usually last for more than 14 days, or
•
Chronic diarrhoea: mostly result from congenital defects of digestion and absorption. This usually last
for more than 30 days (Thapar and Sanderson, 2004; Baldi et al., 2009).
Other methods of classifying diarrhoea include stool characteristics or pathological mechanisms such as watery,
osmotic, altered motility or inflammatory diarrhoea (Ravikumara, 2008) as shown in Fig. 2.1.
•
Watery diarrhoea typically referred to as secretory diarrhoea results from increased chlorine secretion,
decreased sodium absorption and increased mucosal permeability.
•
Osmotic diarrhoea, also a watery form of diarrhoea, is caused by the ingestion of non-absorbable
indigestible material (Baldi et al., 2009) or absence of brush border enzymes required for the digestion
of dietary carbohydrates (Podewils et al, 2004).
5
•
Inflammatory diarrhoea is characterized by the presence of mucus, blood, and leukocytes in the stool,
and is usually induced by an infectious process, allergic colitis or inflammatory bowel disease (IBD)
(Ravikumara, 2008).
Diarrhoea
Medication
or laxative
abuse
Osmotic
Malabsorption
Maldigestion
Secretory
Motility disorder
Inflammatory mediators
Enteric nervous system
Non-digestion
Celiac sprue,
SBBO
Pancreatic
insufficiency
PGE2, PGDFα,
oxidants (NO, HOCl,
H2O2, OH)
Acetylcholine, serotonin,
histamine, substance P,
opioids, dopamine
Lactose intolerance,
sorbitol, lactulose
Infectious
Bacteria, viruses,
parasites, fungi
Inflammatory
Hormonal
Bile salt or fatty acid
Medication or
laxative abuse
Fig.2.1: Classification of diarrhoea and the stimulants (modified from Ebert, 2005)
PGE2= prostaglandin E2, PGDFα= prostaglandin DFα, NO=nitric oxide, HOCl=hydrogen chlorate, H2O2=hydrogen peroxide, OH=hydroxyl radical, SBBO =
small-bowel bacterial overgrowth
2.2. Pathophysiology of Diarrhoea
A “healthy gastrointestinal tract (GIT)” can be defined as one where a balance is reached between the bacteria
colonising the environment and the immune system. Any disturbance in this homoeostasis will result in GIT
disorders like diarrhoea. The general causes of diarrhoea are: microbial infection (bacteria, viruses and
parasites), intestinal inflammation, altered GIT motility as a result of damage to enteric nervous system (ENS)
and immune dysfunctions. The mechanisms of infectious diarrhoea include:
Microbial attachment and localized effacement of the intestinal epithelium: Some enteric pathogens
have the ability to attach and alter the surface of the invaded cell characterized as attaching and
effacing lesion. Attachments of the infectious pathogens to the apical surface of the enterocyte-like cells
create favourable conditions for bacterial growth and multiplication (Ramaroa and Lereclus, 2006). The
mechanism of effacing lesion involves the localized destruction of the adjacent epithelial microvilli and
6
the formation of a pedestal-like structure from the accumulation of cytoskeletal proteins, such as actin,
beneath the site of attachment (Thapar and Sanderson, 2004; Guerrant et al, 1999).
Production of enterotoxins that subvert mucosal transport systems: In the case of intoxication, the
causative organisms may or may not be present in the transmitting medium, but act through preformed
enterotoxins (examples of such organisms are S. aureus (α-haemolysin) and emetic type Bacillus
cereus (cereulide) (Granum, 2006). The toxins may be cytotoxic or haemolytic, thus causing damage to
intestinal epithelial cells. The mechanism of actions used by the enterotoxins to cause diarrhoea include
(Laohachai et al, 2003):
(1) Decrease in intestinal surface area and, hence a decreased fluid absorption rate.
(2) Changes in mucosal osmotic permeability, resulting from mucosal destruction.
(3) Changes in fluid and electrolytes homeostasis through the toxin’s action on ion channels.
Direct epithelial cell invasion: Epithelial cells are the first major cell type encountered by infectious
pathogens in the intestinal mucosa and the main site of host-pathogens interaction (Ramaroa and
Lereclus, 2006). Intestinal mucosal epithelial cells are essential for initiation of the immune response to
different microorganisms (Hodges and Gill, 2010). In addition to forming a physical barrier that protects
the host’s internal organs from the external environment, epithelial cells produce a variety of cytokines
and chemokines in response to microbial infection (MacNaughton, 2006). The survival strategy of some
pathogens is the invasion of epithelial mucosa cells through the activation or inhibition of different signal
transduction pathways and induces cytoskeletal rearrangement within the host cell.
Production of cytotoxins: Microbial cytotoxins degrades of the epithelial cell surface membrane and
consequently results in loss of host epithelial cell layer (Ramaroa and Lereclus, 2006; Thapar and
Sanderson, 2004). Diarrhoea occurs through the destruction of the epithelial cells due to loss of
absorptive surface area and impaired secretion mechanisms.
Immune activation: Some pathogens may induce diarrhoea indirectly via excessively stimulation of the
immune system. The inflammatory mediators such as cytokines (interferon-γ, tumour necrosis factor
(TNF-α), interleukin-6 (IL-6), and IL-1β) (Johnson et al., 2010), reactive oxygen species (ROS), reactive
nitrogen species (RNS) (Sprague and Khalil, 2009) can all interfere with the epithelial tight junctions
(TJs), thereby resulting in diarrhoea.
Immune inhibition: With the direct involvement of the immune system in the protection of the body
against both pathogenic and enteric flora, any descent in functionality in the immune could allow the
opportunity for pathogenic and/or enteric microbes to establish themselves in abundance with resultant
decrease in GIT function and diarrhoea.
2.3. Detailed pathophysiology of diarrhoea
7
2.3.1. Inflammation in diarrhoea
Inflammation is the body’s first line of defence against infection and hazardous stimuli in people and animals
(Iwalewa et al., 2007) with injury or infection in the GIT, resulting in the activation of neutrophils and
macrophages. Once activated, the immune cell (e.g. macrophage) assist with the killing of pathogenic
microorganisms and/ or the removal of harmful and cell debris (Stables and Gilroy, 2010). This task is achieved
through the release of numerous pro-inflammatory cytokines (tumour necrosis factor [TNF]-α and interleukin [IL]1β, IL-3, IL-6); chemokines and chemoattractants (IL-8 and monocyte chemoattractant protein [MCP]-1) (Conforti
et al., 2008) (Fig 2.2).
Infections
Trauma
CSFs
Inflammatory disease
Macrophages
TNF-α
IL-1
Tissue cells
IL-8
Neurokinins
Viral infection
IL-6
Chemokines, CSFs,
Neurokinins, Growth
factor, PGs
IL-12
IL-3
IFN-γ
IFN-β
Fig. 2.2. Cytokines production network in the tissues (modified from Hopkins, 2003)
(IL=interleukin, TNF=tumour necrosis factor, CSF=colony stimulating factor, PGs=prostaglandins, IFN=interferon).
Other inflammatory nediators include ROS and RNS, eicosanoids such as cyclooxygenase products
(prostaglandin E2 (PGE2) or lipoxygenase products (leukotrienes (LTB4) (Nardi et al., 2007) (Fig 2.3), pain
provoking mediators (histamine and bradykinin) (Matu and van Staden, 2003), and/ or cationic antimicrobial
peptides (CAMP). Another antimicrobial properties of inflammation is disruption of the epithelial lining which limit
microbial survival and colonization of the GIT in inflamed intestine due to loss of replication niche
Cell membrane
cPLA2
Arachidonic acid (AA)
COX-1, COX-2
PGG2
5-LOX
5-HPETE
8
COX-1, COX-2
5-LOX
PGH2
PGD-S
PGE-S
PGD2
PGE2
LTA4
PGF-S
PGF2
TXA-S
PGI-S
TXA2
PGI2
12/15-LOX
LTA4-S
LTB4
LXA4
LTC4-S
LTC4
Fig.2.3. Biosynthetic pathways for the eicosanoids (modified from Haeggstrom et al., 2010)
(COX=cyclooxygenase, LOX=lipoxygenase, S=synthase, PG=prostaglandin, LT= leukotriene, TXA=thromboxane, PGI=prostacyclin,
cPLA2=phospholipase A2)
While inflammation process is beneficial to the body as it removes the insulting cause (Lee et al., 2007a;
Pharaoh et al., 2006) the large recruitment and activation of neutrophil and macrophages can induced changes
in gut motility, neuronal functionality, and hydroelectrolyte movement with resultant diarrhoea (Gelberg, 2007).
Some infectious enteric pathogens elicit inflammatory cascade and mediators to manifest diarrhoea (Guttman
and Finlay, 2009).The mechanisms involved in the inflammatory modulated diarrhoea may include several
factors listed below and shown in Fig 2.1.
Epithelial barrier disruption: Gastrointestinal epithelium barrier provide a physical defence against
hostile environment within the intestine lumen (Blikslager, 2010). The intestinal barrier is determined by
interactions among several barrier components including the adhesive mucous gel layer, the mucosal
immune system and the tight junctions (TJs) (Schenk and Mueller, 2008). The intercellular TJs are the
most essential component of the intestinal physical barrier. TJs are multiple protein complexes located
around the apical end of the lateral membrane of the epithelial cells. It performs dual functions as a
selective/semipermeable paracellular barriers allowing movement of ion, solutes and water through the
intestinal epithelium while also preventing the translocation of luminal antigens, microorganisms and
their toxins into the mucosa (Groschwitz and Hogan, 2009; Guttman and Finlay, 2009. Disruption of the
intestinal TJ barrier by inflammatory cytokines, reactive oxygen species and pathogens (Guttman et al.,
2006) impair intestinal TJ function cause an increase in intestinal permeability resulting diarrhoea
(Schenk and Mueller, 2008) as shown in Fig. 2.4.
9
Fig. 2.4. The mechanisms of intestinal epithelial tight junctions as a physical barrier to movement of selected solute
materials across the GIT. The intestinal TJs tightly regulate intestinal paracellular permeability. The barrier impairment
induced by extracellular stimuli, such as inflammatory cytokines and reactive oxygens, allows the lumina bacterial products
and dietary antigens to cross the epithelium and enter circulation (Suzuki and Hara, 2010).
Reduced absorption capacity: Nutrient-coupled absorption of electrolytes takes place in the brush
border microvilli (Dudeja and Ramaswamy, 2006). In an inflamed or infected intestinal tract, the total
absorptive surface area is decreased due to brush border shortening resulting in malabsorption (Cotton
et al., 2011). Small intestinal malabsorption occurs due to impaired absorption of water, glucose and
electrolytes creating an osmotic gradient that draws water into the small intestinal lumen resulting small
intestinal distension and rapid peristalsis, consequently diarrhoea (Schulke et al., 2009; Gelberg, 2007;
Beavis and Weymouth, 1996).
Chloride ion hypersecretion: Diarrhoeal agents such as inflammatory mediators, microbial toxins,
neurotransmitters and endogenous hormones can activate inappropriate chloride ion (Cl−) secretion
from the colonic crypt epithelial cells. Excessive secretion of chloride ion (Cl−) from the intestinal crypt is
the driving force for many diarrhoea aetiologies. The underlying mechanism is the increase in
intracellular levels of cyclic nucleotides (cAMP and cGMP) and/or cytosolic calcium. This process, in
turn, drives the secretion of fluid and electrolytes into the intestinal lumen, which may overwhelm the
intestinal absorptive mechanism, thereby resulting in secretory diarrhoea with potential effect of severe
dehydration (Petri Jr. et al., 2008).
Interference with ability to digest: Inflammatory response in the intestine may negatively affect the ability
of the enterocyte to digest nutritional material. The process causes maldigestion occurs due to a
deficiency in various brush border digestive enzymes, especially for carbohydrate and lipids (Schulke et
10
al., 2009). The high level of undigested carbohydrate and lipids are conversion to short chain fatty acids
by the colonic microbiota and the amount may exceed colonic capacity for their absorption. Excess
short chain fatty acids induced osmotic gradient pulling water and secondarily, ions into the intestinal
lumen resulting in osmotic diarrhoea of colonic origin (Field, 2003). Maldigestion of ingested food
coupled with osmotic diarrhoea ultimately lead to long-term malnutrition in affected host (Ogoina and
Onyemelukwe, 2009).
Inflammatory mediators as secretagogue: The release of cytokines such as interleukin-8 (IL-8) and
eicosanoids into the gastrointestinal tract activated the macrophage of the immune system. The
activated macrophages release inflammatory mediators such as PGE2, LTE4, platelet activating factor
(PAF), histamine, serotonin, adenosine resulting in cell damage mediated by T lymphocytes or
proteases and oxidants generated (H2O2, O2˙–, OH˙, NO) by mast cells. Some of the inflammatory
mediators (PGE2, LTB4, histamine) also serve as secretagogue causing secretory diarrhoea (Field,
2003).
Stimulate enteric nervous system (ENS): Inflammation causes structural changes to the ENS that
ranges from axonal damage to neuronal death (Stanzel et al., 2008). The changes include altered
neurotransmitters synthesis, storage and release, therefore contributing to the altered intestinal motility
during the onset and progression of many GIT disorder (Stanzel et al., 2008) (See section 2.3.3 for
more detailed).
2.3.2.
Oxidative damage in diarrhoea
Excessive generation of reactive oxygen species (ROS) or reactive nitrogen species (RNS) by the intestinal
immunological system as a result of intestinal infection, irritation, inflammation, and depleted endogenous
antioxidant defence causes oxidative stress (Granot and Kohen, 2003). This condition has been implicated as
one of the causes of diarrhoea (Peluso et al., 2002; Granot and Kohen, 2003).
The pathophysiology of oxidative stress (production) is complex and results from the normal immune response in
conditions of disease (infectious and non-infectious), and is initiated by activated mitochondrial of the leukocyte.
The free radicals produced are unstable and highly reactive charged function to destroy invading organism
(Dwyer et al., 2009). The mechanisms of ROS and RNS production involved an incomplete reduction of oxygen
and nitrogen in the electron transfer chain of respiratory process in the mitochondria. In addition, immune
reactions during infection or autoimmune responses through inflammation activation of a variety of inflammatory
cells, which in turn activate the oxidant-generating enzymes including NADPH oxidase, inducible nitric oxide
synthase (iNOS), myeloperoxidase, and eosinophil peroxidase. The ROS generated in the body are superoxide
anions, hydroxyl radical, singlet oxygen and hydrogen peroxide (from leukocyte respiratory burst). The RNS
included nitric oxide (NO) (produced by inducible nitric oxide synthase (iNOS). Other miscellaneous reactive
species are reactive halogen and pseudohalogen species (produced by myeloperoxidase, eosinophil peroxidise,
11
lactoperoxidase). It is well-established in vitro that free radicals may also be generated via transition metalmediated oxidation, the so-called Fenton type chemistry, but due to the limited availability of unbound transition
metals, these reactions are probably unlikely to play a major role as a source of oxidants in vivo (Chen et al.,
2000).
However, since their effect is usually non-specific and aimed at the lipid membrane, the chain reaction initiated
by the immune system will destroy the body’s macromolecules unless scavenged (terminated). At normal
physiological conditions a balance is maintained between amounts of free radicals generate and endogenous
antioxidant defence system that scavenged/quenched the radicals preventing their harmful effects. Cellular
antioxidant endogenous defence mechanisms are divided into three parts depending on their function:
o
Quenching antioxidants: The tissue have inherent antioxidant network capable of donating electrons to
oxidants, thus quenching their reactivity under controlled conditions and the derivatives become
harmless to cellular macromolecules. The antioxidants however become radicals themselves, but far
more stable incapable of inducing cellular damage. The oxidised antioxidants are subsequently recycled
to their active reduced state by a number of efficient cellular processes fuelled by energy from NADPH.
This recycling process is the main key to the efficiency of the antioxidant network.
o
Repairing/removing oxidative damage: This level of antioxidant defence involves the ability to detect
and repair or remove oxidised and damaged molecules before it become a threat to normal
body.physiological process.
o
Encapsulating non-repairable damage: Finally, the body is also equipped with controlled cell suicide or
apoptosis, if the extent of the oxidative damage exceeds the capacity of repair and removal.
However, a shift in favour of the radical generation, increase the burden in the body (oxidative stress) which
causes tissue injury and subsequently diseases. The proposed mechanisms through which these products
induced diarrhoea are presented in Fig 2.5 and discussed below:
Lipid peroxidation are primary mechanisms for intestinal cellular malfunction, and can destroy the
capacity of membranes to maintain ionic gradients resulting in an aberration in ion transport, particularly
affecting potassium efflux and sodium/calcium influx (Dudeja and Ramaswamy, 2006). The production
of arachidonic acid metabolites in the lipid peroxidation process can also contribute to intestinal
dysfunction including diarrhoea. The ROS and RNS-induced lipid peroxidation process involves three
major stages (Catala, 2009): the initiation stage, where the oxidant abstracts hydrogen from
polyunsaturated fatty acids of the cell membrane, forming a radical lipid. The propagation stage may
involve the rearrangement of the lipid radical to form conjugated dienes and can interact with oxygen to
form lipid peroxide radicals.
Enterotoxins
Enteric infection/irritant
12
Pro-inflammatory mediators
Leukocytes
Pro-oxidant
Oxidative burst
ROS/RNS
Inflammation
Oxidative stress
Ca2+ dependent Cl- channel
ENS
Neurotransmitters
Increase Cl¯ secretion
Altered motility
Lipid peroxidation
Intestinal mucosal damage
Increased paracellular permeability
decrease absorption
Diarrhoeal symptoms
Fig.2.5 The integrative pathophysiology and mechanism of diarrhoeal disease
Bold (pathogenesis and areas of possible pharmacological intervention in diarrhoea), italic (mechanisms through which diarrhoea manifest)
The peroxide radicals can in turn abstract hydrogen from lipids to produce lipid hydroperoxide and a
new radical. Lipid hydroperoxides can be oxidized, via reaction with reduced iron (Fe2+) to lipid alkoxy
radical and lipid peroxide, thus continuing the chain reaction of lipid peroxidation. In the final stage, the
lipid peroxide radicals in the presence of reduced metals can be degraded to form highly reactive and
potent toxic aldehydes such as malondialdehyde (MDA) (Fig 2.6 and 2.7). The chain reaction can be
terminated by endogenous antioxidant enzymes and exogenous antioxidant molecules by forming nonreactive substances (Catala, 2009).
Oxidating enzyme + O2
O2˙–
1
2O2˙– + 2H+ (SOD)
2H2O2 + O2
2
H2O2 + Fe2+
OH- + OH˙ + Fe3+
H2O2 + O2˙– + Fe3+
OH- + OH˙ + Fe2+ + O2
2H2O2 + O2˙
2OH- + 2OH˙ + O2
–
2O2˙ + NO
ONOO
(Fenton reaction)
ONOOH
3
(Haber-Weiss reaction)
5
NO2˙ + OH
-
LH + ˙OH → L˙ + O2 → LOO˙ + LH → 2LOOH + 2L˙ + O2 → 2LOO˙+ 2LO˙
LOO˙
DE 1
DE 2 +O2/HS
DE 3
4
MDA
6
7
HAC
8
13
2LO
DA 1 - O2, H. (β-scission)
H20 + 4-HNE
9
Figure 2.6: Lipid peroxidation chain reaction (Valko et al., 2007).
(Equations 1 is generation of superoxide by enzymes such as NAD(P)H oxidase, xanthine oxidase and mitochondria, 2) Superoxide radical is dismutated
by the superoxide dismutase (SOD) to hydrogen peroxide, 3 and 4) hydroxyl radical and hydroxyl ion hydrogen peroxide in the presence of transitional
metal, 5 and 6 chain reaction to generate more radicals, 7 lipid peroxidation of phospholipids, 8 cyclization and scission of the lipid peroxide radical to
generate cytotoxic malonydialdehyde (MDA), hydroxyacrolein (HAC), 4-Hydroxy nonenal (4-HNE) (Fig.2.7).
H
H
O
OH
O
O
O
O
cyclization o f D E 1 (DE. 2)
cyclization o f LO O (D E.1 )
O
O
HO
H
malonydialdehyd e
OH
H
H
O
O
O
cyclization of D E.2 (DE. 3)
O
C
H
H ydroxya crolein
4-Hydroxynone nal
H
Fig.2.7. Chemical structures of lipid peroxidation intermediates outlined in Fig 2.6
Some of the reactive species such as HOCl and NH2Cl can also act as secretagogues on their own or
can evoke the release of acetylcholine or other neurotransmitters, thus stimulating the enteric nervous
system (ENS) to cause increased contractility or motility of intestinal tract (Gaginella et al., 1992). The
reactive species also induce gene expression by stimulating signal transduction such as Ca2+-signalling
and protein phosphorylation.
Increased production of inflammatory mediators: The onset of lipid peroxidation process leads to
changes in the physiological integrity of the cell membrane. The body responds to the process by the
release of pro-inflammatory eicosanoids such as (prostaglandins, prostacyclins and leukotrienes) and
pro-inflammatory cytokines (Nardi et al, 2007) such as interleukins (IL-1B, IL-3,IL-6), interferons (IFN),
tumor nuclear factor (TNF-α) and platelet-activating factor (PAF) (Conforti et al, 2008; Kunkel et al,
1996).
2.3.3. Enteric nervous system in diarrhoea
The enteric neural network is responsible for the control of propulsive transport and segmental peristalsis in the
GIT, as well as secretion and absorption across the intestinal lumen (Wood, 2004; Bohn and Raehal, 2006).
While enteric nervous system (ENS) functions independently of the central nervous system (CNS), it is
modulated by the parasympathetic and sympathetic autonomic nervous system (Farthing, 2003). As a unit, the
ENS is a complicated physiological with autoregulation being mediated by a number of neurotransmitters such
as acetylcholine, serotonin, substance P, histamine and endorphin (Farthing, 2002). Diarrhoea can result from
the alteration of these systems:
14
Smooth muscle contractility: Many agonists and/or antagonists elicit contractility in GIT smooth muscle
(longitudinal or circular) through activation of various receptors located within the muscle (Holzer, 2004).
In some cases the activation of the smooth muscle receptors by neurotransmitters and inflammatory
mediators include reactive oxygen species causes relaxation (spasmolytic). While in other cases, the
process lead to increase in spontaneous or induced contraction (spasmogenic). Ionic channel (Ca2+ and
Cl-) are also known to play important roles in smooth muscle contraction (Giorgio et al., 2007). Anion
and fluid secretion into the intestine lumen are stimulated through activation of the receptors on enteric
secretomotor pathways and epithelial cells, consequently causing secretory diarrhoea.
Motility: Intestinal motility dysfunctions include situations in which movement of material along the GIT is
repetitive and rapid (diarrhoea) and/or too slow (pseudo-obstruction, slow transit constipation) (Talley,
2006; Giorgio et al., 2007) are controlled by activities of neurotransmitters on the ENS. Pathogenic
bacterial overgrowth is common as a result of intestinal hypomotility or low transit time which may lead
to mucosal inflammation, increased accumulation and absorption of toxins which are known
pathophysiology of diarrhoea. The mechanisms may include impaired digestion as in the deconjugation
of bile salts with subsequent fat malabsorption, leading to fatty acid diarrhoea or osmotic effects of
malabsorption of sugars resulting in osmotic diarrhoea. Diarrhoea also results from an increase in the
gut motility (hypermotility) inducing an accelerated transit of food intake. The net fluid absorption from
the food intake is reduced due to less adequate contact time with the GIT epithelial lining for the
absorption of fluids before excretion.
2.3.4. Cystic fibrosis transmembrane conductance regulator (CFTR) regulation
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic adenylate monophosphate (cAMP)activated Cl- channel expressed in epithelial cells in the intestine and other fluid-transporting tissues (Thiagarajah
and Verkman, 2003). Diarrhoeal pathogens and their toxins can induce secretory diarrhoea by simultaneously
stimulation of the active secretion of Cl- and inhibition of Na+ absorption across the apical membrane of
enterocyte with resulting massive fluid and electrolyte loss into GIT (Schuier et al., 2005). The cellular signalling
mechanisms include an increase in cellular cAMP and cyclic guanylate monophosphate (cGMP), which may
result in activation of the CFTR Cl- channel. Pharmacological blocking of CFTR with drugs such as glibenclamide
and CFTRinh-172 inhibits salt and water loss in diarrhoea (Schuier et al., 2005).
2.4. Specific Agents of Diarrhoea
2.4.1. Bacterial causes of diarrhoea
2.4.1.1. Escherichia coli
E. coli is a gram-negative rod shaped bacteria that shares a symbiotic relationship with animal host as part of
normal digestive intestinal flora. Under certain define conditions these organisms or pathogenic strains of these
organisms are known to induce diarrhoea (Clarke, 2001; Le Bouguenec, 2005). There are six main types of
15
pathogenic E. coli associated with diarrhoea, namely enterotoxigenic E. coli (ETEC), enteroinvasive E .coli
(EIEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC)
and diffusively adherent E. coli (DAEC) (Clarke, 2001). While the exact process by which each type of these E.
coli induces diarrhoea symptoms varies significantly, the basic pathophysiology involves their inherent ability to
adhere to epithelial cells and colonize the host tissues (Le Bouguenec, 2005). The characteristics and mode of
actions of each type of the pathological strains in diarrhoea diseases are listed in Table 2.1. Infections from some
of the strains of E. coli are self-limiting and can resolve without pharmacological intervention. However,
symptomatic, supportive and antibiotic, or a combination of the therapies may be beneficial in the
chemotherapeutic management of some cases involving ETEC, EIEC and EPEC infection (Elsinghorst, 2002).
The use of antibiotics recommended, antimicrobial chemotherapeutic agents such as tetracycline, doxycycline,
and ciprofloxacin may be used (Casburn-Jones and Farthing, 2004; Elsinghorst, 2002) in infectious diarrhoea.
16
Table 2.1: The mechanism of actions and symptoms of enteric pathogenic E. coli (Thapar and Sanderson, 2004;
Clarke, 2001).
Strain
Mechanism of action
Enterotoxigenic
coli (ETEC)
E.
Enteroinvasive
coli (EIEC)
E.
Enterohaemorrhagi
c E. coli (EHEC)
Enteropathogenic
E. coli (EPEC)
Enteroaggregative
E. coli (EAEC)
Diffusively adherent
E. coli (DAEC)
Colonization of the small bowel mucosa, followed by
elaboration of heat-labile (LT) and heat stable (ST)
enterotoxins. The ST enterotoxins are classified as STa
and STb. The binding of STa to guanylate cyclase C
receptor results in increased intracellular cyclic
guanylate monophosphate (cGMP) level. The resultant
effect is the stimulation of chloride secretion or
inhibition of sodium chloride absorption causing
intestinal fluid secretion. LT enterotoxins consist of two
serotypes (LT-I and LT-II). LT activate adenylate
cyclase causing intracellular increase in cyclic
adenosine monophosphate (cAMP) levels resulting in
decrease sodium absorption by villous cells and
subsequent active chloride secretion by crypt cells thus
leading to osmotic diarrhoea.
Invasion of the epithelium and mucosal destruction
eliciting inflammatory response accompanied by
necrosis and ulceration of the large bowel with
resultant release of blood and mucosa into the stool.
Adhesion followed by liberation of a potent toxin which
is cytotoxic to Vero cells (referred to as shiga-like
cytotoxin I and II). Other mechanism attributed to the
EHEC virulence includes adhesin
Adherence of the bacterium to the gut epithelium
causing attachment and effacement lesion on intestinal
epithelial cells, alteration of intracellular calcium and
cytoskeleton.
Aggregating pattern of adherence to intestinal mucosa
produces enteroaggregative heat-stable (EAST)
enterotoxins causing cellular damage and function
similar to, but distinct from ST enterotoxins.
Elaboration of α-haemolysin and cytotoxic necrotising
factor 1 (CNF-1).
Symptom
s
Watery diarrhoea
ranging from mild,
self-limiting disease
to severe purging.
Treat
ments
Supportive
therapy
with
antibiotic
in
cases of severe
infection.
Bloody,
diarrhoea
dysentery
Antibiotic
in
cases of bloody
diarrhoea
mucoid
and
Bloody diarrhoea,
fever,
vomiting,
haemorrhagic
colitis, haemolytic
uremic syndrome
(HUB), acute renal
failure, haemolytic
anaemia,
Self-limiting watery
diarrhoea with fever
and vomiting
Symptomatic
therapy
Watery
diarrhoea
Antibiotic
in
severe cases
mucoid
Watery diarrhoea
Antibiotic
in
severe cases
Supportive
therapy
2.4.1.2. Staphylococcus aureus
S. aureus is a gram-positive coccus present in normal intestinal and skin flora of human and homoeothermic
animal. Under define conditions, the pathogenic strains produces heat stable staphylococcal enterotoxins (SETs)
and toxic shock syndrome toxins (TSST-1) (de Oliveira, 2010) both of which are known to induce diarrhoea.
Toxicity from SET results from the consumption of the preformed heat-stable enterotoxins (α-haemolysin) in
contaminated food. Upon ingestion of the food contaminated with the SETs, the toxins results in signs of nausea,
vomiting, fluid accumulation in ileal loops, and diarrhoea associated with fever (Rosengren et al, 2010; Perez17
Bosque and Moreto, 2009). The main sources of S. aureus toxin contaminants are raw material and food
processing unit such as human handling, water and environment (Linscott, 2011).. Serotonin receptor
antagonists have been reported to ameliorate the vomiting, diarrhoea and prostration induced by SETs . The
mechanism behind toxicity results from the activation of autonomous nervous system with resultant
hyperperistalsis as well as activation of central pathways which control vomiting and diarrhoea (Podewils et al,
2004).
In contrast, TSST-1 is characterized by sudden onset of fever, vomiting, diarrhoea, erythematous rash with skin
peels, hypotensive shock, impairment of renal and hepatic functions, and sometime death. Toxicity results via
the production of pro-inflammatory cytokines and chemokines. Toxicity is usually exacerbated by further
interaction between the activated immune system and inflammatory mediators (Krakauer et al., 2001).
2.4.1.3. Campylobacter jejuni
C. jejuni is an invasive Gram-negative, spiral-shaped rod bacterium present in the GIT of mammals, birds and
primates (Lengsfeld et al., 2007). The major source of Campylobacter infection in mammals is from poultry and
poultry products (Podewils et al, 2004). The clinical signs of campylobacter infections include pyrexia, abdominal
pains, watery diarrhoea and dysentery (Podewils et al, 2004). The characteristic mechanisms Campylobacter
infection involves invasion and translocation of the epithelium with a concomitant induction of inflammation (Hu et
al., 2008).
2.4.1.4. Shigella spp
Shigella (Shigella flexneri, Shigella dysenteriae, Shigella sonnei and Shigella boydii) is a Gram-negative rod, non
motile and facultative anaerobic bacterium that invades the colon with resulting inflammation and diarrhoea
(Podewils et al, 2004). Shigella flexneri is responsible for dysenteric symptoms and persistent illness while
Shigella dysenteriae type-1 produces Shiga-toxin like EHEC causes bloody diarrhoea (Podewils et al, 2004).
Shigella sonnei causes bacterial gastroenteritis and bacillary dysentery and Shigella boydii causes fever, chills,
abdominal pain and diarrhoea.
2.4.1.5. Vibrio cholerae
V. cholerae is a motile, facultative anaerobic Gram-negative rod associated with potentially fatal diarrhoea
(Granum, 2006) that results from the ingestion of the cholera enterotoxins (CT) from contaminated water and
seafood (Podewils et al, 2004). Watery, colourless mucous- flecked stool and vomiting are the main clinical signs
associated with cholera which in severe cases can result in a life-threatening fluid and electrolyte imbalance
(Podewils et al, 2004). Pathophysiologically, toxicity results from the CT induction of intestinal hypersecretion
through the activation of the mucosal epithelium cAMP-adenylate cyclase system in the mucosal epithelium
(Casburn-Jones and Farthing, 2004).
18
In addition, it has been speculated that ROS/RNS production in V. cholerae infection could also contribute to
intestinal damage through lipid peroxidation of the cellular and mitochondrial membrane thereby further
increasing membrane permeability and fluid loss (Gorowara et al., 1998). Other species of Vibrio such as V.
parahaemolyticus and V. vulnificus also caused watery diarrhoea, abdominal cramps, nausea, vomiting. These
organisms infect host from raw or undercooked seafood or cooked seafood contaminated with seawater
(Linscott, 2011).
2.4.1.6. Bacillus cereus
B. cereus is a sporulating bacterium that causes both food poisoning and non-gastrointestinal infection (Al-khatib
et al., 2007; Ramarao and Lereclus, 2006). In food poisoning, two main types of diseases namely diarrhoeal and
emetic food poisoning are common. The diarrhoeal type of B. cereus food poisoning is caused by enterotoxins
such as haemolysin BL (HBL), non-haemolytic enterotoxin (NHE) and cytotoxin K (CytK) (Lund et al., 2000).with
clinical signs of abdominal pain with diarrhea. Causes of the diarrhoeal forms always occurs from accidental
contamination of food like meat, vegetables, pasta, deserts cakes, sauces and milk (Linscott, 2011).
In constrast the emetic form is induced by a small preformed heat and acid stable cyclic peptide (cereulide)
(Agata et al., 1995; Ehling-Schulz et al., 2004) with clinical symptoms of sudden onset of nausea and vomiting,
with or without diarrhoea (Linscott, 2011). The major sources include cooked foods, like meat or fried rice that
have not been properly refrigerated. While the other species of Bacillus such as B. subtilis, B. licheniformis, B
pumilus and B. megaterium are usually considered relatively safe, but they can also produce enterotoxins and
emetic toxins involved in foodborne illness (From et al, 2007)
2.4.1.7. Yersinia species
Yersinia species are Gram-negative facultative anaerobic nonsporing rods or coccobacilli bacteria belonging to
the Enterobacteriaceae family. Three human pathogenic species namely: Y. pestis, Y. enterocolitica, and Y.
pseudotuberculosis are recognized (Fallman and Gustavsson, 2005). Y. pestis is the causative agent of bubonic
plague characterized with the onset of fever, chills, headache, and weakness, followed by swelling and
tenderness of lymph nodes while Y. enterocolitica and Y. pseudotuberculosis cause an enteric infection in
humans called yersiniosis with clinical signs such as diarrhoea, vomiting, fever and abdominal pain (may mimic
appendicitis) following ingestion from undercooked pork, unpasteurized milk, tofu, contaminated water,
chitterlings (Linscott, 2011, Damme et al., 2010).
2.4.1.8. Listeria monocytogenes
L. monocytogenes is a Gram-positive bacterium which causes life-threatening invasive diseases referred to
listeriosis in human and animals (Chaturongakul et al., 2008; Todd and Notermans, 2011). Upon ingestion of the
bacteria from contaminated foods such as unpasteurized milk, soft, cheese made with unpasteurized milk
19
Linscott, 2011), the organism may, colonize the intestinal tract with resultant diarrhoea (Chaturongakul et al.,
2008).
2.4.1.9. Clostridium spp
C. difficile is an anaerobic, spore-forming, Gram-positive bacillus widely distributed in the environment and
present in the colon flora of less than 3% of healthy adults (Beaugerie et al., 2003). C. difficile causes a spectrum
of diseases ranging from benign diarrhoea to fatal colitis and most often as a consequent of antibiotics treatment
Most antibiotics predispose C. difficile overgrowth leading to the production and accumulation of and diarrhoea
are Toxins A (enterotoxin) and B (cytotoxin) in the intestine. Both toxins A and B inactivate intracellular Rhoproteins by glycosylation, leading to desorption of the cytoskeleton, production of inflammatory cytokines and
damage to tight junctions The most commonly antibiotics associated with C. difficile overgrowth include
cephalosporins, clindamycin and broad-spectrum penicillins (Wistrom et al., 2001).
In contrast, C. perfringens is an important food poisoning bacterium with clinical sign as diarrhoea, abdominal
cramping and nausea. The main sources of infection include contaminated meat, poultry, gravy and inadequately
reheated food (Linscott, 2011). C. botulinum may also play a role in diarrhoeal diseases when the preformed
botulinum toxin is consumed from improperly canned foods, herb-infused oils, baked and potatoes in aluminium
foil. Symptoms of infection include abdominal cramping, nausea, vomiting, diarrhea, double vision, long term
nerve damage and possible even death from paralysis (Linscott, 2011).
2.4.1.10. Salmonella typhimurium
S. typhimurium is a bacterium that may be associated with mild gastroenteritis to enteric (typhoid) fever,
bacteraemia and septicaemia commonly referred to as salmonellosis (Mastroeni and Maskell, 2006). The clinical
signs of salmonellosis include diarrhoea, fever and abdominal cramps. In people with typhoid fever, Salmonella
spreads systemically from the gut to blood stream and other parts of the body resulting in mortality if not treated
adequately with antibiotics (Castillo et al., 2011). The virulence of the Salmonella bacterium differs among the
different animal species depending on Salmonella serovar involved, strain, infective dose, host animal species,
age and immune status of the host (Castillo et al., 2011). The pathogenesis of Salmonella involves
adhesion/invasion to specific intestinal epithelial cells, mainly in the ileum (Guttman and Finlay, 2009).
2.4.1.11. Enterococcus faecalis
E. faecalis is a gram-positive bacterium that survives symbiotically in the human or animal’s intestinal tract.
However, under conditions such as the disruption delicate host-commensal relationship following antibiotic use,
abdominal surgery or changes in host immunity, the enterococci becomes invaders of the intestinal wall (Butler,
2006) through the production of adhesin, aggregating and binding substances (Butler, 2006). E. faecalis is
known to produce superoxide (O2.-) that can results in hydroxyl radical formation which contributes to oxidative
20
stress in the intestine and membrane lipid peroxidation (Huycke and Moore, 2002; Sun et al, 2010; Peluso et al.,
2002; Granot and Kohen, 2003) (Fig 2.5).
2.5. Fungal induced diarrhoea symptoms
2.5.1. Candida albicans
C. albicans is a yeast fungus and exist as a member of normal flora in the GIT and mucocutaneous membrane
(Forbes et al., 2001). However, following the use of antibiotic therapy that result in sterilization of the GIT flora, C.
albicans can overgrowth to take the place of removed organisms with end result of diarrhoeal symptoms(HenryStanley et al., 2003). Other predisposing factors include altered intestinal permeability and diminished host
immunity response. It has been postulated that this organism produces virulence factors which increases fungal
adherence to host cells, fungal secretion of proteolytic enzymes and fungal morphological switching (ability to
change and grow in several distinct morphological forms: yeast, hyphae, and pseudohyphae, according to
environmental conditions) (Henry-Stanley et al., 2003). Clinical signs associated with enteric candidiasis are
abdominal pain, cramping, rectal irritation and absence of nausea, vomiting, bloody and mucus stool, and
pyrexia (Levine et al., 1995).
2.6. Viral induced diarrhoea
2.6.1. Rotavirus
Rotavirus is a major cause of severe diarrhoea and account for 30% and 80% of all cases of acute
gastroenteritis (Savi et al, 2010). The diarrhoeal mechanism of the organism includes the production of
enterotoxin NSP4 which inducesd Na+-glucose dependent malabsorption and destruction of enterocytes
(cytotoxicity). The toxin also has a direct effect on the intestinal barriers by blocking TJs formation with resultant
diarrhoea through a ‘leak flux’ mechanism in which water is secreted into the lumen of the intestine (Dickman et
al., 2000).
2.6.2. Norovirus
Norovirus is considered one of the major global causes of gastroenteritis (Mattison, 2010) The diseases is
opportunistic and is usually transmitted through faecal contamination of food, water or contact with an infected
host following poor hygiene. The organism has the ability to infect small intestine and induce intestinal TJ
dysfunction, malabsorption through villus surface area reduction and an increased number of cytotoxic
intraepithelial lymphocytes (Troeger et al., 2009). Clinical signs associated with infection are nausea, vomiting,
diarrhoea and abdominal pains (Koopmans, 2008).
2.6.3. Hepatitis A virus
Hepatitis A is a small, non-enveloped spherical with cubic symmetry, thermostable and acid resistant virus. While
the primary target organ of infection is the liver, the resultant hepatitis (Koff, 1998) result in clinical signs dark
21
urine, jaundice, malaise, weakness, fever, anorexia, nausea and vomiting, abdominal pains, and diarrhoea (Koff,
1992). Sources of infection usually result from contaminated water on raw produce, food, and shellfish or
exposure to the water itself (Linscott, 2011).
2.6.4. Human immunodeficiency virus (HIV)
Chronic diarrhoea is one of the complications associated with HIV infection and acquired immune deficiency
syndrome (AIDS) due to multiple enteric opportunistic microbes (DuPont and Marshall, 1995). While HIV is
important in secondary enteric diseases as a result of immune suppression (CD4+ T-lymphocytes destruction),
the virus can result in diarrhoea directly by altering the mucosa structural arrangement viz referred to as HIVenteropathy (Epple et al., 2009). The diarrhoea resulting from HIV appears to be caused by the releases of
cytokines from the infected immune cells (Schmitz et al., 2002).
2.7. Protozoa induced diarrhoea
2.7.1. Giardia intestinalis
G. intestinalis (syn. Giardia doudenalis, Giardia lamblia) is a flagellate protozoa parasite of the upper small
intestine that exists as a motile trophozoite (Cotton et al., 2011). The organism colonizes the small intestinal
lumen and induces non-inflammatory and malabsorptive diarrhoea (Schulzke et al., 2009). The pathophysiology
of Giardiasis involves Na+-dependent D-glucose absorption impairment, active electrogenic anion secretion
activation, mucosal inflammation and leak flux (Buret, 2007; Troeger et al., 2007). Clinical signs of Giardia
infection include bloating, steatorrhea and nausea. Chronic infection cause weight loss, growth retardation and
development in young children
2.7.2. Entamoeba histolytica
E. histolytica is a protozoa parasite which infects the large intestine with resultant intestinal dysfunction
characterized by invasive illness and severe dehydration commonly referred to as amoebiasis (Ralston and Petri,
2011). The pathophysiology of amoebiasis involves villus structural destruction, increased epithelial permeability
and alteration of TJs (Lauwaet et al., 2004). The organism also causes invasive disease such as colitis and
abscess through massive host tissue destruction. The clinical signs are usually similar to S.dysenteriae or
enteroinvasive E. coli with blood and pus contaminated stool. Other related infectious species include E. dispar
and E. moshkovskii (Ralston and Petri, 2011).
2.7.3. Cryptosporidium parvum
C. parvum is an intracellular protozoa parasite that infects epithelia causing cryptosporidiosis (Kenny and Kelly,
2009) which manifest clinically as profuse watery diarrhea, containing mucus, but rarely blood or leukocytes
22
(O’Hara and Chen, 2011). Some other clinical signs of cryptosporidiosis include nausea, vomiting, cramp-like
abdominal pain and mild fever (Linscott, 2011). The period and severity of clinical symptoms of intestinal
cryptosporidiosis depends on the immune status of the infected individual (Linscott, 2011). Cryptosporidiosis in
the healthy individuals is usually a self-limiting illness with approximate duration of 9-15 days while in
immunocompromised patient the infection in severe and life-threatening (O’Hara and Chen, 2011).
2.7.4. Cyclospora cayetanensis
C. cayetanensis is a protozoan parasite which invades the epithelial cells of the small intestine upon ingestion of
sporulated oocysts in contaminated food or water (Chacin-Bonilla, 2010; Manfield and Gajadhar, 2004).
Pathomechanisms of C. cayetanensis infection are intestinal inflammation associated with pathological lesions
such as villus blunting, and malabsorption. The clinical signs of the infection include watery diarrhoea, loss of
appetite, weight loss, abdominal bloating and cramping, increased flatulence, nausea, fatigue, and low-grade
fever (Linscott, 2011).
2.8. Parasitic induced diarrhoea
2.8.1. Trichinella spiralis
T. spiralis is a food-borne zoonotic parasite responsible an enteral phase and a muscular phase (Cui et al.,
2011). The adult worms live in the duodenal and jejunal mucosa of flesh-eating animals and humans, while the
larvae live in skeletal muscles of the same hosts (Cui et al., 2011). The source of infection is raw or undercooked
contaminated meat (pork, bear, walrus and moose), cross-contaminated ground beef and lamb (Linscott, 2011).
The clinical intestinal symptoms manifest one or 2 days after ingesting the contaminated meat due to the
matured adults penetrating the intestinal mucosa, resulting in nausea, abdominal pain, vomiting, and diarrhoea
(Linscott, 2011). T. Spiralis induced changes in intestinal function by hypersensitivity mechanism resulting in an
increased intestinal chloride and fluid secretion (Cui et al., 2011).
2.9. Immune disordered induced diarrhoea
2.9.1. Compromised immune system
The main function of the immune system is to protect against disease through the recognition and removal of
pathogens or their sequelae (Gertsch et al., 2010). To fulfil this role, the body make use of an innate immune
system that defends it in a non-specific manner via molecular interaction and inflammation (Gertsch et al., 2010)
and an adaptive system comprised of specialized effector cells (T and B cells) which not only recognize antigens
but play a role in their active removal (Gertsch et al., 2010). In the GIT, the important protective system which
prevent the normal flora from becoming pathogenic are gut-associated lymphoids tissue (GALT), epithelialderived antimicrobial peptides (AMPs) (such as defensins, cathelicidinand lysozyme present in the secretion
which constantly washes the mucosal surfaces), the mesenteric lymph nodes, the liver’s Kupfer cells, mast cells,
within the intestinal walls and the reticuloendothelial system of the intestinal walls. In diseases characterized by
immune suppression such as HIV, the immune system is destroyed which makes a person more susceptible to
23
infectious diseases (Gertsch et al., 2010). One of the clinical signs that may result is diarrhoea as the abovementioned micro-organisms colonize the compromised GIT.
2.9.2. Hyperactive immune system
The normal response of the immune system during conditions of antigen stimulation is generally an inflammatory
response with the release of numerous inflammatory mediators (interferon-γ, tumour necrosis factors (TNF-α),
interleukin-6 (IL-6), and IL-1β) which in conjunction plays a role in the removal of the causative agents (Sprague
and Khalil, 2009). On the removal of inciting cause, the inflammatory response is usually terminated. However,
on occasion when the body failed to terminate the inflammatory response, the inflammatory agents can be
equally as destructive to the host’s own tissues. The latter usually result from the damage of the epithelial
mucosa cells, from the generated ROS/RNS and subsequent lipid peroxidation. With the destruction of the
epithelial cells, the body loses absorptive capacity with resultant diarrhoea. The produced cytokines also has the
ability to directly increase intestinal mucosa permeability and fluid loss. Inflammatory bowel disease is one of the
diseases caused by up-regulated immune system (Gertsch et al., 2010).
2.10. Antibiotic therapy induced diarrhoea
Diarrhoea develops in some patient following antibiotic chemotherapy with one of the following mechanisms:
Antibiotic toxicity: Some antibiotic (levofloxacin, azithromycin and amoxicillin-clavulanate) may have a
direct negative effect on the GIT with resultant poor absorption or enteropathy characterized by
infiltration of the lamina propria by macrophage (Dobbins, 1968, Hartmut, 2010). In addition, antibiotic
such as erythromycin has prokinetic action on the GIT, mediated through motilin receptor stimulating
potential (Catnach and Fairclough, 1992, Annese et al., 1992).
Alteration of digestive function: The removal of some commensal organisms by antibiotic (Hofmann,
1977) could result in decreased carbohydrate digestion (Saunders and Wiggins, 1981) which leads to
accumulation of these osmotically active substances in the intestinal lumen. The net result is the
accumulation of electrolytes and water from osmotic pull in the intestinal lumen which is evidence as
diarrhoea (Beaugerie and Pettit, 2004).
Overgrowth of pathogenic microorganisms: The use of antibiotic may result in the removal of beneficial
GIT flora. As a result of the disruption of this balance ecosystem, various pathogenic organisms (as
listed above) can overgrow and colonise the GIT (Beaugerie and Pettit, 2004). The predisposing
antibiotic for various diarrhoeal pathogens include cephalosporins, clindamycin and broad-spectrum
penicillins for C. difficile (Wistrom, 2001; Stoddart and Wilcox, 2002); β-lactams or pristinamycin for
Klebsiella oxytoca (Wu et al., 1999) or tetracycline for S. aureus. Salmonella and Candida overgrowth
can be a consequence of wide range of antibiotic with broad base actions (Danna, 1991; Gupta and
Ehrinpreis, 1990).
24
Antibiotic induced diarrhoea
Antibiotic toxicity on the GIT
Changes in the gut flora ecology
Function diarrhoea
Intestinal infection
Fig. 2.8. Mechanism of antibiotic induced diarrhoea
2.11. Diabetic complications induced diarrhoea
Gastrointestinal disorders manifesting as diarrhoea (watery stool) or constipation (dry and hard stool) is a
common symptom in the diabetic patient (Gould and Sellin, 2009) with a prevalence of approximately 12.5 to
32.4% and 60% respectively. In addition the oral hypoglycaemia medications used for the management of
diabetes viz metformin, acarbose, miglitol (Forgacs and Patel, 2011), exenatide and orlistat (Gould and Sellin,
2009) may also induced diarrhoea as side effect while the recommended dietary material such as the nondigestible sweeteners (sorbitol, mannitol and D-xylose) induce an osmotic diarrhoea Forgacs and Patel, 2011).
Bacterial overgrowth may also result in diabetic patient from decreased functioning of the immune system as
described above (Forgacs and Patel, 2011).
2.12. Food allergy induced diarrhoea
Diarrhoea is one of the clinical manifestations of food allergy (Wang et al., 2010). The mechanisms of action
include active ion secretion, altered epithelial barrier function (Groschwitz and Hogan, 2009), and mucosal
damage resulting in malabsorption and osmotic diarrhoea (Wang et al., 2010). The initiation of food induced
intestinal allergy is regulated by numerous inflammatory cells and mediators, including mast cells and TH2
cytokines (IL-4, IL-5, and IL-13) (Wang et al., 2010). The release of neurotransmitters (serotonin, histamine) and
inflammatory mediator (COX-2 and LOX) by mast cell induced ion secretion in the presence of allergen (Schenk
and Mueller, 2008). These neurotransmitters and inflammatory mediators also stimulate intestinal contractions
(altered intestinal motility) which act synergistically with ion secretion to cause diarrhoea.
2.13. Potential mechanisms in the control of diarrhoea
2.13.1. Oxidative damage and antioxidants in diarrhoeal management
Several endogenous strategies are available in human and animal body to combat oxidative damage. These
provide ways for normal oxidative metabolism to occur in the body without damaging the cells and also allow for
normal ROS/RNS-mediated cellular response such as phagocytosis and intracellular signalling (Valko et al,
2007). Therefore, the possibility exists that returning the animal to a more neutral oxidative balance, may
promote repair of damaged membranes (Nose, 2000). As a result antioxidants and/or radical scavengers may be
25
beneficial in the attenuation of diarrhoea. The best known antioxidants as treatment are selenium, vitamin E,
vitamin C and the proanthocyanidins in red wine and resverastrol in commercial grape seed extract.
2.13.2. Inflammation and anti-inflammatory agents in diarrhoea management
As a result of the negative impact the inflammatory cascade can have on the functionality of the GIT, modulation
of these processes through the use of drugs may be of benefit. Possible mechanisms include attenuation of
inflammatory process through the use of anti-inflammatory, antioxidative and radical scavenging mechanisms.
Potential target include drugs with cyclooxygenase and lipoxygenase enzyme inhibitory activity. The drugs that
are used commonly for this are non- steroidal anti-inflammatory drugs (NSAIDs) like indomethacin, aspirin,
ibuprofen, diclofenac and coxibs.
2.13.3. Enteric nervous system in diarrhoea symptoms and treatment
The ENS is an important target for pharmacological intervention in diarrhoea through the use of agonists and
antagonists that target these ENS endogenous receptors. Numerous pharmaceutical agents are currently
available for alleviating many of the clinical signs of diarrhoea. The effects and possible sites of pharmacological
intervention against the activities of neurotransmitters in diarrhoeal symptoms are presented in Table 2.2.
Table 2.2: Neurotransmitters and modulators of ENS causing intestinal secretion in diarrhoea
Neurotransm
itters
Effects on GIT
Receptors
Acetylcholine
Main
endogenous
excitatory
neurotransmitter in the
GIT.
Nicotinic
and
muscarinic
receptor
subtypes
M1
and M3.
Serotonin
Modulate
muscular
contraction
and
relaxation,
intestinal
fliud secretion and
enhanced
colonic
transit.
Transmitter of enteric
neurones and extrinsic
afferent fibre, control of
GI motility, secretion,
vascular permeability,
immune function and
pain sensitivity
Modulation of GIT
motility, enhancement
of gastric acid secretion,
increases mucosal Clion secretion, and
5-HT3, 5-HT4
Substance P
Histamine
Potential
pharmacological
intervention
Non selective nicotinic
acetylcholine receptor
antagonist or specific
muscarinic
acetylcholine receptor
antagonist
5-HT3
receptor
antagonist (diarrhoea)
and 5-HT4 receptor
agonist (constipation)
NK1, NK2 and
NK3
NK1 and NK2 receptor
antagonist
H1, H2, H3
H1, H2 antagonist
modulator
atropine
Metoclopramide, granisetron,
ondansetro,
tropisetron,
palonsetron,
alosetron,
cisapride
Cimetidine and ranitidine
26
Opioid peptide
Nitric oxide
Dopamine
Motilin
modulator of immune
functions.
decrease
motility,
increase transit time,
increase
fluid
absorption from the
intestine
ENS neurotransmitter,
pro-absorptive
and
secretory agent
Prokinetic
and
antiemetic,
enhance
antral contractility and
inhibit fundus receptive
relaxation
induced
antral
contractility
Mu (µ),delta (δ)
and kappa (ĸ)
µ, δ agonist
iNOS inhibitor,
modulator
D2
antagonist
loperamide
diphenoxylate
NO
racecadotril,
NG-nitro-l-arginine
ester (L-NAME
methyl
Domperidone
Erythromycin,
motilactides
motilides,
2.14. Plants as potential source of therapeutic agents in alleviating diarrhoeal symptoms
Due to the widespread occurrence of diarrhoea as a disease together with the prevalence coinciding with human
development, plants and fungi have featured widely in the management of the disease. Their use has become so
common in human and veterinary medicines that a number of compounds considered to be allopathic are of
natural origin. A non exhaustive list includes:
•
Antioxidant: The natural vitamins and red pigments present in plant.
•
Anti-inflammatory: Salicylic acid from willow bark.
•
ENS modifiers: Atropine from Atropa belladonna, tincture of opium from Papaver somniferum.
•
Antibiotic: By definition all antibiotics are natural product produced by fungi. Almost all the available
classes of antimicrobials are of fungal origin.
Alongside these naturally derived allopathic medicines, medicinal plant have been widely use in alleviating
diarrhoeal symptoms in humans and animals (Brijesh et al., 2006; Gutierrez et al., 2007). Numerous species of
these plants have been screened and validated for their use in treatment of diarrhoea (Gutierrez et al., 2007).
2.14.1. Anti-infectious mechanisms of plant secondary metabolites against diarrhoeal pathogens
•
Antimicrobial: Many plant metabolites are known to exhibit some level of toxicity toward
microorganisms. Numerous mechanisms of actions have been hypothesized to explain their
antimicrobial activity such as microbial enzymes inhibition, deprivation of essential growth substrates,
cell membrane disruption (Cowan, 1999) or direct interference with metabolic pathways.
•
Antiadhesion: Adhesion of some enteric pathogen to the mucosa epithelium of the host cells is the first
important step in intestinal infections that may lead to the development of diseases (Ofek and Sharon,
1990). Application of antiadhesives chemotherapy can be effective only against microorganisms that
27
depend on the surface contact with host cells as prerequisite for survival, multiplication and virulence
(Lengsfeld et al., 2007).
•
Antitoxin: Since enteric pathogens may induce diarrhoea through the production of toxin (endotoxin or
cytotoxin) the neutralization with plant antidiarrhoeal compounds may beneficial in the management of
diarrhoea. Activated charcoals processed from plants are also used as toxin binders. Other binder
includes pectin obtained from apples.
•
Immunomodulatory: With immune suppression being a pre-disposing, drugs or medicinal plant
preparations with immune stimulating activities may help in attenuating many infectious diseases.
2.14.2. Antioxidative mechanisms of plant phytochemical as potential antidiarrhoeal agents:
Free radical scavenging: Many plant preparations and phytochemicals have strong antiradical activities
which can antagonize the deleterious action of free radical. The mechanism may be electron transfer or
hydrogen donating to stabilized the free radical molecules generating radicals that are relatively stable
due to delocalization resulting from resonance and unavailability of site for further attack by molecular
oxygen (Mello et al, 2005).
Complexation of catalytic metallic ion: Metallic ions such as ferrous ion (Fe2+), cuprous ion (Cu2+),
Manganese ion (Mg2+) and Zinc ion (Zn2+) can also generate free radicals (Kane, 1996). Many plant
molecules moderate oxidation activity by complexing with the free transition metal thereby inactive their
capacity to catalyze oxidative process.
Pro-oxidation enzymatic pathways: With the generation of oxidant being enzyme driven, the antioxidant
activities of plant phytochemical may be able to inhibit these enzymatic pathway or inactivation of the
enzyme.
Lipid peroxidation inhibition: Scavenging of free radicals is one of the major antioxidation mechanisms
to inhibit the chain reaction of lipid peroxidation and reduction of the deleterious effect of the cytotoxic
products.
Inhibition of nitric oxide (NO): NO generated by inducible NOS (iNOS) can act synergistically with other
inflammatory mediators in the development of diarrhoea. The inhibition or down-regulation of iNOS
expression may be beneficial to reduce the inflammatory response.
Immune system optimization: Over expression of immune system may cause damage to surrounding
tissues and consequently results in a host of diseases and illness including diarrhoea. Many medicinal
plants and phytochemical compounds protect against oxidative stress due to immunomodulatory activity
(Wang et al., 2002).
28
2.14.3. Anti-inflammatory mechanisms of plant phytochemical in diarrhoea management:
Cyclooxygenase inhibition: Compounds with COX enzyme inhibitory potential could be use as antiinflammatory agents. Some plant secondary metabolites such as alkaloids, phenols, , terpenoids and
their derivatives have potential to inhibit the formation of pro-inflammatory signalling molecules such as
prostaglandin (Polya, 2003)
Lipoxygenase inhibition: Lipoxygenase metabolites are critical mediators of inflammation and thus
important in the pathogenesis of abdominal distress and diarrhoea associated with intestinal
inflammation. Plant phytochemicals with lipoxygenase inhibitory potential are candidate for antiinflammation and the resulting diarrhoea.
Modulation of cytokines activity: A number of diarrhoea pathogenesis causes severe intestinal
inflammatory with hypersecretion of pro-inflammatory cytokines (MacNaughton, 2006). Inhibition of the
pro-inflammatory cytokine mediator’s can remove their negative activities associated with
gastrointestinal disorders including diarrhoea.
2.14.4. Antidiarrhoeal mechanisms of plant phytochemicals
Antispasmodic: Spasmolytic agents are used in the treatment hypermotility of the GIT while prokinetic
agents are used in treatment of hypomotility (Gilani, 2005). Many phytochemicals demonstrated various
range of spasmolytic or antispasmodic activities against spontaneous or agonist induced contraction on
isolated parts of the GIT.
Antisecretory: Microbial enterotoxins cause diarrhoea by disturbing the balance between intestinal
absorption and secretion in favour of the later. Therefore, inhibition of the intestinal secretion is one
therapeutic model for treating diarrhoea (Velazquez et al., 2009).
2.15.0. Classification of phytochemicals with antidiarrhoea potential
2.15.1. Terpenoids
Terpenoids are the most structurally diverse groups of natural products formed by fusion of isoprene monomers
in plants. This class of plant secondary metabolites are grouped according to the number isoprene units or
numbers of carbon in their skeletal structure (Zwenger and Basu, 2008). The group include monoterpenes which
contain two units of isoprene with C10 and are present as essential or volatile components of herbs, spices and
flowers. Sesquiterpenes are derivatives of three isoprene units containing 15 carbon atoms in their structures
and are present in essential oil. This group of compounds acts as phytoalexins, antimicrobial and antifeedant in
plants. Diterpenes contains 20 carbon atoms derived from four units of isoprene and pharmacological activities
such as taxol (anticancer) and forskolin (for treating glaucoma). Triterpenes are contains 30 carbon atoms
skeleton formed by the head-to-head joining of two C15 chains, each of which contains three isoprene units joined
head-to-tail. Tetraterpenes such as carotenoids are 40 carbon atoms made of 8 isoprene units.
29
Several terpenoids have been identified so far to have good activity in one or more of the antidiarrhoeal
mechanisms described above. Some of the compounds include:
Niloticane from Acacia nilotica (L.) Willd ex Del. subsp. kraussiana (Benth) Brenan (Fabaceae) had
inhibitory effect on (Bacillus subtillis, Staphylococcus aureus, and Escherichia coli at 4.0, 8.0 and 33.0
µg/ml respectively) (Eldeen et al, 2010).
1,3-Dihydroxy-12-oleanen-29-oic acid; 3,30-Dihydroxyl-12-oleanen-22-one; 1,3,24-Trihydroxyl-12olean-29-oic
acid;
1,23-Dihydroxy-12-oleanen-29-oic
acid-3-O-2,4-di-acetyl-1-rhamnopyranoside
isolated from Combretum imberbe have been reported to have MIC of 16.0 µg/ml against E. coli (Angeh
et al., 2007).
Oleanolic acid, ursolic acid, and betulinic acid from Chaenomeles speciosa have the potential of
blocking the binding of virulence heat labile unit B (LTuB) of E. coli enterotoxin to ganglioside receptor
(GM 1). The IC50 values enterotoxin binding activity were 202.8±47.8, 493.6±100.0, and 480.5±56.9
µM respectively (Chen et al., 2007).
Glycyrrhizin from Glycyrrhiza uralensis have been reported to have LTuB-binding inhibitory activity with
IC50 of 3.26±0.17 mM, therefore, can suppress LT-induced intestinal fluid accumulation (Chen et al.,
2009).
Oleanolic acid and echinocystic acid both isolated from Luffa cylindrica (cucurbitaceae) were reported to
increase phagocyte index, stimulate macrophage, increase humoral and cell-mediated immune
responses (Khajuria et al., 2007).
30
H
H
HO
H
OH
H
OH
H
HO
O
Ursolic acid
OH
OH
H
H
HO
HO
OH
H
HO
OH
1,3,24-Trihydroxyl-12-olean-29-oic acid
3,30-Dihydroxyl-12-oleanen-22-one
O
Betulinic acid
O
HO
OH
H
O
H
H
H
H
Oleanolic acid
HO
O
H
H
O
H
O
H
HO
H
H
H
HO
O
OH
H
OH
HO
R
OH
H
Asiatic acid
OH
H
H
HO
1,3-Dihydroxy-12-oleanen-29-oic acid
R= O-2,4-di-acetyl-1-rhamnopyranoside
1,23-Dihydroxy-12-oleanen-29-oic acid-3-O-2,4-di-acetyl-1-rhamnopyranoside
OH
H
HO
H
O
O
H
H
H
H
HO
H
H
OH
H
O
HO
OH
Arjunolic acid
Betulonic acid
Niloticane
OH
CH3
CH3
H
HO
H
O
Sugiol
H
HO
H
O
OH
H
H
OH
HO
Maslinic acid
Erythrodiol
Fig. 2.9. Chemical structures of bioactive terpenoids against diarrhoeal mechanisms
Sugiol (a diterpenoid) isolated from Amentotaxus formosana reported to exhibit good xanthine oxidase
inhibitory activity with IC50 of 6.8±0.4 µM compared to standard allopurinol with IC50 of 2.0±0.7 µM (Lin
et al., 2010).
31
Epibetulinic acid and betulonic acid isolated from Maytenus cuzcoina Loesener demonstrated nitric
oxide inhibition of 89.1±4.4% (IC50 of 0.7 µM) and 69.2±5.1% (IC50 of 0.3 µM) respectively in vitro
(Reyes et al., 2006).
Betulonic acid isolated Maytenus cuzcoina Loesener to have demonstrated PGE inhibition activity of
58.4±3.9% (IC50 of 2.7 µM) (Reyes et al., 2006) in vitro.
Maslinic acid, oleanolic acid, erythrodiol and uvaol isolated from olive pomace oil was shown to have
concentration dependent IL-6, TNF-α modulatory effects in a human mononuclear cells culture assay
(Marquez-Martin et al., 2006).
Ganoderic acids C and D isolated from Ganoderma lucidum have been reported to have anti-allergic
properties through histamine release inhibitory mechanisms (Rios, 2008). Friedoolean-type triterpenoid
(bryonolic acid) (Rios, 2005) and cucurbitacin structure, including dihydrocucurbitacin B (Escandell et
al., 2007) and cucurbitacin R (Escandell et al., 2010) from Cayaponia tayuya (Cucurbitaceae) were
reported to exhibit anti-allergic activities through inflammatory responses modulation.
2.15.2. Alkaloids
Alkaloid refers to a group of heterocyclic nitrogen compounds and many exhibit remarkable physiological and
pharmacological activities (Samy and Gopalakrishnakone, 2008). Most alkaloids are derived from amino acid
precursor and are classified based on their structure as pyridine, tropane or pyrrolizidine alkaloids. Though,
alkaloids have many pharmacological mechanisms such as microbiocidal effect on diarrhoeagenic pathogens,
the main antidiarrhoea effect is probably that of delayed intestinal transition of bowel materials (Cowan, 1999).
Some of the pharmacological important antidiarrhoeal alkaloids include:
Kurryam, koenimbine and koenine isolated from Murraya koenigii Spreng (Rutaceae) seed were
reported to be active against various diarrhoeal mechanisms such as castor-induced diarrhoea, GIT
motility, PGE2 induced enterpooling (Mandal et al., 2010).
8-acetonyldihydronitidine and 8-acetonyldihydroavicine both isolated from Zanthoxylum tetraspermum
had strong antistaphyloccocal activities with MICs of 1.56 and 3.12 µg/ml respectively. However, the
two compounds were reported to have no inhibitory activity against E. coli (Nissanka et al., 2001).
Boldine isolated from Peumus boldus Molina and Corydalis ternate Nakai had good antioxidant
properties, indicating the effectiveness of the compound in preventing various oxidative-stress related
illnesses like inflammatory cascades, immune dysfunctions. However, at high concentration, it causes
cellular damage and potentiates lipoperoxidation, which is a pro-oxidant property.
32
CH 3
O
CH 3
HO
CH 3
HO
O CH 3
CH 3
N
H
H3C O
O CH 3
Koenimbine
Kurryam
CH 3
CH 3
O
HO
O CH 3
O
H3C
CH 3
N
H
Koenine
O
H3C
H
N CH
3
OH
H3C
(S)-boldine
H3C
CH 3
N
H
O
N H
H
O
H3C
H3C
HO
HO
N
H
CH 3
O
HO
O
CH 3 OH
O
N
H
CH 3
H3C
CH 3
O
(S)- reticuline
Laurotetanine
Magnocurarine
O
O
CH 3
O
O
CH 3 O
OH
O
N
CH 3
H
8-acetonyldihydronitidine
O
O
O
O
N
CH 3
H
8-acetonyldihydroavicine
Fig. 2.10. Chemical structures of bioactive alkaloids against diarrhoeal mechanisms
2.15.3. Phenolic
33
Phytophenolic compounds are widely distributed as secondary metabolites of medicinal plants, as well as in
some edible plants (Naczk and Shahidi, 2004). The consumption of diet rich in phenolic compounds has been
hypothesized to be important in health promotion and disease prevention in humans and animals (Ramful et al,
2010). Phenolic compounds are characterized as aromatic metabolites that have one or more acidic hydroxyl
groups attached to the phenyl ring. The sub-class of phenolics compounds are presented in Fig. 2.8. This group
of compounds exhibits numerous biological activities directly or indirectly on intestinal epithelium which contribute
to alleviation of diarrhoea symptoms.
Phenolic
Polyphenolic
Phenolic acid
Tannins (3 or more
phenol subunit)
Flavonoids: Flavone,
flavonol, flavonol, flavonone,
isoflavone, chalcone,
anthocyanin
Hydrolysable tannin
Condensed tannin
Gallic acid with sugar moieties:
gallotannin, ellagitannin
Miscellaneous such as
coumarins, stilbenes, lignin
Hydroxycinnamic acid
Hydroxybenzoic acid
Catechin and epicatechin polymers:
proanthocyanidin, procyanidin, prodelphinidin
Fig. 2.11 Sub-classes of biologically important group of phenolic compounds.
Phenolic compounds with antidiarrhoeal activities against some of the mechanisms described above include:
2(S) -5′- (-1′″,1″′-dimethylallyl) -8- (3″,3″-dimethylallyl) -2′,4′,5,7-tetrahydroxyflavanone; 2(S) -5′- (1″′,1″′dimethylallyl) -8- (3″,3″-dimethylallyl) -2′-methoxy-4′,5,7-trihydroxyflavanone and 5′- (1″′,1″′-dimethylallyl)
-8- (3″,3″-dimethylallyl) -2′,4′,5,7-tetrahydroxyflavone] from Dalea scandens var. paucifolia with MIC of
1.56 µg/ml against standard and Methicillin-resistant S. aureus (MRSA) (Nanayakkara et al., 2002).
Moracin T isolated from Morus mesozygia was reported to exhibit antimicrobial activities with MIC of 5.0
µg/ml against E. coli, S. dysenterica, P. aerigunosa, S. typhi, and 10 µg/ml against S. aureus, C.
albicans (Kuete et al., 2009).
Isoquercitrin, catechin and epicatechin isolated from Chiranthodendron pentadactylon flowers have
antisecretory effect on Vibrio cholerae toxin induced intestinal fluid accumulation with ID50 of 19.2, 51.7
and 8.3 µg/ml against loperamide (positive control) with ID50 of 6.1 µg/ml.
34
Davidigenin isolated from Mascarenhasia arborescens inhibits histamine induced contractile response
of rat ileum and Ach induced contractile response on rat duodenum. The compound was reported to
have non-competitive concentration dependent inhibitory activity (Desire et al., 2010).
Vitexin isolated from Aloysia citriodora has also been reported to have antispasmolytic activities
(Ragone et al., 2007).
Quercetin has 90.7±0.3% and 79.6±2.3% inhibition of Ach and the depolarized KCl induced
contractions on guinea pigs isolated ileum at IC50 <0.1 µg/ml respectively (Cimanga et al., 2010).
Quercitrin was reported to have spasmolytic activities of 82.3±2.3% and 72.1±0.6% against Ach and
the depolarized KCl induced contractions on guinea pigs isolated ileum at IC50 <0.01 µg/ml respectively
(Cimanga et al., 2010).
Spasmolytic activities of rutin were also reported as 93.4±1.6% and 86.3±2.1% against Ach and the
depolarized KCl induced contractions on guinea pigs isolated ileum at IC50 <0.01 µg/ml respectively
(Cimanga et al., 2010).
Luteolin isolated from Pogonatherum crinitum has iNOS inhibitory activity with Emax equals
100.00±0.00% and IC50 of 10.41 µM while Kaempferol isolated from the same plant has iNOS inhibitory
activity with Emax of 95.12±1.15% and the IC50 equals 10.61±0.44 µM (Wang et al., 2008)
Stilbenoids such as r-2-viniferin, trans-amurensin and cis-amurensin isolated from Vitis amurensis have
LOX inhibitory activity with IC50 of 6.39±0.08 µM, 12.1±0.32 µM and 16.3±0.52 µM respectively (Ha et
al., 2009).
Laurentixanthone A isolated from Vismia laurentii has good activities with MIC of 4.88 µg/ml against S.
dysenterica, S. flexneri, B. substilis (Nguemeving et al., 2006).
5,7-dihydroxy-2-[14-methoxy-15-propyl phenyl]-4H-chromen-4-one isolated from Leuca aspera has
superoxide radical scavenging activity of 75.4±0.31% and lipid peroxidation inhibition of 68.7±0.41% at
a concentration of 40 ppm (Meghashri et al., 2010).
Lipid peroxidation inhibitory activity of foeniculoside X isolated from Foeniculum vulgare at concentration
of 10-5M was reported to be 6.30 nm TBARS/mg LDL (Marino et al., 2007).
An in vivo lipid peroxidation inhibitory activity of arzanol isolated from Helichrysum italicum has also
been reported (Rosa et al., 2011).
35
H
O
OH
O
H
H
HO
HO
H
O
O
OH
CH3
O
OH
R
R=Glc 1
OH O
(5, 7-dihydroxy-2-[14-methoxy-15-propyl phenyl]-4H-chromen-4-one
6 Glc
OH
OH
Foeniculosde X
HO
OH
O
OH
O
OH
Moracin T
HO
O
OH
OH
OH
OH
O
Quercetin
O
OH
HO
OH
O
OH
HO
OH
O
arzanol
O
OH
HO
O
OH
O
Rutin
OH
OH
HO
H
O
OH
H
O
OH
O
Luteolin
HO
H
HO
H
O
rutinosyl
O
H
O
HO
H
OH
H
H
OH
OH
OH
OH
Cis-amuresin B
OH
OH
Trans-amuresin B
Fig. 2.12. Chemical structures of bioactive phenolics against diarrhoeal mechanisms
2.16. Ethnobotany and scientific investigation of plant species used traditionally in treating diarrhoea in
South Africa
36
A survey on traditional practice in South Africa indicates that diarrhoea is one of the most prominent ailments
being treated with medicinal plants (Dambisya and Tindimwebwa, 2003). At this stage, the scientific validation of
their therapeutic potential, standardization, safety and mechanisms of actions of most of the plants is still lacking
(Havagiray et al., 2004). Ethnopharmacological properties and phytochemistry of the medicinal plants used for
the treatment of diarrhoea in South Africa is reviewed and presented in Appendix 1.
2.17. Conclusion
Many medicinal plants are used in various traditional cultures of South Africa to treat diarrhoea and the
associated complications. The traditional recipes include infusions, decoctions, and tinctures of the leaves,
stems, roots, seeds and bark of medicinal plants. Several scientific methods have been used to evaluate and
validate the traditional use of some of the plants as antidiarrhoeal agents. Such properties investigated as the
potential antidiarrhoeal mechanism are the antimotility, antipropulsive, antioxidant, anti-inflammation,
antispasmolytic, antimicrobial, antiprotozoal, and immunomodulatory activity of the medicinal plant preparations.
Several of these aspects will be examined for some selected medicinal plants in this study.
37
CHAPTER THREE
Plant selection, collection, extraction and analysis of selected species
3.1.
Introduction
Renewed interest in the therapeutic potential of medicinal plants means that researchers are concerned not only
with validating ethnopharmacological usage of plant, but also with identifying, isolating and characterizing the
active components (Fennell et al., 2004). However, the presence of numerous inactive components makes the
screening and isolation of the target component(s) extremely cumbersome (Sticher, 2008). In choosing medicinal
plants for scientific evaluation of their biological activities and validation of ethnopharmacological usage, some
criteria such as
•
Evidence of ethnopharmacological usage by the native population.
•
The ailment(s) which the plant(s) is used to cure.
•
The availability of the plant in its natural habitat.
•
The sustainable use of the part(s) of the plant (root, leaves, stem, bark or whole plant) (Baker et al.,
1995; Van der Watt and Pretorious, 2001).
•
Mode of preparation and administration by traditional healers must also be considered.
Plant quality and pre-treatment are also important determinant of the phytochemical constituents and invariably
the biological activities of an extract. These factors depend on plant parts used, genetic variation, geographical
location, climatic conditions, collection period, drying methods, and storage conditions. Due to these possible
variations, plant material from recognized botanical gardens or herbaria is usually recommended because they
are protected, correctly identified and serve as reliable sources for subsequent collections. Preparation of
voucher specimens is also an important aspect of medicinal plant research. Standard procedures for pretreatment of plant materials have been developed (Eloff et al., 2008). The basic steps include pre-washing if
necessary, air drying under shade at room temperature, grinding into powder and storage in an air tight container
at appropriate temperature (room or refrigerated).
In view of limited resources and the large number of potential medicinal plants to be studied, efficient systems of
evaluation need to be developed for rapid phytochemical and biological screening. The first step is the use of
appropriate extraction process to remove the phytochemical from the plant cellular matrix (Sticher, 2008).
Extraction processes need to be exhaustive, efficient, simple, rapid and inexpensive in extracting targeted
compound(s). A number of extraction methods such as soxhlet, percolation, maceration, digestion, reflux, and
steam distillation have been developed over the years (Sticher, 2008). However, solid-liquid extraction with a
suitable range of solvents remains the most viable, convenient and effective procedure widely in use. The plant
extracts are usually qualitatively analyzed for chemical composition (phytochemical fingerprint) and biological
activities (for example bioautography for antimicrobial assay, antioxidative profiling with DPPH radical solution,
acetyl cholinesterase inhibition) on thin layer chromatography (TLC). These plant pre-treatment methods,
38
extraction and analyses were employed in this chapter to determine qualitatively the phytochemical constituents
and biological activities of selected plant extracts. The plants studied were selected based on literature
documentation of their use in South African traditional medicine (SATM) as antidiarrhoeal agents and results
from preliminary antimicrobial studies on some of the species in the phytomedicine tree project.
3.2. Solid-liquid extraction
Extraction is first pre-purification step in the isolation and characterization of active compound(s) of a medicinal
plant (Sticher, 2008). Selective removal of interfering components from solid plant material involves a five-unit
operation:
Mixing of plant material and extractant.
Solubilisation of the solute with the aid of a shaker or sonicator.
Filtration of the mixture to remove solutes and extractant from the plant residue.
Drying of sample using technique such as freeze drying, evaporation under vacuum (rota-evaporation)
or air drying.
Recovery of the solute extract.
The type of extractant may range from non-polar to polar solvent depending on the targeted class of bioactive
component(s). Though the method is relatively simple, some of the drawbacks include: long extraction time,
labour intensivity, high solvent consumption and inadequate reproducibility. In traditional medicine practice,
ethanol and water are the most widely used extractants. The bioactive components of medicinal plants are
usually unknown, and the nature of the extractant used affects the composition of the crude extract. Therefore,
solvents such as hexane, dichloromethane, ethyl acetate, acetone, methanol, propanol, water or a combination
of solvents are used in laboratory settings. Acetone has been adjudged to be the best extractant of plant extract
for bioassay because it extracts a broad spectrum of components (polar and non-polar), is miscible with all other
solvents, is highly volatile, and exhibit low toxicity to biological organisms in various assays (Eloff, 1998).
Temperature is also an important factor in extraction, drying and storage of plant extracts because of varying
compound stability due to chemical degradation, losses by volatilization and oxidation. Milder extracting and
drying temperatures are required to avoid loss of activity by plant extracts possibly due to thermal decomposition.
Storage of plant extracts, fractions or isolated pure compounds should be done at 4oC in the dark to avoid any
negative influence of temperature and light.
3.3. Liquid-liquid fractionation
Solvent partitioning of extracts allows a finer separation of the plant constituents into fractions of different
polarity. Bioactivity-guided fractionation, where the fractions are tested following separation to quickly identify
and isolate the agents responsible for bioactivity is a desirable step in medicinal plant research. The solvent
partition process involves the use of two immiscible solvents of different polarities. Various solvents are used
39
starting with non-polar (hexane, dichloromethane, diethyl ether) to medium polar (chloroform, ethyl acetate), and
finally more polar solvent (acetone, methanol, butanol and water).
3.4. Thin layer chromatography (TLC)
3.4.1. Phytochemical fingerprints
TLC is widely used in natural product extract analysis, stability tests of extracts and finished products, and in
sample quality control (Cimpoiu, 2006). TLC fingerprints of medicinal plants and extracts can be used for
identification and quality control of medicinal preparations. The identification of separated components can be
achieved on the basis of retention factor (Rf) values and colour spots. In relation to other chromatographic
methods, TLC offers the simplest and cheapest means of detecting natural product constituents, requiring little
sample clean-up and equipment (Nyiredy and Glowniak, 2001). Characteristic features of TLC include: analysis
of many samples and comparison of their phytochemical profiles on the same plate; results can be stored and
communicated as images (picture, video or scanned) and flexibility in the choice of mobile and stationary phase
(Cimpoiu, 2006). Identification of compounds can be done using three different mobile phases on the same
stationary phase or three different stationary phases with one mobile phase to develop the fingerprint of the
extracts and standards. If the difference in Rf values is less than 0.03, then the compounds is identified without
further isolation (Nyiredy and Glowniak, 2001). However, position isomeric compounds such as ursane and
oleanane derivatives can have superimpose or close Rf values, making them inseparable.
Visualization of separated compounds is achieved by natural colour in daylight or by fluorescent quenching on
254 nm (for conjugated double bonds or extended π electron systems) or 366 nm UV light. Some commercial
plate absorbents contain fluorescent dye that lights when placed under UV light and compounds are indicated
with blue, green, brown, red or purple areas against a fluorescent background. Visualization of chromatogram
under UV light at 366 nm shows orange-yellow bands for flavonoids and blue fluorescent bands for phenolic
acids (Males and Medic-Saric, 2001). Many chromogenic spray reagents are also available for specific classes of
compounds or serve as indicators for broad classes of compounds. Examples are vanillin/sulphuric acid solution,
anisaldehyde and ferric chloride-potassium ferricyanide given intense blue bands for phenolic compounds
(Wettasinghe et al., 2001).
3.5. Materials and Methods
3.5.1. Selection of South Africa medicinal plants for antidiarrhoeal screening
For this project, 27 plant species from nine families (Table 3.1) were selected for preliminary screening based on
the following criteria:
1. Ethnopharmacological use of the plant in the management of diarrhoea locally,
2. Phylogenetic relationship to other plants used in treatment diarrhoea due to the possibility of their
producing related chemical compounds (chemotaxonomy),
40
3. Medicinal plants reportedly used in countries other than South Africa but naturalized or endogenous in
South African flora,
4. Preliminary pharmacological evaluation of the medicinal plant from the phytomedical laboratory of the
Department of Paraclinical Sciences (University of Pretoria),
5. Absence of published literature describing antidiarrhoea and biological studies, and
6. Their availability for evaluation.
A literature review on the selected plants for antidiarrhoea and other biological studies yielded little or no
previous research work.
3.5.2. Collection of plant materials
The leaves of the 27 plants were collected from the Marie van der Schijff Botanical Garden University of Pretoria
Main Campus at Hatfield, Pretoria or from Phytomedicine Programme tree project stored samples. The plants
were identified and authenticated by Ms. Lorraine Middleton and Magda Nel at the University of Pretoria
Botanical Garden. Voucher specimens were maintained at the HGWJ Schweikert Herbarium of the Department
of Plant Science, University of Pretoria, Hatfield Campus, Pretoria, South Africa.
3.5.3. Preparation of plant material and optimization of phenolic-enriched extraction process
Plant leaves collected were pre-treated according to Phytomedicine programme (University of Pretoria) standard
protocol. In brief, the leaves were sorted from the stem, packed in a well perforated bag and air dried under
shade at room temperature for 2 week. The dried leaves were ground, powdered and kept in an air tight
polyethylene bag until needed for the extraction process. Simultaneous extraction and fractionation of the leaves
using a mixture of 70% acetone acidified with 0.1% HCl and hexane. The chlorophyll, fat and wax-enriched
hexane fraction was decanted from the phenolic-enriched 70% acetone fraction.
Table 3.1: Medicinal plants selected for antidiarrhoeal investigation in this study
Family/Species
syn
Voucher
specimen
information
Reasons for
selection
Ozoroa mucronata (Bernh.ex C.Krauss) R.fern &
Ozm
PRU 068928
2, 4, 5, 6
Ozoroa paniculosa (Sond.) R.fern & A. Fern
Ozp
PRU 66851
1, 2, 4, 5, 6
Searsia leptodictya Diels
Sle
PRU 70151
2, 4, 5, 6
Searsia pendulina Jacq.
Spd
PRU 84141
2, 4, 5, 6
Searsia pentheri Zahlbr.
Spt
PRU 709769
2, 4, 5, 6
Carissa macrocarpa (Eckl.) A.DC
Cam
PRU 37819
2, 4, 5, 6
Genera
Anacardiaceae
A. Fern
Apocynaceae
41
Burseraceae
Commiphora harveyi (Engl.) Engl.
Com
PRU 49952
2, 4, 5, 6
Celastraceae
Maytenus peduncularis (Sond.) Loes.
Mpd
PRU 76382
2, 4, 5, 6
Maytenus probumbens (L.f.) Loes.
Mpr
PRU 77119
2, 4, 5, 6
Maytenus senegalensis (Lam.) Exell
Mse
Maytenus undata (Thunb.) Blakelock
Mun
PRU 18576
1, 2, 3, 4, 5, 6
Combretum bracteosum (Hochst.) Brandis ex
Cob
PRU 117443
1, 2, 4, 5, 6
Combretum padoides Engl. & Diels
Cop
PRU 115416
1, 2, 4, 5, 6
Combretum vendae A.E. van Wyk
Cov
PRU 50800
1, 2, 4, 5, 6
Combretum woodii Dummer
Cow
PRU 20544
1, 2, 4, 5, 6
Euclea crispa (Thunb.) Gurke
Euc
PRU 76444
2, 4, 5, 6
Euclea natalensis A.DC.
Eun
PRU 66327
1, 2, 4, 5, 6
Bauhinia bowkeri Harv
Bab
PRU 44967
2, 4, 5, 6
Bauhinia galpinii N. E. Br
Bag
PRU 28944
1, 2, 4, 5, 6
Bauhinia petersiana Bolle
Bap
PRU 66874
2, 4,5
Bauhinia variegata L.
Bav
PRU 38533
1, 2, 3, 4, 5, 6
Erythrina latissima E. Mey
Erl
PRU 16349
2 , 4, 5, 6
Indigofera cylindrical sensu E. Mey
Inj
Schotia brachypetala Sond.
Scb
PRU 55333
1, 2, 4, 5, 6
Ficus craterostoma Warb.ex Mildbr. & Burret
Fic
PRU 38554
2, 4, 5, 6
Ficus glumosa Delile
Fig
PRU 48293
1,2, 4, 5
Syzygium paniculatum Gaertner
Syp
PRU 115417
2, 3, 5, 6
Combretaceae
1, 2, 3, 4, 5
Engl.
Ebenaceae
Fabaceae
Moraceae
Myrtaceae
2, 3, 4, 5, 6
(1) Ethnopharmacological use of the plant in the management of diarrhoea locally, (2) phylogenetic relationship to other
plants used in treatment diarrhoea due to the possibility of their producing related chemical compounds (chemotaxonomy),
(3) medicinal plants reportedly used in countries other than South Africa but naturalized or endogenous in South African
flora, (4) preliminary pharmacological evaluation of the medicinal plant from the Phytomedicine Programme of the
Department of Paraclinical Sciences (University of Pretoria), (5) absence of published literature describing antidiarrhoea and
biological studies, and (6) their availability for evaluation.
The acetone residue was removed by evaporation under vacuum using a rotary evaporator at 40oC. The residual
water fractions were divided into two portions (A and B). Portion A was freeze dried and served as the crude
extract while portion B was fractionated using solvents of increasing polarities as presented in Figure 3.1. The
crude extracts and fractions were reconstituted in various suitable solvents for the biological assays.
Plant material
Hexane fraction (Hf)
Simultaneous extraction and
fractionation with acidified
70% acetone and n-Hexane
Crude extract
42
Portion A (dried as crude
extract) (CRE)
Phytochemical evaluation of phenolic
components (total phenolics, non tannin
phenolics, total tannins, condensed
tannin, gallotannin, total flavonoids,
flavonol, anthocyanidin)
Portion B (Liquid-liquid
partition using solvent of
different polarities
Dichloromethane
fraction (DCMf)
Ethyl acetate
fraction (EAf)
n-Butanol
fraction (Bf)
Residual water
fraction (RWf)
Fig. 3.1. Flow chart for the extraction, phytochemical analysis and fractionation of plant material
3.5.4. Phytochemical profiling
The phytochemical profiles of the crude extracts and fractions were determined using thin layer chromatography
(TLC) by spotting 10 µl of solution at a concentration of 10 mg/ml. The plates were developed with various
combinations of hexane (H), ethyl acetate (E), formic acid (F), acetic acid (A), chloroform (C), methanol (M),
water (W), benzene (B) and ammonia (Am) at different ratio to create eluting solvent of varied polarities. The
combination with ratios in parenthesis that were used:
(1) E: F: A: W (70:5:5:10)
(2) E: F: A: W (70:5:15:10)
(3) E: M: Am (90:20:15)
(4) H: E: F (90:10:2),
(5) H: E: F (70:30:2)
(6) H: E: F (50:50:2)
(7) (B: E: Am (90:10:1)
(8) C: E: F (50:40:10)
(9) E: M: W: F (50:6.5:5:2)
(10) H: E: F (20:80:2)
The developed TLC plates were sprayed with vanillin/H2SO4 solution and heated at 100oC to allow colour
development (FAO/IAEA, 2000). Other reagents such as ferric chloride-potassium ferricyanide and panisaldehyde/H2SO4 (acetic acid, 5 ml; conc. H2SO4, 25 ml; ethanol, 425 ml; water, 25 ml) (Kubata et al., 2005)
were also used.
3.6. Quantification of the phenolic constituents of the extracts
3.6.1. Determination of total phenolic constituents
The total phenolic constituents of the extracts were determined using Folin-Ciocalteau method as described by
Makkar 2003, with some modifications. The crude extracts at concentration of 1:1 (mg/ml) plant material:
extracting solvent (50 µl) was dispensed into a test tube and made up to 500 µl with distilled water. FolinCiocalteau reagent (250 µl) diluted with distilled water (1:1) and 1250 µl of 20% sodium carbonate solution were
43
added to the extract. The mixture was vortexed and absorbance recorded at 725 nm after 40min incubation at
room temperature. The amount of polyphenols (expressed as mg Gallic /g dry weight) was calculated from a
prepared standard curve for gallic acid (0.0019-0.25 mg/ml gallic acid). The standard curve equation is y =
4.9022x + c, where y is absorbance, x is mg Gallic acid, c=0, R2=0.9804)
3.6.2. Determination of total tannin
The total tannin content of the extracts was determined using polyvinylpyrrolidone (PVPP) binding method
(Makkar, 2003). The bound mixtures were prepared by mixing 100 mg of PVPP, 1.0 ml of distilled water and 1.0
ml of tannin-containing extracts in a centrifuge tube. The mixtures were mixed thoroughly and kept at 4oC for 15
min and then filtered. The filtrate (100 µl) was transferred into a test tube and the phenolic content was evaluated
as described in section 3.7.1 above. Non-tannin phenolic constituents were determined from the standard curve
of catechin expressed as catechin equivalent in mg/g dry material. The standard curve equation is y = 4.9022x +
c, where y is absorbance, x is mg Gallic acid, c=0, R2=0.9804). The tannin content was calculated as the
difference between the total phenolic and non-phenolic content of the extracts because the tannin was bound
and precipitated by PVPP.
3.6.3. Determination of proanthocyanidin
The proanthocyanidin content of the extracts was determined using the butanol-HCl assay as described by
Makkar, 2003. The extract (500 µl) was dispensed into a test tube and diluted to 10 ml with 70% acetone. To this
3 ml of butanol/HCl (95/5%) and 100 µl of 2% ferric ammonium sulphate in 2N HCl were added. The test tubes
were loosely covered and heated in a boiling water bath for 50min. The absorbance was recorded at 550 nm
after the tubes were allowed to cool to room temperature. Absorbance of the unheated mixture was used as
blank.
3.6.4. Determination of condensed tannin
The condensed tannin content of the extracts was determined using vanillin/HCl assay as described by Heimler
et al, 2006. To 0.5 ml of the extract measured into a test tube, 3 ml of vanillin reagent containing 4%
concentrated HCl and 0.5% of vanillin in methanol was added. The mixture was allowed to stand for 15 min. The
absorbance was recorded at 500 nm against methanol as blank. The amount of condensed tannin in the extracts
was expressed as catechin equivalent (CE)/g dry plant material. The standard curve ranged from 0.0019 to 0.25
mg/ml (Absorbance= 0.1791 mg catechin + 0.0504, R2=0.944).
3.6.5. Determination of hydrolysable tannin (gallotannin)
The gallotannin content of the extracts was determined using the potassium iodate assay (Vermerris and
Nicholson, 2006). To 3 ml of the extract, 1 ml of saturated solution of potassium iodate was added and allowed to
stand at room temperature for 40 min. The absorbance was read at 550 nm. A standard curve was prepared
44
using gallic acid under the same conditions as the extracts and results were expresses as gallic acid equivalent
(GAE)/g dry plant material (Absorbance= 0.8264mg catechin + 0.0392, R2=0.9155).
3.6.6. Determination of total flavonoids and flavonol
The total flavonoids content of the extracts was determined by aluminium chloride method as described by
Abdel-Hamed et al (2009) with some modification. Briefly 100 µl of the extract was mixed with 100 µl of 20%
AlCl3 and two drops of glacial acetic acid. The mixture was diluted with methanol to 3000 µl. Absorbance was
read at 415 nm after 40 mins. Blank samples were prepared with the extract without AlCl3. Standard curve was
prepared using quercetin (3.9-500 µg/ml) in methanol under the same condition. The amount of flavonoids was
expressed as mg quercetin equivalent/g of dry plant material (Absorbance= 4.9747 mg quercetin, R2=0.9846).
The flavonol content of the extracts was determined by aluminium chloride method as described by AbdelHamed et al (2009) with some modification. One ml of the extract was mixed with 1 ml of 20 mg/ml of AlCl3 and 3
ml of 50 mg/ml of CH3COONa. Standard curve was prepared using quercetin (0.0019 - 0.0312 mg/ml) in
methanol under the same condition. Absorbance was read at 440 nm after 2.5 hr. The amount of flavonol was
expressed as mg quercetin equivalent/g of dry plant material (Absorbance= 34.046mg quercetin, R2=0.9853).
3.6.7. Determination of anthocyannin
Total anthocyanin content of the extracts was determined by a pH differential method with 96 well microplate
(Lee et al, 2008, Lee et al., 2005) using spectrophotometer. Absorbance was measured at 520 nm and 700 nm
in buffers at pH 1.0 and 4.5 using a molar coefficient of 29,600. Results were expressed as mg cyanidin-3glucoside equivalent/g dry plant material using equation 1 and 2 (Lee et al., 2005).
Equation 1
Equation 2
A= (A520-A700) pH 1.0 - (A520-A700) pH 4.5
Anthocyanin (cyanidin-3-glucose equivalent mg/L) = A × MW × DF × 103/ × l
Where MW (molecular weight) = 449.2 g/mol for cyanidin-3-glucose (cyn-3-glu); DF = dilution factor; l =
pathlength in cm;
= 26900 molar extinction coefficient in L × mol-1× cm-1 for cyn-3-glu; 103 factor for conversion
from g to mg.
3.7. Results
3.7.1. Yield of extractions and fractionations processes
The yield of the phenolic-enriched crude extracts and the fractions of various polarities using hexane,
dichloromethane, ethyl acetate, butanol and residual water are presented in Table. 3.2. The 70% acetone was an
extremely efficient extractant with an average of 34.61±5.84% extracted. The maximum yield was obtained for
the crude extracts of S. leptodicya (48.50±12.47% g/g dried plant material) followed by O paniculosa
45
(43.87±6.60% g/g dried plant material) while S. pentheri (21.13±2.67 g/g dried plant material) yielded the least.
There was a surprisingly high standard deviation between the three repetitions with a single extraction with new
plant material. This may have been caused by a difference in the particle size of samples. The extraction process
efficiently removed the chlorophyll from the bulk 70% acetone extractant into hexane fraction. In most cases
there was a difference between the percentage extracted and the total percentage of all the fractions. This loss
may be ascribed to solubility difficulties encountered with the dried residual water fraction which could not be
reconstituted due to the formation of insoluble complexes between the polyphenolics and other high molecular
weight components such as polysaccharides and possibly alkaloids. Unfortunately at that stage a freeze drying
was not available. This problem may have been partially resolved if the water fraction was freeze dried. To
evaluate the degree to which the different plant species contain compounds of different polarity the percentafe of
quantiy present in the crude extract into the different fractions was calculated (Table 3.2).
46
Table 3.2. The percentage yield of the crude extracts and various fractions (g/g dried plant material)
Plant spp
Crude
Hexane
DCM
ETOAc
Butanol
Residual
Insoluble
Water
ppt
Bab
33.25±0.83
3.44±0.15
1.39±0.16
2.45±0.34
9.91±1.09
7.47±0.41
8.27±1.87
Bag
38.83±6.18
2.38±0.35
1.31±0.27
2.70±0.21
15.05±1.16
8.93±0.18
10.90±2.94
Bap
32.23±2.84
1.67±0.17
1.71±0.17
3.30±0.78
11.82±0.48
8.71±1.23
7.69±2.53
Bav
31.62±5.46
1.83±0.07
1.90±0.23
2.98±0.17
10.65±1.68
8.07±0.13
Erl
22.12±0.32
1.35±0.55
0.34±0.05
0.26±0.06
6.63±2.20
10.44±0.96
2.77±1.87
Inc
36.15±1.62
1.58±0.17
0.78±0.08
1.71±0.32
12.10±0.66
9.31±1.51
7.84±2.47
Scb
30.15±3.47
1.53±0.49
1.14±0.40
1.58±0.06
11.54±1.00
7.39±1.93
6.21±1.30
Cob
34.24±3.08
1.27±0.21
3.39±1.13
3.28±0.44
8.15±0.30
8.81±1.40
Cop
39.96±0.78
2.33±0.51
3.31±0.68
3.56±0.18
17.42±0.79
7.43±1.06
1.84±0.96
Cov
38.77±0.48
1.33±0.47
2.86±0.24
3.13±0.51
14.82±2.53
12.08±0.16
2.74±1.06
Cow
36.88±3.39
3.95±1.75
2.07±0.38
2.67±0.41
12.35±2.99
8.39±0.52
7.41±3.29
Ozm
30.65±2.44
2.0±0.20
0.86±0.11
1.03±0.01
7.28±1.53
14.25±2.08
2.66±1.02
Ozp
43.87±6.60
6.57±0.55
1.55±0.38
4.30±0.82
14.54±0.96
8.81±2.02
8.62±1.77
Sle
48.50±12.47
5.85±0.61
1.49±0.30
4.25±0.52
10.30±1.82
9.29±0.89
13.28±3.34
Spd
33.76±0.28
5.05±0.69
0.98±0.28
3.03±0.35
12.21±0.81
11.40±3.31
1.96±0.53
Spt
21.13±2.67
2.96±0.30
1.50±0.25
1.04±0.28
6.34±0.17
8.80±1.50
1.62±0.89
Mpd
33.12±1.07
3.80±0.04
1.22±0.14
1.32±0.08
8.89±0.92
13.39±1.92
4.24±1.98
Mpr
35.10±4.77
3.05±0.28
1.18±0.31
0.90±0.28
8.50±1.31
12.41±0.47
4.78±1.35
Mse
37.89±3.05
3.75±0.40
1.20±0.14
1.30±0.08
8.77±0.91
13.21±1.89
10.08±2.71
Mun
36.89±4.67
1.42±0.18
1.63±0.48
1.08±0.46
10.88±0.46
12.08±1.77
3.13±1.57
Euc
34.97±1.90
2.76±0.56
1.49±0.32
2.05±0.16
13.44±0.86
10.81±0.30
2.84±0.73
Eun
32.83±3.19
2.05±0.82
1.73±0.21
2.35±0.45
10.89±1.39
12.`6±1.96
2.77±1.87
Fic
25.68±3.22
1.50±0.06
0.63±0.07
0.91±0.07
7.59±1.93
9.39±0.30
2.69±1.36
Fig
35.22±4.04
1.82±0.11
1.13±0.16
1.44±0.17
12.84±1.43
8.35±0.58
10.54±2.55
Cam
40.80±1.57
2.21±0.13
0.90±0.08
2.73±0.42
10.77±2.67
11.04±1.92
9.77±3.54
Com
33.09±1.19
1.01±0.04
1.06±0.28
1.64±0.08
10.39±4.81
7.80±0.59
5.37±1.85
Syp
36.80±8.10
1.44±0.20
0.78±0.06
1.10±0.17
8.34±1.89
10.60±0.40
5.95±2.64
3.7.2. Phytochemical screening (fingerprints)
The TLC phytochemical profiles of the crude extracts and fractions of the 27 plant species investigated are
presented in Figs 3.1–3.4. Figures 3.1, 3.2, 3.3, and 3.4 are the TLC fingerprints of the crude, hexane fraction,
dichloromethane fraction, ethyl acetate fraction developed with three mobile phases of different polarities for
each fraction.
In each chromatogram the order from left to right was Bab (Bauhinia bowkeri), Bag (Bauhinia galpinii), Bap
(Bauhinia petersiana), Bav (Bauhinia variegata), Erl (Erythrina latissima), Inc (Indigofera cylindrica), Scb
47
(Schotia brachypetala), Cob (Combretum bracteosum), Cop (Combretum padoides), Cov (Combretum vendae),
Cow (Combretum woodii), Ozm (Ozoroa mucronata), Ozp (Ozoroa paniculosa), Sle (Searsia leptodictya), Spd
(Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr (Maytenus procumbens), Mse
(Maytenus senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi),
Syp (Syzygium paniculatum).
Fig.3.2. Chromatograms of 100 µg of crude extracts of different plant species developed with ethyl actetate: acetic acid:
formic acid: water (75:5:5:10) (top), ethyl actetate: acetic acid: formic acid: water (70:5:15:10) (middle) and ethyl acetate:
methanol: ammonia (90:20:15) (bottom) and visualized with vanillin sulphuric acid. For identity of plant species see under
section 3.7.2 or under abbreviations used.
The chromatograms revealed complex mixture of compounds which exhibited different coloured reactions with
the vanillin/H2SO4 spray reagent. The classes of compounds in the extracts include terpenoids (purple or bluish
purple) (Taganna et al., 2011) and phenolics such as flavonoids (yellow, pinkish or orange), stilbenes (bright red
to dark pink colour), and proanthocyanidins (pink colour). The phenolic components were confirmed by blueblack spots with ferric chloride-potassium ferric cyanide reagents (Wettasinghe et al., 2001) while the flavonoids
were confirmed by yellow spot (Rijke et al., 2006) with aluminium chloride/acetic acid spray reagent
(AlCl3/CH3COOH).
48
Fig. 3.3: Chromatograms of the hexane fractions of different plant species developed with hexane: ethyl acetae: formic acid
(90:10:2) (top), hexane: ethyl acetae: formic acid (70:30:2) (middle), and benzene: ethyl acetate: ammonia (90:10:1)
(bottom) and visualized with vanillin sulphuric acid. For identity of plant species see under section 3.7.2 or under
abbreviations used.
Characterization of the phytochemical profiles of the extracts indicated that the extraction method and
extractants used resulted in splitting the complex mixtures into polar components concentrated in the 70%
acetone component (crude extracts) and non-polar compounds concentrated in the hexane component. From
the chromatogram, the crude extracts contained phenolics (especially flavonoids and proanthocyanidin) and
terpenoids. The hexane and dichloromethane fractions contained prominent spots for terpenoids while the ethyl
acetate fractions had prominent spots typical of flavonoid and other phenolic compounds.
49
Fig 3.4: Chromatograms of the dichloromethane fractions of different plant species developed with hexane: ethyl acetae:
formic acid (70:30:2) top, hexane: ethyl acetae: formic acid (50:50:2) (middle) and chloroform: ethyl acetate: formic acid
(50:40:10) bottom and visualized with vanillin sulphuric acid. For identity of plant species see under section 3.7.2 or under
abbreviations used.
50
Fig 3.5 Chromatograms of the ethyl acetate fractions of different plant species developed with chloroform:ethyl
acetate:formic acid (50:40:10) (top), hexane: ethyl acetate:formic acid (10:90:10) (middle) and ethyl
acetate:methanol:water:formic acid (100:13:10:2) (bottom) and visualized with vanillin sulphuric acid. For identity of plant
species see under section 3.8.2 or under abbreviations used.
3.7.3. Phenolic composition of the crude extracts
In this study, the total phenolic, total tannin, condensed tannin, proanthocyanidin, hydrolysable tannin as
gallotannin, flavonoids and flavonol constituents of the phenolic-enriched crude extracts were evaluated using
various standard protocols.The total polyphenolic and non-tannin phenolic constituent of each crude extract was
evaluated using the Folin-Ciocalteau reagent. All the 27 extracts contain significant amount of polyphenols and
non-tannin compounds; however, the quantity varied widely between the species (74.91 ± 1.26 – 467.0± 15.8
mg GAE/g plant material) (Fig.3.6).
Among the different extracts tested, the highest content of polyphenols was Combretum padoides (467.0±15.8
mg GAE/g plant material) which did not differ significantly (P<0.05) to Combretum vendae with (444.20±15.4 mg
GAE/g plant material). These two plant species were followed by Carissa macrocarpa (354.15±3.01 mg GAE/g
plant material), Commiphora harveyi (362.60±2.10 mgGAE/g plant material), Euclea natalensis (204.98±1.89 mg
GAE/g plant material), Ozoroa paniculosa (370.89±4.80 mg GAE/g plant material) and Searsia pendulina
(339.80±5.10 mg GAE/g plant material) all of which are significantly similar. The lowest content of polyphenols
was Ozoroa mucronata with (74.91±1.26 mg GAE/g plant material) which is not significant different from that of
51
the Erythrina lattisima with (76.08±2.59 mg GAE/g plant material) followed by Maytenus procumbens
(112.71±1.51 mg GAE/g plant material) significantly similar to Maytenus undata (123.82±1.45 mg GAE/g plant
material). The non-tannin phenolic constituent of the crude extracts ranges from 31.45±1.16 to 174.72±0.39 mg
GAE/g plant material (Fig 3.6). The plant with the highest non-tannin phenolics was C. macrocarpa (174.72±0.39
mg GAE/g plant material) followed by C. vendae (155.80±6.40 mg GAE/g plant material) which was not
significantly different to O. paniculosa (139.93±5.93 mg GAE/g plant material) p< 0.05.
Total phenolic constituents of the plant extracts
500
f
mgGAE/g plant material
f
400
e
e
e
e ,k
e,k
k
300
a,b
a
b
c
b,g
g
c,g
200
k
a
c,g
c,d
d
c,g
i
i
100
c,g
d
h
h
S
yp
O
zp
S
le
S
p
d
S
p
t
S
cb
C
o
b
C
o
p
C
o
v
C
o
w
C
o
m
E
uc
E
u
n
E
rl
F
ic
F
ig
In
c
M
p
d
M
p
r
M
se
M
u
n
O
zm
B
ab
B
ag
B
ap
B
av
C
am
0
Plant species
Non tannin phenolic constituents of the crude extracts
200
d
mgGAE/g plant material
f
150
f,g
g
g
e ,g
a,c
a
a
a
a,g
a,c
a,c
100
c
a,c
a,c
a,c
a,c
b,c
b
b,j
b,h
b,i
b,i
50
i
h,i,j
i
S
yp
O
zp
S
le
S
p
d
S
p
t
S
cb
C
o
b
C
o
p
C
o
v
C
o
w
C
o
m
E
uc
E
u
n
E
rl
F
ic
F
ig
In
c
M
p
d
M
p
r
M
se
M
u
n
O
zm
B
ab
B
ag
B
ap
B
av
C
am
0
Plant species
Fig. 3.6. Total phenolic and non tannin constituent of the crude plant extracts
The plant with lowest content of non-tannin phenolic was M. procumbens (31.46±1.16 mg GAE/g plant material)
which was not significantly different to M. undata (35.64±2.12 mg GAE/g plant material) p< 0.05.
The total tannin content of the extracts ranged from 25.55±0.81 to 359.40±8.30 mg GAE/g plant material
(Fig.3.6). The highest tannin constituent was C. padoides (359.40±8.30 mg GAE/g plant material) and was
52
mainly hydrolysable gallotannin (305.80±19.09 mg GAE/g plant material) (Fig 3.7). This was followed by C.
vendae (288.40±8.30 mg GAE/g plant material) which also contained high hydrolysable gallotannin
(197.60±12.79
mg
GAE/g
plant
material)
Total tannin constituents of the crude extracts
400
mgGAE/g plant material
g
h
300
j
j
j,k
k
k
200
e
e
e
a
i
c
c
100
i,n
n
b
b
f
c,f
f,m
f
f
m
d
l
l
S
le
S
p
d
S
p
t
S
cb
S
yp
In
c
M
p
d
M
p
r
M
se
M
u
n
O
zm
O
zp
F
ig
E
rl
F
ic
B
ab
B
ag
B
ap
B
av
C
am
C
o
b
C
o
p
C
o
v
C
o
w
C
o
m
E
u
c
E
u
n
0
Plant species
Condensed tannin composition of the crude extracts
200
mgCE/g plant material
b
150
a
a
a
100
c
c
c
d
50
d
d,e
d,k
d,g
e ,f,k
d,k
d,h
d,k
d,l
g,h,j,l
j
i,j
g,h,j,l
i,j
i
i
i,j
i
In
c
M
p
d
M
p
r
M
se
M
u
n
O
zm
O
zp
S
le
S
p
d
S
p
t
S
cb
S
yp
E
rl
F
ic
F
ig
C
o
b
C
o
p
C
o
v
C
o
w
C
o
m
E
u
c
E
u
n
B
ab
B
ag
B
ap
B
av
C
am
0
Plant species
Fig. 3.7. Total tannin and condensed tannin of the crude extracts
The hydrolysable gallotannin constituents of C. vendae were not significantly different (p< 0.05) to that for Euclea
crispa and Indigofera cylindrical at 199.36±17.61 and 185.21±11.50 mg GAE/g plant material respectively. E.
latissima had the lowest tannin content at 25.55±0.81 mg GAE/g plant material followed by O. mucronata at
27.17±0.18 mg GAE/g plant material. For both these plants the tannin content was mainly proanthocyanidin at
33.42±3.76 and 19.88±2.51 mg LE/g plant material respectively (Fig 3.8).
53
Proanthocyanidin composition of the crude extracts
250
e
mgLE/g plant material
200
150
b
b
b
a
100
f
c,j
f,j
f,j
j
c
f,j
c,l
k ,l,m ,n
d,k
50
d
d,g
d,m
d,n
d
d,h
d
d,i
d,i
g,h,i
i,m
i
B
ab
B
ag
B
ap
B
av
C
am
C
o
b
C
o
p
C
o
v
C
o
w
C
o
m
E
u
c
E
u
n
E
rl
F
ic
F
ig
In
c
M
p
d
M
p
r
M
se
M
u
n
O
zm
O
zp
S
le
S
p
d
S
p
t
S
cb
S
yp
0
Plant species
Gallotannin composition of the crude extracts
q
mgGAE/g plant material
300
f
f
200
f
c,d,e
100
a,c
a
a,e
a,g,m
a,g
a,j
a,c
b
c,d,e ,m
c,e ,g
j
b,j,k
b,i
b,l
i,k ,l b,l b,l b,l
b,l
b,l
b,l
B
ab
B
ag
B
ap
B
av
C
am
C
o
b
C
o
p
C
o
v
C
o
w
C
o
m
E
u
c
E
u
n
E
rl
Fi
c
F
ig
In
c
M
p
d
M
p
r
M
se
M
u
n
O
zm
O
zp
S
le
S
p
d
S
p
t
S
cb
S
yp
0
Plant species
Fig. 3.8. Proanthocyanidin and gallotannin constituent of the crude extracts
The condensed tannin content ranged from 6.99±0.32 to 183.53±10.10 mg CE/g plant material. Bauhinia galpinii
had the highest condensed tannin at 183.53±10.10 mg CE/g plant material. This was followed by C. macrocarpa,
Bauhinia bowkeri, and Combretum bracteosum at 125.0±2.72, 120.02±8.37 and 121.90±5.50 mg CE/g plant
material respectively, which are not significantly different (p<0.05) from each other. O. mucronata had the lowest
condensed tannin at 6.99±0.32 mg CE/g plant material followed by M. pendulina at 7.32±1.20 mg CE/g plant
material and E. latissima at 11.90±0.8 mg CE/g plant material.
The highest proanthocyanidin content was found in C. macrocarpa at 213.10±7.00 mg LE/g plant material
followed by S. leptodictya, B. galpinii, and Searsia pendulina at 126.54±6.46, 121.08±2.20 and 117.83±2.24 mg
LE/g plant material respectively. Statistically, S. leptodictya, B. galpinii, and Searsia pendulina were not
54
significantly different (p<0.05) for their proanthocyanidin content. M. procumbens and S. pentheri had the lowest
proanthocyanidin content at 10.46±1.76 and 10.08±2.24 mg LE/g plant material respectively.
Total flav onoid composition of the crude e xtracts
200
h
mgQE/g plant material
h
f
150
e
e
c,e
b,c
100
c,d
b,c
b,c
b,d
b,c
b,c
b,c
b,c
b
b
50
b,c
b
a
a
a
a
a
a
a,g
g
S
p
t
S
cb
S
yp
S
le
S
p
d
O
zp
In
c
M
p
d
M
p
r
M
se
M
u
n
O
zm
F
ig
E
rl
F
ic
E
u
c
E
u
n
B
ab
B
ag
B
ap
B
av
C
am
C
o
b
C
o
p
C
o
v
C
o
w
C
o
m
0
Plant species
Flavonol composition of the crude extracts
20
15
10
f
h
i
i
g
a
d
d
b
b
c
B
ab
B
ag
B
ap
B
av
C
am
0
m
b,m
m
b,m
d
e
C
o
b
C
o
p
C
o
v
C
o
w
C
o
m
E
u
c
E
u
n
c
a
a,g
d
d
S
yp
5
i
k
j
E
rl
F
ic
F
ig
In
c
M
p
d
M
p
r
M
se
M
u
n
O
zm
O
zp
S
le
S
p
d
S
p
t
S
cb
mgQE/g plant material
l
Plant species
Fig. 3.9. Total flavonoids and the flavonol constituent of the crude extracts
The total flavonoid ranged from 11.27±3.37 to 176.87±5.96 mg QE/g plant material. The highest flavonoid
content was present in Schotia brachypetala at 176.87±5.96 mg QE/g plant material followed by O. paniculosa
168.27±5.96 mg QE/g plant material. No significant difference (p<0.05) was present between the two plant (Fig
3.9). The lowest flavonoid content was present in M. senegalensis at 11.27±3.37 mg QE/g plant material. The
highest flavonol content was present for M. procumbens at 17.85±0.20 mg QE/g plant material followed by C.
padoides at 8.81±0.13 mg QE/g plant material. The lowest flavonol content was C. bracteosum at 0.13±0.07 mg
QE/g plant material.
3.8. Discussion
3.8.1. Yield
55
The extraction of phenolic constituents from plant matrix is complex and is influenced by their chemical nature,
extraction method, sample particle size, solvent as well as presence of interfering substances. Phenolics can
also complex with carbohydrates, proteins, and other plant components like alkaloids. High molecular weight
phenolics and their complexes are usually insoluble and solubility is also a function of the solvent polarity Naczk
and Shahidi, 2004). Consequently, phenolic extracts composed of varied classes of phenolics present in different
proportion with the degree of solubility in the solvent system as the primary determinant. In this experiment,
simultaneous extraction and fractionation using acidified 70% acetone and n-hexane was adopted. Two
immiscible phases of phenolic-enriched acetone solution (low phase) and chlorophyll-enriched terpenoids
containing hexane (upper phase) were obtained.
The extraction process is an important factor for assessing the biological activity of medicinal plant extracts
(Berlin and Berlin, 2005) as it influence yield of the extracts, extractive capacity of an extractant, and quality
parameter of the herbal preparations (Albuquerque and Hanazaki, 2006). Low polar solvent extractants such as
hexane, petroleum ether and dichloromethane extract non polar compounds mainly of terpenoids or highly
methoxylated phenolics. In contrast, medium and high polar solvents such as ethyl acetate, acetone, methanol,
ethanol water or mixture of these solvents extract the polar compounds ranging from simple phenolics to
complex polymeric phenolics (tannins).
3.8.2. Thin layer chromatogram
TLC fingerprints of the plant leaf crude extracts and fractions showed complex mixtures of non-polar to polar
compounds. TLC was used as qualitative method to characterize and document the phytochemical profiles of the
extracts as a fingerprint. The phytochemical constituents of plants depend on several factors including seasonal
changes, biotic (genetic) and abiotic (climatic stress, infection and soil fertility) factors (Moure et al., 2001). TLC
analyses help in monitoring composition of the extracts and fractions to ensure that no component(s) are lost
during processing. It also provides a means of comparing phytochemical composition of different plant extracts
developed side by side.
When comparing TLC fingerprints of the hexane fractions and the crude extracts from the extraction process,
hexane fractions were enriched with non-polar components while the crude extracts were enriched with polar
components, mostly of phenolic compounds. Solvents (2) E: F: A: W (70:5:15:10), (5) H: E: F (70:30:2), (8) C: E:
F (50:40:10) and (9) E: M: W: F (50:6.5:5:2) were the best mobile phase obtainable for preparing TLC fingerprint
of the crude extract, hexane fractions, dichloromethane fractions and ethyl acetate fractions respectively in this
work.
Polyphenolic compounds are important bioactive component of medicinal plant extract exhibiting various
pharmacological properties (Vundac et al., 2007). Phenolic-enriched extracts have been reported to correlate
with a wide range of physiological and health benefits which include antiallergenic, antiviral, antibacterial,
antifungal (Pietta, 2000), antisecretory, antispasmolytic, antimotility (Yue et al., 2004), anti-inflammatory,
56
immunomodulatory and parasitic activities. In traditional medicine preparation of plant extract recipe, water or
ethanolic solutions are the main extractants.
3.8.3. Phenolic constituents of the crude extracts
Polyphenolic compounds are important bioactive component of medicinal plant extract exhibiting various
pharmacological properties (Vundac et al., 2007). Phenolics form one of the main classes of secondary
metabolites and several thousand (among them over 8,150 flavonoids) different compounds have been identified
with a large range of structures: monomeric, dimeric and polymeric phenolics (Lattanzio et al., 2006). Several
classes of phenolics have been categorized on the basis of their basic skeleton. These groups of phytochemicals
are primarily natural antioxidants which act as reducing agent, metal chelators and single oxygen quenchers.
Phenolic-enriched extracts have been reported to correlate with a wide range of physiological and health benefits
other than antioxidative activity.
Polyphenolic compounds have antidiarrhoea properties exhibiting one or more activities against diarrhoea
pathogenesis. These may include antiallergenic, antiviral, antibacterial, antifungal (Pietta, 2000), antisecretory,
antispasmolytic, antimotility (Yue et al., 2004), anti-inflammatory, immunomodulatory and parasitic activities. In
traditional medicine preparation of plant extract recipe, water or ethanolic solutions are the main extractants.
These extractants extract more or less polar compounds made majorly of phenolic compounds. Specific types of
phenolic compounds present in the crude extracts are therefore evaluated.
Flavonoids are C6-C3-C6 polyphenolic compounds present in food, beverage and medicinal plants. They have
been reported to have useful pharmacological properties including anti-inflammatory activity, enzyme inhibitors,
antiallergic, anti-inflammatory, antiviral, antispasmolytic, pro-secretory (Yue et al., 2004) and antimicrobial
activity. Flavonoids are known to act on the inflammatory response via many routes and block molecules like
COX, iNOS, cytokines, nuclear factor-кB and matrix metalloproteinases. In addition, flavonoids have good
antioxidant, free radical scavengers that donate hydrogen, inhibit lipid peroxidation (Rauha, 2001; Havsteen,
2002) and metal ion chelators. However, the antioxidant power of flavonoids depends on some important
structural prerequisites such as the number and the arrangement of hydroxyl groups, the extent of structural
conjugation and the presence of electron-donating and electron-accepting substituents on the ring structure
(Miliauskas et al., 2005). These groups of phytochemicals are known to play some beneficial roles in the
prevention of many oxidative and inflammatory diseases (Arts and Hollman, 2005) inhibiting oxidative and
inflammatory enzymes (Middleton et al., 2000).
Gallotannins are complex sugar esters of gallic acid and together with the related sugar esters of ellagic acid
(ellagitannins) made up the hydrolysable tannins. Gallotannins exhibit biological activities including antimicrobial,
antiviral, anti-inflammatory to anticancer and antiviral properties (Erde‘lyi et al., 2005). The mechanisms
underlying the anti-inflammatory effect of tannins include the scavenging of radicals, and inhibition of the
57
expression of inflammatory mediators, such as some cytokines, inducible nitric-oxide synthase, and
cyclooxygenase-2 (Polya, 2003; Erde‘lyi et al., 2005).
Condensed tannins also referred to as proanthocyanidins are oligomers or polymers essentially derived from
flavan-3-ol and their derivatives via carbon to carbon (C–C).or rarely C-O-C links. They differ structurally
according to the number of hydroxyl groups on both aromatic rings (rind A and B) and the stereochemistry of the
asymmetric carbons of the heterocyclic ring (ring C). Condensed tannins are classified according to their
hydroxylation pattern into several subgroups including procyanidins (3,5,7,3’,4’-OH), prodelphinidins,
(3,5,7,3’,4’,5’-OH), propelargonidins (3,5,7,4’-OH), profisetinidins (3,7,3’,4’-OH), prorobinetinidins (3,7,3’,4’,5’OH), proguibourtinidins (3,7,4’-OH), proteracacinidins (3,7,8,4’-OH), and promelacacinidins (3,7,8, 3’,4’-OH) (Cos
et al., 2003). As with other polyphenols, tannin structures are suitable for free radical scavenging activities
serving as an excellent hydrogen or electron donors to form radicals that are relatively stable due to
delocalization resulting from resonance and unavailability of site for attack by molecular oxygen (Mello et al,
2005). Tannins can also binds to some free radical producing enzymes forming an insoluble tannin-protein
complex (astringent characteristic), complex with catalytic metallic ions making it unavailable to initiate oxidation
reaction, and inhibiting lipid peroxidation process (Russo et al, 2005; Mello et al, 2005). These compounds are
antagonists of hormone receptors or inhibitors of enzymes such as cyclooxygenase enzymes (Polya, 2003).
Tannins have the ability to protect renal renal cells against ischemia reperfusion injury (Yokozawa et al, 1997)
characterized by an overproduction of O2˙–due to both an electron leak in the mitochondrial respiration chain and
the conversion of xanthine dehydrogenase to xanthine oxidase (Wernes and Lucchesi, 1990). The protective
action of tannins in this process is related to direct inhibition of enzymatic function of xanthine oxidase activity
(Russo et al, 2005).
Production of reactive species (H2O2, O2˙–, and OH˙) and per-oxy-nitrite occurs at the site of inflammation and
contributes to the exacerbation of inflammatory disease and tissue damage. In acute inflammation or chronic
inflammations, the production of O2˙– is increased at a rate that overwhelms the capacity of the endogenous SOD
enzyme defense to dissipate. Reduction in the O2˙– generation can decrease side-effects of the radical in
inflammatory conditions. Tannins have been demonstrated to exhibit anti-inflammatory activity by exerting antioxidative properties in reducing O2˙– and malondialdehyde (MDA) production, plasma extravasations and cell
migration mainly of leukocytes and potentates the activity of SOD in radical scavenging (Nardi et al, 2007). It
shows that reactive species are most important mediators that provokes or sustain inflammatory processes and
consequently, their annihilation by antioxidants and radical scavenger can alleviate inflammation (Delaporte et al,
2002; Geronikaki and Garalas, 2006).
3.9.
Conclusion
The extraction methods used optimally extract the phytochemical constituent from the powdered leaves. The
extraction process adopted in this work separated the phytochemicals into non-polar hexane portion and polar
58
water soluble portion in the first step. In addition to taxonomic identification and authentication of medicinal plant,
Chemical characterization is also an important and useful means of quality control as it directly correlate with
pharmacological functions. The TLC fingerprints revealed the complexity of plant extracts and fractions with
chemical compositions of a wide range of polarities. For the optimization of the TLC fingerprinting more than one
mobile phase were used to obtain a representative chromatogram of the extracts. In this study, combination of
fingerprint with multicomponent quantification of the phenolic compositions was adopted as a good method for
chemical profiling of the plants.
There was a strong similarity in the chromatograms of Erythrina latissima, Combretum vendae and Combretum
woodii. Erythrina and Combretum are not closely related and the similarity may be an example of convergent
evolution. Combretum vendae and C. woodii are however, closely related as part of the subgenus Combretum
and the results indicate the potential use of chemical markers in taxonomy.
59
CHAPTER FOUR
Antimicrobial activities of the plant extracts against potential diarrhoeal pathogens
4.0. Introduction
Infectious disease defined as an illness caused by a specific pathogen or its toxins that result from transmission
of the causative agent or its virulence effectors from an infected person, animal or reservoir to a vulnerable host.
The susceptibility of host to infectious pathogens, disease development, progression and severity depends on
the age, gender, genetic, immune and nutritional status. Infectious diseases represent a leading cause of
morbidity and mortality worldwide despite the advancement in orthodox medicine accounting for more than 26%
of all death with developing countries carrying the major burden (Becker et al., 2006). Infections are also
considered to be a major contributing factor associated with reduced performance in food animals during growth.
Particularly, persistent infections account for slow growth, suboptimal feeding efficiency and economic loss in the
livestock industry (Borghetti et al., 2009). The infective pathogens include bacteria, fungi, viruses, protozoa and
parasites which manifest their virulence through different mechanisms (see section 2.2 for detailed discussion).
The discovery of antibiotics in 1928 and subsequent development in 1940 as medical treatment provides
effective and efficient therapeutic agents for controlling almost all infectious diseases including many feared and
contagious infections. Antibiotics are effective in curing many infectious diseases, but they also enhance
selection of resistant microbes as some pathogens rapidly became resistant to many of the originally susceptible
drugs (Barbour et al, 2004).
At present, the pharmaceutical drugs available to control antibiotic-resistant bacteria are becoming limited. The
indiscriminate use and abuse of antibiotics has led to the development of antimicrobial resistance strains and
toxicity of some drugs to human and animals (Barton, 2000; Parekh and Chanda, 2007). As a result of these
problems, European Union (EU) with EU-directive 1831/2003 imposed ban on the use of antibiotics as growth
factor in animal production with effect from 2006 to avoid cross resistance problem with human pathogens and
chemical residues in foods (Makkar et al, 2007).
Drug resistance of human and animal pathogenic microbes and parasites has created a serious problem
worldwide as previously treatable ailments such as diarrhoea (including dysentery and cholera), and tuberculosis
are now more difficult and expensive to treat. The mechanisms of microbial resistance to antibiotic include
(Dwyer et al., 2009):
Genetic alterations which involved the physical exchange of genetic material with another organism (via
plasmid conjugation, phage-based transduction, or horizontal transformation), the activation of latent
mobile genetic elements (transposons or cryptic genes), and the mutagenesis of its own DNA.
60
Chromosomal mutagenesis arises directly from interaction between the chromosome and the
antibacterial agent or antibiotic-induced oxidative stress, or indirectly from the bacterium’s error prone
DNA polymerases during the repair of a broad spectrum of DNA lesions.
The situations have complicated by the treatment of infectious diseases in immunocompromised patients. These
negative health trends necessitate for a new prevention and treatment of infectious diseases including diarrhoea.
Medicinal plants have also featured as therapeutic agents used by the world population for basic health care
needs and to combat many kinds of infectious diseases worldwide (Voravuthikunchai and Limsuwan, 2006).
Medicinal plants have curative properties due to the presence of complex mixture of phytochemicals acting
individually or synergistically to exert the associated therapeutic effects. Some of the plant compounds may be
novel bioactive substances that can be effective as therapeutic agents for treating ailments such as infectious
diarrhoea. These phytochemicals exhibit their antidiarrhoeal effects through various mechanisms such as
antimicrobial (Lutherodt et al, 1999), increasing colonic water and electrolytic re-absorption, inhibition of intestinal
motility (Oben et al, 2006) and anti secretory effects (Rao et al, 1997). There is considerable research in the
screening of natural products from extracts of edible and medicinal plants for the development of alternative
drugs to prevent and curtail the emergence of drug-resistance pathogens or other forms of ailments.
4.1. Qualitative antimicrobial (Bioautography) assay
This refers to the direct bioactivity test on developed TLC plates as a means of localizing the biological activity
such as microbial growth inhibition, enzymatic inhibition or antioxidative properties of extracts to the particular
active compound(s). This helps in focusing attention on the relevant components of an extract (Saxena et al.,
1995). Fractionation of medicinal plant extracts in combination with bioautography provides an efficient and
relative cheap method for bioactivity-guided isolation of target compound(s) (Hostettman et al., 1997). Practical
application of bioautography in activity guided isolation includes enzyme inhibition assay such as the Ellman
method for cholinesterase inhibitors (Ellman et al., 1961). In this method, the developed TLC plate is sprayed
with a substrate, enzyme and indicator to determine the inhibition by colour variation (white zone against yellow
background) (Rhee et al., 2001). In the antimicrobial bioassay, two bioautography methods are available. Firstly,
the agar diffusion method involves pouring a layer of inoculated agar solution of the microbes on the developed
TLC plate and allowed to set, and the bioactive zone(s) are transferred to the agar gel by diffusion where they
can inhibit the growth of the microorganism (Fig 4.1). Secondly, the direct method involves spraying of
microorganism broth inocula onto the TLC plate (Homans and Fuchs, 1970) and incubating in humid conditions
61
to
facilitate
the
growth
of
the
organism.
Fig 4.1. The classification of microbiological methods for biological detection (Adopted from Choma and Grzelak,
2011)
Microbial growth inhibitions are recognized based on the ability of the living microorganism to transform
tetrazolium salts to a coloured formazan product. White spots against an intense purple coloured background
indicate the compound(s) that kill the tested microorganism (Hostettman and Martson, 2002).
4.2. Quantitative antimicrobial activity (Minimum inhibitory concentration (MIC)) assay
Among the quantitative antimicrobial methods used in evaluating plant extract activity, agar diffusion assay
(Greenwood, 1989) and two-fold serial micro-dilution assay (Eloff, 1998) are the most common in phytomedicine
research. Sensitivity of the two protocols and their mechanisms varied widely. The mechanism of agar diffusion
is the movement of bioactive compounds through the solid agar medium to kill or inhibit the growth of organism it
may come in contact with. However, agar diffusion assays may sometimes lead to a false negative result, due to
influence of the agar type, salt concentration, incubation temperature molecular size of the antimicrobial
components (Greenwood, 1989), and limited diffusion of bioactive component in agar medium. The two-fold
serial micro-dilution assay depend on direct contact between the test sample and organism is adjudged to be 30
times more sensitive than the other methods used to screen plant extracts for antimicrobial activity (Eloff, 1998).
Although, the effective solubility and miscibility of the bioactive component in the test medium such as the nonpolar compounds like terpenes, alkaloid and highly methoxylated phenolics is a limiting factor.
4.3. Selection of microorganisms used in the study
The selection of the microorganisms for antibacterial evaluation in this study was based on their known
pathogenic effects in both human and animals with emphasis on diarrhoeal pathogens. Pathogenic E. coli has
been implicated in diseases such as diarrhoea, hemorrhagic colitis, haemolytic uremic syndrome and
thrombocytopenic purpura (Voravuthikunchai and Limsuwan, 2006). Enterococcus faecalis has been implicated
in causing enteric infection with diarrhoeal effects (Butler, 2006). Pseudomonas aeruginosa strains cause
diseases such as mastitis, abortions and upper respiratory complications (Masika and Afolayan, 2002).
Staphylococcus aureus is one of the prominent microbes causing skin infection such as boils, abscesses,
carbuncles and sepsis of wounds and it also produces toxins causing diarrhoea and vomitting (Maregesi et al,
62
2008). Candida albicans is a typical opportunistic pathogen causing diarrhoea (Gambhir et al, 2006), oral and
vaginal candidiasis (Shai et al, 2008) especially in immunocompromise individuals due to unexpected opportunity
by a failure of host defence. Cryptococcus neoformans has been implicated in causing life-threatening
meningoencephalitis (Xue et al, 2007) and pneumonia in immunocompromise individuals (Hamza et al, 2006).
4.4. Material and Methods
4.4.1. Microorganism strains
Two standard strains of Gram-positive bacteria (Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa
ATCC 25922) and two standard strains of Gram-negative bacteria (Escherichia coli ATCC 27853, Enterococcus
faecalis ATCC 29212) were used for antibacterial assay. Three clinical pathogenic fungi namely yeasts (Candida
albicans, Cryptococcus neoformans) and mould (Aspergillus fumigatus) (All fungal strains obtained from the
Department of Veterinary Tropical Diseases, Faculty of Veterinary Sciences, University of Pretoria) were used.
4.4.2. Culturing of the Bacteria
The bacterial strains were maintained in Mueller Hinton agar (MHA) (Fluka, Spain) while the fungi were
maintained in Sabouraud dextrose agar (Merck, Germany) at 4oC under anaerobic conditions. All the organisms
were subcultured every 2 weeks. Before testing, the bacterial inoculums were prepared and cultivated in Mueller
Hinton broth for 12 h at incubation temperature of 37oC. The fungi inoculums were prepared in Sabouraud
dextrose broth (SDB,). The microbial cultures were serially diluted (10 fold increments) in sterile broth to obtain
the cell suspension of 1.0 ×105 CFU/ml.
4.4.3. Bioautography against some pathogenic microorganisms
Bioautography was undertaken to ascertain the number of active compound(s) present in crude extracts and
fractions. TLC plates were developed as described in section 3.5.4 (Pp 43 - 44), and sprayed with overnight
cultures of E. coli, S. aureus, P. aeruginosa or E. faecalis and incubated at 37oC for 12-16 h prior to being spray
with tetrazolium violet (INT). The inhibitory activity of any components was evident as clear white zones against
the purple/red background.
4.4.4. Determination of Minimum Inhibitory Concentration (MIC) against the bacteria pathogens
The minimum inhibitory concentration (MIC) for the crude extract and fractions against bacteria were evaluated
using the twofold serial dilution assay with tetrazolium violet added as growth indicator (Eloff, 1998). The extracts
(100 µl) at an initial concentration of 1.0 × 104 µg/ml was serially diluted with distilled water up to 50% in 96-well
microtitre plate to prepare solution range between 5000 µg/ml first well and 40 µg/ml last well. The bacterial (100
µl) inoculants from 12 h broth cultures (section 4.4.2) diluted to 1:100 were added to each well to obtain final
extract concentration range of 2500 µg/ml first well and 20 µg/ml last well. Gentamicin (25 µg/ml first well and
0.18 µg/ml last well) was used as positive control and the solvent used in dissolving the extract was used as
63
negative control. Final volume in each well was 200 µl. The plates were incubated for 24 h at 37oC and 100%
relative humidity. The inhibition of the bacteria were visualised by adding 40 µl of aqueous p-iodonitrotetrazolium
violet (INT) (Sigma) to each well (concentration 200 µg/ml). The plates were incubated for another 1 h and MIC
was determined as the lowest concentrations of test sample before purple formazan colour were observed.
4.4.5. Determination of Minimum Inhibitory Concentration (MIC) against the fungal pathogens
Minimum inhibitory concentrations (MIC) against three pathogenic fungi were determined using twofold serial
dilution assay as described above by Eloff, 1998 with the following modification of Masoko et al, 2005. The
fungal inoculants (100 µl) were in fresh Sabouraud dextrose broth and positive controls was amphotericin B (50
µg/ml first well and 0.4 µg/ml last well) and negative controls was 70% acetone, final visualization of inhibitory
activity was obtained after an incubation for 24 h at 37oC, and 100% relative humidity.
4.5. Results
4.5.1. Microbial bioautography
The TLC bioautography of the crude extracts and fractions of the 27 plant species tested against standard strain
bacteria and clinical fungal isolates are presented in Fig 4.2-4.12.
Fig. 4.2. Bioautography of hexane (upper) fraction of different plant species against S. aureus (Bab (Bauhinia bowkeri),
Bag (Bauhinia galpinii), Bap (Bauhinia petersiana),Cam (Carissa macrocarpa), , Cop (Combretum padoides), Cov (Combretum vendae), Cow (Combretum
woodii), Com (Commiphora harveyi), Euc (Euclea crispa), Eun (Euclea natalensis), Erl (Erythrina latissima), Fic (Ficus craterestoma), Fig (Ficus glumosa)
developed with hexane: ethyl acetae: formic acid (70:30))
The antimicrobial activities of the extracts were concentrated on the non-polar-enriched hexane fraction while the
polar enriched components no sign of microbial inhibition.
64
Fig. 4.3. Bioautography of dichloromethane fractions of different plant species against S. aureus (Bab
(Bauhinia
bowkeri), Bag (Bauhinia galpinii), Bap (Bauhinia petersiana),Bav (Bauhinia variegata) Cam (Carissa macrocarpa), , Cop (Combretum padoides), Cov
(Combretum vendae), Cow (Combretum woodii), Com (Commiphora harveyi), Euc (Euclea crispa), Eun (Euclea natalensis), Erl (Erythrina latissima), Fic
(Ficus craterestoma), Fig (Ficus glumosa) developed with chloroform: ethylacetate: formic acid (100:13:10)).
Fig. 4.4. Bioautography of hexane fractions of different plant species against E. faecalis (Bab (Bauhinia bowkeri), Bag
(Bauhinia galpinii), Bap (Bauhinia petersiana),Cam (Carissa macrocarpa), Cop (Combretum padoides), Cov (Combretum vendae), Cow (Combretum
woodii), Com (Commiphora harveyi), Euc (Euclea crispa), Eun (Euclea natalensis), Erl (Erythrina latissima), Fic (Ficus craterestoma), Fig (Ficus glumosa)
developed with hexane: ethyl acetae: formic acid (70:30))
65
Fig. 4.5. Bioautography of dichloromethane fractions of different plant species against E. coli (Bab (Bauhinia bowkeri),
Bag (Bauhinia galpinii), Bap (Bauhinia petersiana),Bav (Bauhinia variegata) Cam (Carissa macrocarpa), , Cop (Combretum padoides), Cov (Combretum
vendae), Cow (Combretum woodii), Com (Commiphora harveyi), Euc (Euclea crispa), Eun (Euclea natalensis), Erl (Erythrina latissima), Fic (Ficus
craterestoma) developed with chloroform: ethylacetate: formic acid (100:13:10)).
Fig. 4.6. Bioautography of dichloromethane fractions of different plant species against E. faecalis (Bab
(Bauhinia
bowkeri), Bag (Bauhinia galpinii), Bap (Bauhinia petersiana),Bav (Bauhinia variegata) Cam (Carissa macrocarpa), , Cop (Combretum padoides), Cov
(Combretum vendae), Cow (Combretum woodii), Com (Commiphora harveyi), Euc (Euclea crispa), Eun (Euclea natalensis), Erl (Erythrina latissima), Fic
(Ficus craterestoma), Fig (Ficus glumosa) developed with chloroform: ethylacetate: formic acid (100:13:10)).
66
Fig. 4.7. Bioautography of hexane of different plant speicies against C. neoformans
(Ozm (Ozoroa mucronata), Ozp
(Ozoroa paniculosa), Sle (Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr (Maytenus
procumbens), Mse (Maytenus senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi), Syp (Syzygium
paniculatum)).
Fig. 4.8. Bioautography of dichloromethane fractions against C. neoformans
(Ozm (Ozoroa mucronata), Ozp (Ozoroa
paniculosa), Sle (Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr (Maytenus procumbens), Mse
(Maytenus senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi), Syp (Syzygium paniculatum))
67
Fig. 4.9. Bioautography of hexane fractions against A. fumigatus
(Ozm (Ozoroa mucronata), Ozp (Ozoroa paniculosa), Sle
(Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr (Maytenus procumbens), Mse (Maytenus
senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi), Syp (Syzygium paniculatum))
Fig. 4.10. Bioautography of dichloromethane fractions against A. fumigatus
(Ozm (Ozoroa mucronata), Ozp (Ozoroa
paniculosa), Sle (Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr (Maytenus procumbens), Mse
(Maytenus senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi), Syp (Syzygium paniculatum))
68
Fig. 4.11. Bioautography of hexane fractions against C. albicans
(Ozm (Ozoroa mucronata), Ozp (Ozoroa paniculosa), Sle
(Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr (Maytenus procumbens), Mse (Maytenus
senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi), Syp (Syzygium paniculatum)).
Fig. 4.12. Bioautography of dichloromethane fractions against C. albicans
(Ozm (Ozoroa mucronata), Ozp (Ozoroa
paniculosa), Sle (Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr (Maytenus procumbens), Mse
(Maytenus senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi), Syp (Syzygium paniculatum))
4.5.2.
Minimum inhibitory concentration (MIC)
The antibacterial activities of the phenolic-enriched extracts, fractions and sub-fractions of different polarities of
27 plant species evaluated against 4 microorganisms (two gram positive and two gram negative bacteria) are
presented in Table 4.1. The results are presented as minimum inhibitory concentrations (µg/ml) against tested
bacteria and the fungi. The extracts and fractions of the plant species tested exhibited average to good degree of
69
inhibition against the growth of all the tested bacteria and fungi strains. The extracts and fractions exhibited high
potency or growth inhibition at concentration between 19 and >2500 µg/ml for the various different organisms. In
this investigation, hexane and dichloromethane fractions had significant and broad-spectrum antimicrobial
activities against all tested microbial strains.
The most susceptible bacterium to the crude extract is E. faecalis with MIC ranged from 78 to 1250 µg/ml and
the least susceptible bacterium to the crude phenolic-enriched extract is E. coli with MIC ranging from 312 to
2500 µg/ml. However, all the microorganisms tested are highly susceptible to hexane and dichloromethane
fractions with MICs ranging from 19 to 1250 µg/ml. Some of the interesting results include the hexane and
dichloromethane fractions of C. padoides, C. vendae, C. woodii, B. galpinii, M. penducularis, M. procumbens, S.
leptodictya and S. pendulina with MICs of 19 - 39 µg/ml against E. coli. The hexane and dichloromethane
fractions of C. padoides, C. vendae, C. woodii, M. penducularis, M. procumbens, M. senegalensis, O. mucronata,
O. paniculosa, S. leptodictya and S. pentheri also exhibited good microbial growth inhibitory activity against E.
faecalis with MICs between 19 - 78 µg/ml.
Growth inhibition activities against S. aureus of interest include hexane, dichloromethane, ethyl acetate, butanol
fractions of B. galpinii and C. vendae (MIC 39 - 156 µg/ml); hexane and dichloromethane fractions of C.
padoides, O. mucronata and S. pentheri (19 - 78 µg/ml), and hexane, dichloromethane, ethyl acetate fractions of
O. paniculosa, S. leptodictya and S. pendulina (19 - 39 µg/ml).
Pseudomonas aeruginosa is also susceptible to growth inhibition by hydrogen and dichloromethane of C.
padoides, E. latissima, M. pendicularis, M. procumbens, and M. senegalensis with MICs of 19 - 78 µg/ml;
hydrogen, dichloromethane, ethyl acetate M. undata, O. paniculosa, S. leptodictya, S. pendulina and S. pentheri
with MICs of 19 - 78 µg/ml. The reference antibiotic (gentamicin) exhibited good antibacterial activity against the
four tested bacterial strains with MIC ranged between 0.18 and 1.56 µg/ml.
70
Table 4.1. Minimum inhibitory concentration (MIC) of the crude extracts and fraction against E. coli and E.
faecalis
Plant
spp
Bab
Bag
Bap
Bav
Cam
Cob
Cop
Cov
Cow
Cmh
Erl
Euc
Eun
Fic
Fig
Inc
Mpd
Mpr
Mse
Mun
Ozm
Ozp
Sle
Spd
Spt
Scb
Syp
E. coli
CRE
625
312
312
625
312
312
156
312
625
625
1250
312
312
2500
1250
1250
312
1250
2500
1250
1250
1250
1250
1250
2500
625
625
Hf
DCMf
ETOAc
Butanol
Water
39
39
78
78
156
39
19
39
39
312
156
625
312
312
156
156
156
156
156
156
19
39
39
39
19
78
312
78
312
312
312
156
156
312
312
156
312
78
156
78
78
312
1250
>2500
312
312
312
39
39
78
312
78
78
39
39
39
312
312
39
39
78
156
39
39
39
39
39
156
312
78
156
156
156
156
39
78
78
156
39
39
156
156
625
156
312
156
39
156
156
312
156
156
156
312
312
312
625
1250
1250
>2500
312
625
2500
1250
2500
156
156
>2500
>2500
312
>2500
1250
1250
312
E. faecalis
CRE
Hf
78
156
78
312
312
156
78
156
156
156
156
156
625
2500
312
1250
>2500
2500
312
625
1250
625
625
1250
1250
1250
DCMf
312
156
156
312
312
312
19
39
39
156
78
312
312
78
312
39
312
625
312
312
19
78
78
156
19
156
312
312
39
39
39
156
39
39
39
156
39
78
312
39
39
39
156
19
19
19
19
19
312
156
ETO
Ac
39
156
78
39
78
39
156
78
78
78
78
156
156
312
625
156
156
312
312
625
156
156
312
156
39
39
Butanol
Water
78
78
39
39
156
78
78
78
78
156
78
78
156
312
156
312
1250
625
625
2500
1250
78
625
625
1250
78
156
312
312
312
312
312
312
39
312
1250
2500
1250
71
Table 4.1. Cont.......Minimum inhibitory concentration (MIC) of the crude extracts and fraction against S. aureus
and P. aeruginosa
S. aureus
P. aeruginosa
Plant spp
Bab
Bag
Bap
Bav
Cam
Cob
Cop
Cov
Cow
CRE
625
625
312
625
312
625
156
156
312
Hf
78
39
156
78
312
78
19
39
39
DCMf
312
78
312
1250
156
312
39
78
78
ETOAc
625
39
78
156
156
156
78
156
Butanol
78
78
78
78
78
156
39
156
Water
312
312
78
312
312
39
312
CRE
312
312
78
156
312
312
156
156
625
Hf
78
312
312
78
156
78
78
156
312
DCMf
312
78
625
625
625
312
39
156
156
ETOAc
156
312
625
625
312
625
312
156
312
Butanol
312
156
312
312
78
156
19
39
312
Cmh
312
78
156
78
156
Erl
Euc
Eun
1250
156
312
78
312
312
312
312
625
78
78
312
312
312
312
Fic
2500
156
312
1250
Fig
625
312
Inc
625
Mpd
>2500
78
39
Mpr
1250
39
Mse
625
Mun
Ozm
625
2500
Ozp
Water
312
312
625
625
2500
1250
312
312
156
312
2500
156
625
78
312
625
78
625
625
156
156
312
78
39
78
1250
2500
156
312
625
156
625
1250
312
78
156
625
312
78
312
19
19
156
312
78
312
625
312
78
39
156
625
78
39
156
625
625
39
39
156
1250
156
19
156
19
156
625
312
1250
312
312
78
156
78
39
78
312
2500
2500
156
19
19
78
156
156
39
39
78
156
Sle
312
19
19
78
312
625
39
39
39
625
Spd
312
78
78
39
625
625
78
78
19
2500
Spt
312
19
19
156
312
156
19
19
78
2500
Scb
156
1250
156
156
156
1250
156
312
156
156
156
1250
Syp
156
39
156
312
312
2500
156
156
156
78
312
1250
312
312
312
312
1250
312
4.5.3. Minimum inhibitory concentration (MIC)
The phenolic-enriched crude extracts and fraction exhibited good to moderate growth inhibitory activities against
the three fungal strains of different morphology with MICs ranging from 19 to 2500 µg/ml (Table 4.2). Candida
albicans demonstrated resistance to all the crude extracts and fractions with the exception of dichloromethane
and butanol fractions which had MICs of 19 - 78 µg/ml. In contrast, Cryptococcus neoformans was sensitive to
majority of many crude extracts and fractions at the concentration ranging from 19 - 78 µg/ml. The fungi were
susceptible to amphotericin B with the MIC ranges from 0.78 - 6.25 µg/ml.
72
Table 4.2. Minimum inhibitory concentration (MIC) of the crude extracts and fraction against C. albicans, C. neoformans and A. fumigatus Values below 100 µg/ml in different colour
Plant spp
Bab
Bag
Bap
Bav
Cam
Cob
Cop
Cov
Cow
Cmh
Erl
Euc
Eun
Fic
Fig
Inc
Mpd
Mpr
Mse
Mun
Ozm
Ozp
Sle
Spd
Spt
Scb
Syp
C. albicans
CRE
625
312
1250
312
625
625
312
1250
1250
625
1250
625
625
2500
625
1250
312
1250
1250
1250
625
312
312
625
2500
625
312
H
625
625
156
156
625
312
625
625
156
156
156
156
78
78
78
312
156
78
625
312
156
39
39
625
156
156
DCM
78
78
78
625
312
19
312
19
39
156
156
78
39
39
19
78
39
39
78
156
78
39
78
78
312
156
ETOAc
156
156
156
156
312
156
312
78
156
156
But
39
78
156
78
19
156
156
312
78
39
156
78
156
78
156
312
78
78
39
39
156
312
156
312
156
625
625
312
312
625
156
625
312
156
312
156
156
Water
39
625
625
625
156
1250
625
312
625
625
625
156
625
312
312
312
625
625
1250
625
625
625
625
1250
625
625
C. neoformans
CRE
H
156
312
78
315
78
625
78
78
78
78
625
39
156
78
19
78
312
156
39
312
78
78
156
78
78
312
78
78
78
78
78
78
312
625
156
156
156
156
1250
156
312
78
312
156
156
312
156
312
1250
78
312
156
78
DCM
78
78
39
156
625
625
156
312
19
19
78
39
19
19
39
78
39
39
78
156
39
39
39
19
78
39
ETOAc
78
39
39
78
19
39
19
78
39
39
But
39
39
156
78
19
19
312
156
156
19
39
156
156
78
39
78
39
78
39
39
39
39
1250
156
78
312
156
156
312
156
312
78
39
78
78
39
39
Water
39
312
78
78
1250
39
312
312
312
1250
625
156
625
625
1250
2500
2500
1250
2500
1250
1250
625
1250
2500
1250
A. fumigatus
CRE
H
2500
156
625
156
625
312
625
625
19
312
312
156
156
156
156
156
156
625
39
1250
156
312
625
312
625
1250
312
625
78
1250
78
312
78
1250
78
625
156
1250
625
625
39
312
78
625
78
625
78
2500
78
312
78
312
156
DCM
312
156
78
1250
156
78
156
78
19
156
156
625
156
78
156
156
78
78
625
156
78
39
39
19
19
19
ETOAc
312
156
78
312
625
156
78
156
78
1250
312
625
312
156
78
But
156
78
156
156
312
312
156
156
78
156
156
625
312
156
1250
625
156
156
156
78
156
1250
2500
156
156
39
312
156
78
312
625
Water
625
156
156
156
156
625
625
625
625
312
625
156
312
156
312
625
625
312
625
1250
625
312
312
625
625
312
73
4.6. Discussion
4.6.1. Antimicrobial bioautography
The crude extracts and various fractions were screened qualitatively for growth inhibitory activity against 4
bacteria and 3 fungi representing different morphologies as yeasts and moulds. Many compounds present in the
non-polar enriched hexane fractions inhibited the growth of the organisms tested with several zones of
inhibitions.
However, the polar fractions exhibited poor individual inhibitory activities, and this may be due to the high
solubility of polar compounds in water, like flavonoids resulting in washing and spread of the compounds on the
TLC plate surface. Therefore, reducing the threshold inhibitory concentrations of the bioactive compounds on the
spot of the chromatogram against the organisms tested. Other factors may include the disruption of synergistic
effects of the individual compounds separated on TLC plates or the concentration of the bioactive components is
not sufficient to inhibit microbial growth.
A number of methods have been developed for effective and quick screening of microbial growth inhibitory
properties of compounds like the disc or agar diffusion assay adapted as agar-overlay methods (Rasoanaivo and
Ratsimamanga-Urverg, 1993). However, the differential diffusion of the bioactive compounds from the TLC plate
to the agar layer make the method unsuitable for certain class of compounds, especially the water-insoluble
types like terpenoids and non-polar compounds (Eloff, 1998). The direct bioautography method allows the
localization of a number of components with significant individual inhibitory activities against the tested
organisms. The characteristic features of this method are its quickness, efficiency, simplicity, high sample
throughput, small test sample size and no sophisticated equipment required. The method is adaptable and
applicable to all extracts that can be separated on TLC, against any organism capable of growing directly on TLC
plate surfaces.
4.6.2. Minimum inhibitory concentration (MIC)
In this investigation, in vitro antimicrobial efficacy of the crude 70% acetone leaf extracts and fractions derived
from 27 plants (13 genera across 9 families) used in South African traditional medicine for treating diarrhoea and
related ailments was quantitatively assessed on the basis of minimum inhibitory concentration (MIC). All the
plants evaluated exhibited varying degree of inhibitory effect against the standard strain of human and animal
pathogenic bacteria (Gram-positive as well as Gram-negative) and clinical isolate of pathogenic fungi. There
have been no specific cut-off values as a reference or standard for categorizing antimicrobial activity of plant
extracts and fractions. In this study, crude extracts and fractions with an MIC value less than 500 µg/ml were
considered to have good activity and MIC value less than 100 µg/ml were considered to have significant
antimicrobial activity of pharmacological interest according to the criterion by Rios and Recio (2005). A lower MIC
values indicated high effectiveness of the compound as antimicrobial agent as little quantity which may be below
toxicity level of the extracts can be applied without being harmful to the host.
74
Crude extracts of 4 out 27 had an MIC less than a 100 µg/ml (Bauhinia bowkeri, Bauhinia galpinii, and
Combretum padoides) against E. faecalis and Bauhinia petersiana against P. aeruginosa (78 µg/ml). However,
the antimicrobial activities were potentiated in the fractions with the non-polar fractions of hexane (MIC ranges
from 19 to 312 µg/ml) and dichloromethane (MIC ranges from 19 to 625 µg/ml) enriched with terpenoids
exhibiting more broad-based potency compared with the polar fractions of ethyl acetate (MIC ranges from 39 to
1250 µg/ml) and butanol (MIC ranges from 39 to <2500 µg/ml). The MIC value less than 100 µg/ml obtained for
some fractions were significant although much higher than that of the control antibiotic (gentamicin with an MIC
ranged from 0.18 to 1.56 µg/ml against bacteria and amphotericin B with an MIC ranged from 0.78 to 6.25 µg/ml
against fungi).
The water fractions have relatively low antimicrobial activities (MIC ranges from 312 to <2500 µg/ml) except for
the C. vendae with an MIC value 39 µg/ml against E. faecalis and S. aureus. In traditional medicine plant
preparation, water is used as the major extractant. The poor antimicrobial activity of water fractions of most of the
plants indicated that decoction or infusion may be less effective in infectious diseases. These observations are
consistent with most of the findings in other studies.
From the phytochemical evaluation, the crude extracts contain high level of polyphenolic compounds. The
activity of the extracts and polar fractions (ethyl acetate, butanol, and residual), though not exclusive to
polyphenolic compounds only would be expected to correlate to the respective constituents, the structural
configuration, functional groups and possible synergistic effects among the constituents. Members of this class of
compounds are known to have either bacteristatic or bactericidal properties against most microorganisms
depending on the structure and concentration used. The mechanism of their antimicrobial activity may be related
to their fundamental properties of having the ability to form complex with protein and polysaccharides, thus the
capacity to inactivate microbial adhesions, enzymes, and cell envelope transport protein. The presence and
position of hydroxyl group in the phenolic structure determine and influence the antimicrobial activities of this
class of compounds (Taguri et al, 2004). Phenolic compounds including tannins and flavonoids were found to
have high antimicrobial activity (Majhenic et al, 2007; Vaquero et al, 2007). Some mechanisms of antimicrobial
activity of phenolic compounds includes their ability to denature microbial proteins as surface-active agents
(Sousa et al, 2006), ability to react with cellular membrane component which impairs both function and integrity
of cells (Raccach, 1984), and the reducing property of phenolics can influence the redox potential (Eh) of
microbial growth causing growth inhibition (Jay, 1996).
However, two methods are widely used in quantitative evaluation antimicrobial activities of plants extracts: Agar
disc diffusion method (NCCLS, 2002) and serial dilution method (Eloff, 1998). Both methods depend on the
effective solubility of the extracts in the test medium in order to obtain a maximum efficacy against the
organisms. However, some phenolic compounds form complex with proteins and other macromolecules present
in the test medium, therefore, get precipitated. While some extract components especially the non-polar are not
readily soluble in test medium which is more than 90% water in most cases. These factors may at times cause
75
reduction in the effectiveness of the plant extracts to inhibit microbial growth. The antimicrobial profiles indicated
that the extracts and fractions there from were active against Gram-positive and Gram negative bacteria, yeast
and mould fungi. The susceptibility of both bacteria and fungi to the extracts may be indicative of the presence of
broad-based bioactive compounds or general metabolic toxins.
Pathogenic enteric microorganisms present in contaminated food and water produces enterotoxins or irritants
that cause intestinal disorder such as diarrhoea. In vitro antimicrobial assays against standard strains of the
intestinal pathogens using the polyphenolic-rich crude extracts and fractions have demonstrated various degree
of microbial growth inhibition. The plant extracts and fractions investigated have moderate to good activities
against diarrhoeal standard strains such as E. coli, S. aureus and C. albicans and P. aeruginosa, thus validating
their use in traditional medicine for treatment of diarrhoea symptoms. The mechanisms involved in diarrhoea
symptoms are multifaceted and interwoven. It is, therefore, possible that extracts and fractions with moderate
antimicrobial activities could still have good antidiarrhoeal effects by elaborating other biological activities such
as antioxidant, anti-inflammatory, antisecretory, binding of toxins, and antimotility effects on the gastrointestinal
tract.
4.7. Conclusion
In infectious diarrhoea many bacteria, protozoa, virus and parasites have been implicated as causative agents.
These agents include Vibrio cholera, Escherichia coli, Shigella dysenteriae, Bacillus cereus, Stapylococcus
aureus, Clostridium difficile, Entamoeba histolytica, Salmonella typhi and Giardia lamblia. Some viruses such as
Rotavirus and adenovirus have also been implicated as causative agent of diarrheal diseases. The infectious
mechanisms of the pathogenic strains of the enteric microbes include microbial adhesion and attachment,
localized effacement of the epithelial mucosa lining, production and elaboration of secretory enterotoxins,
production of cell-destroying cytotoxins, and direct epithelial cell invasion.
In this study, the emphases were on E, coli, S. aureus, E. faecalis, C.albicans as diarrhoeal pathogens. Seven
virulence groups of diarrheagenic E. coli, namely enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli
(EHEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC),
diarrhoea-associated haemolytic E. coli (DHEC) and cytolethal distending toxin (CDT)-producing E. coli have
been classified (Clarke, 2001). On a global scale EPEC, EHEC, ETEC and EIEC are the most important
diarrhoeal agents accounting for 4-8%, 0-1%, 12-20% and 0-2% respectively in terms of total episodes (Bhan,
2000). The virulence mechanisms of ETEC, EHEC, S. aureus, E. faecalis and some strains of V. cholerae
include production of endotoxin, cytotoxins and reactive species. The use of antimicrobial therapy with
microbicidal or microbistatic mechanisms may not be effective in the diarrhoea cases involving these organisms
because the toxins if already present in contaminated food or water does not need the pathogens to exert
activity. Therefore, non-antimicrobial therapy may be required in such cases but antitoxins which can antagonize
76
toxin and receptor interactions. More work is needed in evaluating the antitoxin and antiadhesion of medicinal
plant extracts as other forms anti-infectious mechanisms.
Many of the plant extracts and fractions used have good activities especially the non-polar fractions of hexane
and dichloromethane against the pathogens tested, and this may explain the traditional use of these medicinal
plants.
Considering importance of oxidative burst such as ROS/RNS in the immune mechanisms and possible
consequences of cellular damages, if the resultant oxidative stress is not resolved by the endogenous
antioxidant system, the antioxidant potentials of the plants will be evaluated in the next chapter.
77
CHAPTER FIVE
Free radical scavenging and antioxidant activities of the extracts and fractions as antidiarrhoeal
mechanism.
5.1. Introduction
The intestinal mucosa lining is constantly exposed to the oxidants and toxins from the diet, as well as to
endogenous free radicals and other highly reactive species commonly referred to as reactive oxygen species
(ROS) or reactive nitrogen species (RNS). These reactive species are generated endogenously in many basic
biochemical processes of the body from the respiration (Stojiljkovic et al., 2009) and some cell-mediated immune
functions (activated neutrophils and macrophages) in response to microbial infection (Neish, 2009). The
enzymatic sources of ROS/RNS include NAD(P)H oxidase, xanthine oxidase, uncoupled endothelial nitric oxide
(NO) synthase (eNOS), arachidonic acid metabolizing enzymes such as cytochrome P-450 enzymes,
lipoxygenase and cyclooxygenase, and the mitochondrial respiratory chain (Griendling, 2005; Mueller et al.,
2005). Exogenous hazards such as exposure to ionizing radiation, smoke and toxins can also generate free
radicals (Masoko and Eloff, 2007; Li and Trush, 1994). Free radicals such as trichloromethyl (CCl·3), superoxide
(O2˙–), hydroxyl (•OH), peroxyl (ROO-), and nitric oxide (NO-) are produced metabolically in living organisms. In
addition, some non-radical derivatives of oxygen molecules (hydrogen peroxide (H2O2), hypochlorous acid
(HOCl)), are also generated in biological systems. The formation of ROS/RNS have been implicated in the
pathogenesis of several human and animal diseases such as atherosclerosis, diabetes mellitus, chronic
inflammation, neurodegenerative disorders, gastrointestinal disorders and certain types of cancer (Catalá,
2006).The mechanisms involves in diseases initiation by free radical or oxidative species are outline in Fig 5.1. In
physiological conditions, the epithelial mucosa cell integrity and homeostasis are protected from deleterious
effects of ROS by antioxidant defence system consisting of nonenzymatic antioxidants (glutathione (GSH),
vitamins A, C, E, carotenoids) and antioxidant enzymes superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GPx) and glutathione reductase (GR) (Krishnaiah et al., 2011). However, in pathological
conditions excessive oxidation (oxidative stress) in the intestinal tract result in lipid peroxidation of the membrane
phospholipids. The peroxidation of membrane phospholipids is basically damaging because the formation of lipid
peroxidation products leads to the spread of free radical reactions and cytotoxic aldehydes by-products. The
general process of lipid peroxidation consists of three stages: initiation, propagation, and termination (Catalá,
2006). The initiation phase of lipid peroxidation includes hydrogen atom abstraction. Several species can
abstract the first hydrogen atom and include the radicals: hydroxyl (•OH), alkoxyl (RO•), peroxyl (ROO•), and
possibly HO2• but not H2O2 or O2−• (Gutteridge, 1988).
Cell injury
Disturbance to thiol-dependent
enzymes and changes in thiol:
disulphide status
Mutation
DNA damage
Destruction of nucleotide coenzyme
activities and change in redox status of
NAD(P)H
78
Covelent binding to
protein and lipid
ROS/RNS
Membrane damage to proteins,
transport disturbances
Lipid peroxidation, changes in
membrane structure and function
Changes in enzyme activities and
lipid metabolism
Damage to proteins,
increased protein turnover
Secondary metabolites causing disturbances at a
distance, such as to the functions of other membranes
Fig. 5.1. Deleterious reactions from the production of reactive free radicals in biological system (amended from
Slater et al., 1987)
The initial reaction of •OH with polyunsaturated fatty acids produces a lipid radical (L•), which in turn reacts with
molecular oxygen to form a lipid peroxyl radical (LOO•). The LOO• can abstract hydrogen from an adjacent fatty
acid to produce a lipid hydroperoxide (LOOH) and a second lipid radical (Catalá, 2006). The LOOH formed can
suffer reductive cleavage in the presence reduced metals, such as Fe2+, producing lipid alkoxyl radical (LO•).
Both alkoxyl and peroxyl radicals stimulate the chain reaction of lipid peroxidation by abstracting additional
hydrogen atoms (Buettner, 1993) (See Fig. 2.6 for detailed reaction mechanisms). Peroxidation of lipids can
disturb the assembly of the membrane, causing changes in fluidity and permeability, alterations of ion transport
and inhibition of metabolic processes (Nigam and Schewe, 2000). Injure to mitochondria induced by lipid
peroxidation causes further ROS generation (Green and Reed, 1998). In addition, LOOH break down, frequently
in the presence of reduced metals or ascorbate, to reactive aldehyde products, including malondialdehyde
(MDA), 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (4-HHE) and acrolein (Esterbauer et al., 1991; Parola
et al., 1999; Uchida, 1999; Kehrer and Biswal, 2000; Lee et al., 2001).
Lipid peroxidation and the metabolites are the main oxidative biochemical processes contributing to the
disruption of detoxifying pathways in intestine and to dysfunction of enterocytes, which may cause various
disorders of digestive tract including diarrhoea. GSH redox cycle plays the main role in lipid peroxidation
scavenging in the intestine. Enzyme GPx reduces LOOH using GSH as a reducing factor, while GR regenerates
GSH from its oxidized form glutathione disulfide (GSSG), with simultaneous oxidation of nicotinamide adenine
dinucleotide phosphate (NADPH). GSH acts not only as an enzyme cofactor, but can react directly with free
radicals and is involved in recycling other cellular antioxidants. Excessive generation of ROS/RNS and depleted
endogenous antioxidant defences have been implicated in the pathogenesis and perpetuation of intestinal
damage which can clinically manifest as diarrhoea. Under disease conditions, more ROS/RNS is generated by
the body enhancing the oxidative stress. ROS are also effective in activating redox-responsive pro-inflammatory
transcription factors, nuclear factor (NF)-β and activator protein (AP)-1 (Rahman and Adcock, 2006).
79
Supplementary therapy with antioxidant compounds provides an additional relief against deleterious effect of
ROS/RNS.
5.1.1. Superoxide ion
Superoxide radical anion (O2˙–) generated from an electron leakage in the mitochondrial respiration chain and the
conversion of xanthine dehydrogenase to xanthine oxidase (Wernes and Lucchesi, 1990) as a result of electron
donation to oxygen molecule is regarded as the primary ROS in biological system. Although O2˙– is not very
active but the radical interact with other molecules to produce highly potent secondary ROS either directly or
indirectly through enzyme and/or metal catalyzed mechanisms (Valko et al., 2005). In acute inflammation or
chronic inflammations, the production of O2˙– is increased at a rate that overwhelms the capacity of the
endogenous SOD enzyme defence to dissipate.
5.1.2. Hydrogen peroxide
The generation of hydrogen peroxide (H2O2) by activated phagocytes plays an important part in the killing of
several bacterial and fungal strains (Sanchez-Moreno, 2002). Additionally, H2O2 is generated in vivo under
physiological conditions by peroxisomes and several oxidative enzymes including glucose oxidase, d-amino acid
oxidase, and dismutation of superoxide radical, catalysed by superoxide dismutase. There is increasing evidence
that H2O2, either directly or indirectly via its reduction product (hydroxyl ion (OH-)), acts as a messenger molecule
in the synthesis and activation of inflammatory mediators (Auroma et al., 1989).
5.1.3. Hydroxyl radical
Hydroxyl radical (•OH) is the neutral form of hydroxyl ion and the most reactive free radical in biological systems
generated from free metal ions (copper or iron) catalyzed breakdown of H2O2 (Fenton reaction) or superoxide ion
reaction with H2O2 (Haber-Weiss reaction, Fig 2.6). Hydroxyl radicals have short half of 10-9 s with the highest 1electron reduction potential of 2310 mV, and is primarily responsible for the cytotoxic effect in aerobic organism.
The radical reacts with every cell components in living organisms at the second-order rate constants of 109–1010
mol/s (Siddhuraju and Becker, 2007) such as lipid, polypeptides, proteins, and DNA, especially thiamine and
guanosine. Unlike O2˙– and H2O2, which can be enzymatically eradicated by the activity of superoxide
dismutases (2O2˙– + 2H+ → H2O2 + O2) and catalases/peroxidises (2H2O2 → 2H2O + O2), respectively, there
exists no known enzyme that catalyzes the cellular detoxification of •OH (Dwyer et al., 2009).
5.1.4. Peroxyl radical
Peroxyl radicals are important reactive species in living systems formed by a direct reaction of oxygen with alkyl
radicals or the protonation of the superoxide ions. Peroxyl radicals are potent oxidants with standard reduction
potential of more than 1000 mV (Decker, 1998). The radicals abstract hydrogen from other molecules with lower
standard reduction potential to perpetuate chain reaction such as propagation stage of lipid peroxidation. Cell
80
membranes including intestinal epithelial mucosa are phospholipid bilayers with extrinsic proteins and are the
primary target of lipid peroxidation (Girotti, 1998) causing cell dysfunction and tissue injury. Lipid peroxidation
cytotoxic by-products such as malonaldehyde can react with free amino group of proteins, phospholipid, and
nucleic acids leading to structural modification, which induce dysfunction of immune systems.
5.1.5. Hypochlorous acid
Hypochlorous acid (HOCl) is also a strong oxidant generated in vivo by neutrophil myeloperoxidase (MPO)
catalyzed oxidation of chloride ions and H2O2. The cytotoxicity of this reaction contributes to the phagocytosis of
infectious microorganisms in the host defence system. However, HOCl generated by MPO also inactivate some
enzymes such as α-antiproteinase contributing to proteolytic damage of healthy human tissues in inflammatory
disease (Halliwell and Gutteridge, 1990; Hippeli and Elstner, 1999). The oxidant has also been implicated as a
secretagogue.
5.1.6. Nitric oxide
Biological tissues generate nitric oxide (NO•) by specific nitric oxide synthases (eNOS, iNOS) metabolization of
arginine to citrulline via a five electron oxidative mechanisms. In normal physiological processes, nitric oxide
(NO•) acts as an important oxidative biological signalling molecule in neurotransmission, blood pressure
regulation, defence mechanisms, smooth muscle relaxation and immune regulation (Bergendi et al, 1999). Nitric
oxide (NO•) has greater stability in an environment with a lower oxygen concentration compared to the hydroxyl
radical with half life >15 s.
Cells of the immune system produce both the superoxide anion and nitric oxide in the oxidative burst inducing
inflammatory processes. Under these conditions, nitric oxide and the superoxide anion may react together to
produce significant amounts highly reactive oxidative molecule (peroxynitrite anion (ONOO−)). This potent
oxidising agent that can cause DNA fragmentation and initiate lipid peroxidation (Carr et al., 2000):
NO• + O2•−→ ONOO−
The NO• toxicity is predominantly linked to its ability to combine with superoxide anions with the rate constants
known for reactions of NO•, 7.0×109M−1 s−1.
5.2. Antioxidant assays
Several standardized methods have been proposed to analyze the antioxidant potential of a substrate including
plant extract and isolated compounds from it. Criteria for the standard methods include (i) measurement of the
chemical process actually occurring in potential applications; (ii) utilization of biological relevant molecules; (iii)
technically simple; (iv) with a defined endpoint and chemical mechanism; (v) readily available instrumentation;
(vi) good repeatability and reproducibility; (vii) adaptable for assay of both hydrophilic and lipophilic antioxidants;
(viii) and adaptable to high-throughput analysis (Prior, et al., 2005). The assays are based on scavenging
capacity against specific biological ROS/RNS and/or against stable, non-biological radicals and evaluation of
81
total reduction capacity such as 1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging assay (BrandWilliams et al., 1995); 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging method
(Re et al., 1999); β-carotene linoleic acid bleaching assay (Siddhuraju and Becker, 2003); inhibition of linoleic
acid peroxidation (Osawa and Namiki, 1981); ferric reducing antioxidant power (FRAP) (Benzie and Szeto,
1999); total radical trapping antioxidant potential (TRAP) assay (Leontowicz et al., 2002); hydroxyl radical
scavenging activity (Jodynis-Liebert et al., 1999); hydrogen peroxide scavenging activity (Ruch et al., 1989); nitro
blue tetrazolium (NBT) reduction assay or superoxide anion scavenging activity (Kirby and Schmidt, 1997) and
oxygen radical absorbance capacity (ORAC) assay (Silva et al., 2007). Each method has its own merit and
demerit in evaluating antioxidant capacity of plant extracts and their components. Based on the criteria
enumerated above, the most common and reliable methods are the ABTS and DPPH methods.
5.2.1. Antioxidant bioautography
For the qualitative detection of free radical scavengers and the number of antioxidant compounds present, DPPH
or β-carotene is usually the spraying reagents (Martson, 2011) of TLC chromatograms. DPPH is a purplecoloured free radical that turns yellow on reduction by an antioxidative component of an extract. Yellow spots on
TLC plates spayed with DPPH solution against the purple background indicate the presence of an active
compound(s). In the β-carotene assay, the TLC plate is sprayed with a solution of β- carotene, dried and
exposed to 254 nm UV light to bleach the β- carotene. Areas where antioxidants inhibit degradation of βcarotene appear as orange zones on a pale background.
5.2.2. The chemistry of some common antioxidant assays
5.2.2.1. Hydroxyl radical
Hydroxyl radical is the most reactive species and source of many other secondary free radicals in biological
systems; thus, it is important to evaluate hydroxyl radical scavenging capability. Hydroxyl radical (HO•)
scavenging is usually evaluated using the ‘‘deoxyribose assay’’: a mixture of ferric chloride (FeCl3) and
ethylenediamine tetraacetic acid (EDTA) in the presence of ascorbate reacts to form iron(II)-EDTA plus oxidized
ascorbate, H2O2 then reacts with iron(II)-EDTA to generate iron(III)-EDTA plus HO• from the Fenton reaction
(Fe2+H2O2→Fe3+ HO•+ HO-). The radicals not scavenged by other components of the reaction mixture attack
the sugar deoxyribose, and degrade it into a series of fragments, some or all of which react on heating with
thiobarbituric acid at low pH to give a pink chromogen. Thus the scavenging activity towards HO- of a substance
added to the reaction mixture is measured on the basis of the inhibition of the degradation of deoxyribose.
Another spectrophotometric method developed for assessment of hydroxyl radical scavenging capacity of
antioxidants includes Fenton reaction as the hydroxyl radical generation system and salicylate as a
spectrophotometric indicator. Attack by •OH radicals on salicylate produce 2, 3-dihydroxybenzoate, 2, 4dihydroxybenzoate, and 2, 5-dihydroxybenzoate as major products. The hydroxylated products can be identified
and quantified by Beer’s law testing the additivity of absorbances of the hydroxybenzoates. This method is able
82
to measure the hydroxyl radical scavenging capability of individual antioxidants with a wide dynamic activity
range, i.e., 635-637 nm. These spectrophotometric methods may not be the most sensitive, but they are simple,
reproducible, and cost effective method valuable in antioxidant studies.
5.2.2.2. Hydrogen peroxide scavenging
Hydrogen peroxide-scavenging activity is measured by using a peroxidase-based assay system. The most
common used peroxidase is horseradish, which uses H2O2 to oxidize scopoletin into a nonfluorescent product. In
the presence of a putative scavenger, the oxidation of scopoletin is inhibited and the H2O2 scavenging can be
monitored by decay in H2O2 concentration spectrophotometrically from absorption at 230 nm using the molar
optical density of 81 M-1cm-1.
5.2.2.3. Superoxide scavenging capacity
The scavenging activity towards superoxide by antioxidants is measured by the inhibition of generation of
superoxide with the hypoxanthine–xanthine oxidase (HX-XO) system. The superoxide generating from HX–XO
reduces nitro-blue tetrazolium (NBT) to formazan at pH 7.4 and room temperature which can be followed
spectrophotometrically at 560 nm. Any added molecule capable of reacting with superoxide inhibits the
production of formazan and the reduction of the absorbance is estimated as superoxide scavenging activity
compared to the value obtained with no test added sample.
5.2.2.4. 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH• assay)
The DPPH radical scavenging assay is hydrogen atom transfer processes widely used evaluate the antioxidant
activity of reductants (plant extracts, phytochemical or pharmaceutical drugs) (Kaviarasan et al., 2007). Although
DPPH assay has no direct biological relevance, the process is related to the inhibition of lipid peroxidation
(Rekka and Kourounakis, 1991). The DPPH free radical is reduced to the corresponding hydrazine when it reacts
with hydrogen donors (antioxidant) using decolouration mechanisms (purple to yellow), which are monitored by
the decrease in absorbance at 515–528 nm. From the methodological point of view the assay conducted in
ethanol or methanol solution of DPPH is considered a valid, easy and accurate assay to evaluate radical
scavenging activity of antioxidants, since the radical compound is stable and does not have to be generated as in
other radical scavenging assays. The results are highly reproducible and comparable to other free radical
scavenging methods such as ABTS.
5.2.2.5. 2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTS•+) free radical-scavenging
method
The ABTS radical scavenging method is based on the reduction of blue/green ABTS˙+ chromophore generated
from the reaction between ABTS and potassium persulphate (K2S2O7) by an electron-donating antioxidant. The
83
decolourization of the ABTS˙+ chromophore is measured spectrophotometrically at 734 nm in both lipophilic and
hydrophilic medium.
ABTS (white) + K2S2O7 (white) → ABTS˙+ (blue-green)
5.2.2.6. Ferric reducing antioxidant power (FRAP)
The FRAP method is based on the reduction at low pH, of a colourless ferric complex to an intense bluecoloured ferrous complex by an electron donating antioxidant. The reduction of ferric complex is monitored by
measuring the increase in absorbance at 750 nm. The chemistry of FRAP assay can be summarized with
equation (1) with oxidant accepting an electron from antioxidant to be reduced an intense coloured molecule.
Fe(CN)63− + antioxidant → Fe(CN)64− + oxidized antioxidant
Fe(CN)64− + Fe3+→ Fe[Fe(CN)6]−
The FRAP assay is a robust and potentially useful test using inexpensive reagents and equipment and a speedy
reaction applicable over a wide concentration range.
5.3. Materials and Methods
5.3.1. Qualitative antioxidant assay using TLC-DPPH method
This was done to determine the number of active compound(s) present in the crude extracts and fraction(s) with
free radical scavenging capacity against DPPH radical. Chromatograms were developed as described in section
3.6.4 and sprayed with 0.2% methanolic DPPH solution. The presence of yellow spots against a purple
background indicated component(s) with antioxidant activities.
5.3.2.1. DPPH• radical-scavenging assay
The antioxidant activities of the samples were measured in term of radical scavenging ability using the stable
radical (DPPH.) of Brand-Williams et al, (1994) with some modifications. Methanol solutions (40 µl) of the
samples at various concentrations (19–2000 µg/ml), and positive control (trolox and ascorbic acid) at
concentration (19 to 250 µg/ml) were added to 160 µl of DPPH in methanol (25 µg/ml) in a 96 well-microtitre
plate. The change in absorbance (516 nm) measured after at 1, 10, 20 and 30 min (At) with a microtitre plate
reader (Versamax). The sample concentrations were corrected for the dilutions.
5.3.2.2. ABTS•+ radical-scavenging assay
The free radical-scavenging activity as a measure of hydrogen donating capacity was determined by using ABTS
cation decolourization method of Re et al., 1999 with some modifications. ABTS radical solution (7 µM) was
prepared by dissolving 1.32× 104 µg of ABTS in 10 ml of 50% methanolic solution and 7.68 ×104 µg of
potassium persulphate (K2S2O4) in 10 ml of distilled water. The two solutions were mixed together and made up
to 200 ml with 50% methanolic solution, and kept in the dark at room temperature for 12 h. Prior to running the
assay, the ABTS radical solution was diluted with a 50% methanolic solution to an absorbance (Ao2) between
84
0.7- 0.8 at 734 nm. The extracts were serially diluted (40 µl) (19 to 2000 µg/ml) in 96 well-microtitre plate and
160 µl of ABTS radical solution added to each well. The absorbance were taken exactly after 6 min of reaction
(At2) and blank absorbance (Ab2) were prepared using the respective extracts without ABTS radical.
5.3.2.3. FRAP assay
The FRAP of the samples was determined by direct reduction of potassium ferric cyanide (K3Fe3(CN)6) to
potassium ferrocyanide (K3Fe2(CN)6) (electron transfer process from the antioxidant). The increase in
absorbance from the formation of Pearl’s Prussian blue complex following the addition of excess ferric ion was
measured as described by Berker et al., (2007) with some modification. The reaction medium (200 µl) containing
40 µl of the test samples or positive controls (trolox and ascorbic acid) (concentration range between 19 -2000
µg/ml), 100 µl of 1.0M hydrochloric acid, 20 µl of 1% (w/v) of SDS, 30 µl of 1% (w/v) of potassium ferric cyanide
was incubated for 20 min at 50OC, cooled to room temperature and finally 20 µl of 0.1% (w/v) of ferric chloride
was added. The absorbance at 750 nm was read and blank absorbance was taken by preparing the reaction
medium the same way except the addition of ferric chloride. The reducing capacities were taken as slope
obtained from the line of best fit of the absorbance against concentration using the linear regression curve.
5.3.2.4. Hydroxyl radical scavenging assay
The hydroxyl radical scavenging activities of the test samples were measured by the salicylic acid method. The
hydroxyl radical scavenging activity of the extracts was determined according to method of Smirnorff and
Cumbes, (1996) with some modifications. The hydroxyl radical was generated by using Fenton reaction which
contains 50 ml of FeCl3 (8.0 mM), 80 ml of H2O2 and 50 ml of distilled water was allowed to stand for 1h. The
mixture was filtered to remove the debris. Hydroxyl radical was determined by mixing 120 µl of the hydroxyl
radical solution with 66µl of the extracts followed by 14 µl of salicylic acid (20 mM). The mixture was incubated
for 30min at 37oC and absorbance taken at 510 nm.
5.3.2.5. Lipid peroxidation inhibition assay
Lipid peroxidation of linoleic acid was determined as described by Kishida et al., 1993. Each reaction mixture
contained 4.1 ml of 2.5% linoleic acid in ethanol and 10 ml of 0.2M phosphate buffers (pH 7.4), 1.0 ml of 0.0025
mg/ml FeSO4 was added as catalyst. Different concnetartions of the samples (10-500 ug/ml) were added to the
reaction mixture in a centrifuge tube. The reaction mixture was incubated at 40oC for 2 h. The reaction was
terminated by adding 1.0 ml of 25% trichloroacetic acid and 1.0 ml of 0.67% thiobarbituric acid. The reaction
mixture was heated at 95oC for 30 min and cooled for 15 min. The mixture was extracted with butanol and
absorbance measured at 500 nm.
85
5.4. Results
5.4.1. TLC-DPPH analyses (Antioxidant bioautography)
The qualitative antioxidant screening of spraying DPPH on TLC plate indicated the presence of a number of
antioxidant compounds in the crude extracts and fractions (hexane, dichloromethane ethyl acetate, and butanol).
Antioxidant compounds were visualized as yellow spot against the purple background of DPPH as shown in Fig.
5.2 - 5.5. The numbers of active compounds identifiable on a plate depend on the mobile phase used in the
development of the plate. For the crude extracts, solvents 1and 2 separated the antioxidative compounds but
solvent 3 revealed close similarity between Combretum vendae and Combretum woodii, both exhibiting activities
at three different spots of same with Rf values of 0.94, 0.53 and 0.48 respectively.
Fig.5.2. TLC-DPPH profiles of the crude extracts of extracts of different plants
(left to right Bab (Bauhinia bowkeri), Bag
(Bauhinia galpinii), Bap (Bauhinia petersiana), Bav (Bauhinia variegata), Erl (Erythrina latissima), Inc (Indigofera cylindrica), Scb (Schotia brachypetala),
Cob (Combretum bracteosum), Cop (Combretum padoides), Cov (Combretum vendae), Cow (Combretum woodii), Ozm (Ozoroa mucronata), Ozp
(Ozoroa paniculosa), Sle (Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr (Maytenus
procumbens), Mse (Maytenus senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi), Syp (Syzygium
paniculatum) developed with ethyl actetate: acetic acid: formic acid: water (75:5:5:10) (top), ethyl actetate: acetic acid: formic acid: water (70:5:15:10)
(middle) and ethyl acetate: methanol: ammonia (90:20:15) (bottom)).
For the hexane fraction using eluent 5, the antioxidant compound was present only in Erythrina latissima sample
with Rf value of 0.26. The dichloromethane fraction of the extract exhibited antioxidant activity at many spots with
some being minor while E. latissima, C. vendae, and C. woodii revealed major antioxidant spots at Rf values of
0.6, 0.46 and 0.33 using solvent 5. Spots at Rf values of 0.53 and 0.40 were peculiar to C. vendae, and C. woodii
respectively. Eluent 6 separated more antioxidant components in all the plants tested with Bauhinia bowkeri, E.
86
latissima, C. vendae, and C. woodii exhibiting activity at 0.8, E. latissima, C. vendae, and C. woodii at 0.66, B.
bowkeri, B. galpinii, B. petersiana, B. variegata, E. latissima, C. vendae, and C. woodii at 0.51, C. vendae and C.
woodii at 0.17 and C. vendae at 0.1.
Fig. 5.3. TLC-DPPH profile of the hexane fractions of different plants
(Bab (Bauhinia bowkeri), Bag (Bauhinia galpinii), Bap
(Bauhinia petersiana), Bav (Bauhinia variegata), Erl (Erythrina latissima), Inc (Indigofera cylindrica), Scb (Schotia brachypetala), Cob (Combretum
bracteosum), Cop (Combretum padoides), Cov (Combretum vendae), and Cow (Combretum woodii) developed with hexane: ethyl acetae: formic acid
(70:30:2))
Fig.5.4 TLC-DPPH profiles of the dichloromethane fractions of different plants
(left to right Bab (Bauhinia bowkeri), Bag
(Bauhinia galpinii), Bap (Bauhinia petersiana), Bav (Bauhinia variegata), Erl (Erythrina latissima), Inc (Indigofera cylindrica), Scb (Schotia brachypetala),
Cob (Combretum bracteosum), Cop (Combretum padoides), Cov (Combretum vendae), Cow (Combretum woodii), Ozm (Ozoroa mucronata), Ozp
(Ozoroa paniculosa), Sle (Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr (Maytenus
procumbens), Mse (Maytenus senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi), Syp (Syzygium
paniculatum) developed with hexane:ethyl acetae: formic acid (70:30:2) top, hexane:ethyl acetae: formic acid (50:50:2) (middle) and chloroform:ethyl
acetate:formic acid (50:40:10) bottom.
87
Fig. 5.5. TLC-DPPH profiles of the ethyl acetate fractions of different plants left tor right
(left to right Bab (Bauhinia
bowkeri), Bag (Bauhinia galpinii), Bap (Bauhinia petersiana), Bav (Bauhinia variegata), Erl (Erythrina latissima), Inc (Indigofera cylindrica), Scb (Schotia
brachypetala), Cob (Combretum bracteosum), Cop (Combretum padoides), Cov (Combretum vendae), Cow (Combretum woodii), Ozm (Ozoroa
mucronata), Ozp (Ozoroa paniculosa), Sle (Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Mpd (Maytenus peduncularis), Mpr
(Maytenus procumbens), Mse (Maytenus senegalensis), Mun (Maytenus undata), Cam (Carissa macrocarpa), Com (Commiphora harveyi), Syp
(Syzygium paniculatum) developed with chloroform:ethyl acetate:formic acid (50:40:10) (top), hexane: ethyl acetate:formic acid (10:90:10) (middle) and
ethyl acetate:methanol:water:formic acid (100:13:10:2) (bottom).
5.4.2. Effective concentration required to reduce DPPH radical (oxidant) by half (EC50)
The phenolic-enriched crude extracts and fractions exhibited strong radical scavenging activity against DPPH
radicals in a dose dependent manner (Table 5.1). There are significant variations in the capacity of the test
samples to scavenge the DPPH radical with EC50 ranging from 0.21±0.03 to 303.65±3.84 µg/ml. Butanol
fractions of Combretum padoides had the highest anti DPPH radical activities compared to crude extracts and
other fractions with EC50 0.21±0.03 µg/ml followed by butanol fractions of Combretum vendae and Combretum
woodii with EC50 0.25±0.06 and 0.33±0.01 µg/ml respectively. The EC50 of these fractions are lower than the
EC50 of the positive controls (trolox 1.18±0.06 – 1.31±0.07 µg/ml and ascorbic acid 1.50±0.06 – 1.68±0.07
µg/ml). As expected the non-polar compounds present in the hexane and dichloromethane fractions had poor
radical scavenging activities compared to the controls.
Table 5.1. DPPH radical scavenging potential of the crude extract and fractions expressed as EC50 (µg/ml)
88
Plant
species
Bab
Crude
Hexane
DCM
ETOAc
Butanol
19.53±4.83a
11.14±3.59b
5.21±1.04b,c
1.25±0.23c
0.64±0.05c
Residual
Water
4.99±0.56c
Bag
14.39±0.48a
79.58±13.14b
9.92±1.16a,c
2.82±0.44a,c
1.02±0.06c
2.86±0.42a,c
Bap
43.29±5.05a
47.45±2.91a
8.18±1.11b
3.21±1.01b,c
1.51±0.07c
15.20±1.66d
Bav
123.60±11.05a
97.02±30.03a
8.40±0.62b
1.88±0.10b
0.89±0.05b
23.07±3.83c
Erl
2.54±1.40
76.71±20.25
6.02±2.0
5.61±0.37
2.24±0.05
57.98±13.94
Cob
5.72±1.21
85.04±10.56
20.53±0.40
7.76±0.68
4.97±0.19
35.65±4.78
Cop
4.44±0.35
12.65±1.3
3.33±0.30
0.44±0.06
0.21±0.03
0.84±0.15
Cov
1.65±0.20
16.88±2.66
1.02±0.14
0.25±0.06
0.96±0.12
Cow
3.88±1.78
9.41±3.51
2.16±0.284
1.24±0.13
0.33±0.01
1.10±0.07
Ozm
15.82±4.02
132.13±5.0
63.48±4.00
7.92±1.63
-
-
Ozp
1.29±0.07
31.95±5.6
9.77±0.71
1.22±0.44
11.79±1.12
-
Sle
1.81±0.09
54.88±2.53
10.14±1.51
0.91±0.04
38.93±0.28
-
Spd
1.19±0.15
138.5±9.50
16.51±0.77
1.26±0.03
41.8±3.37
-
Spt
4.26±0.40
139.63±10.62
4.91±0.69
2.09±0.32
-
-
Mpd
3.81±0.03
113.4±12.60
28.20±4.14
2.33±0.21
29.95±4.01
76.79±10.30
Mpr
7.39±0.32
111.2±10.69
30.80±4.56
2.52±0.30
20.71±0.90
189.50±7.56
Mse
13.46±0.52
253.0±29.69
121.46±11.0
4.73±0.06
24.01±0.13
81.17±11.39
Mun
6.99±0.14
160.4±31.4
42.88±6.16
2.23±0.15
7.86±15
303.67±3.84
Euc
3.00±0.37
134.46±10.8
4.70±0.72
0.84±0.00
0.91±0.19
2.04±0.24
84.88±9.74
7.23±4.40
1.66±0.22
1.34±0.00
2.62±1.19
Inc
Scb
Eun
Fic
Fig
Cam
5.85±0.55
138.46±35.73
11.03±0.77
1.85±0.27
2.55±0.23
10.4±2.36
Com
19.31±1.40
103.22±30.37
17.08±0.27
0.90±0.00
1.13±0.22
310.53±12.0
Syp
Bab (Bauhinia bowkeri), Bag (Bauhinia galpinii), Bap (Bauhinia petersiana), Bav (Bauhinia variegata), Cam (Carissa macrocarpa), Cob (Combretum
bracteosum), Cop (Combretum padoides), Cov (Combretum vendae), Cow (Combretum woodii), Com (Commiphora harveyi), Erl (Erythrina latissima), Euc,
(Euclea crispa), Eun (Euclea natalensis), Fic (Ficus craterestoma), Fig (Ficus glumosa), Inc (Indigofera cylindrica), Mpd (Maytenus peduncularis), Mpr
(Maytenus procumbens), Mse (Maytenus senegalensis), Mun (Maytenus undata), Ozm (Ozoroa mucronata), Ozp (Ozoroa paniculosa), Sle (Searsia
leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Scb (Schotia brachypetala), Syp (Syzygium paniculatum)
Surprisingly the residual water fractions of many of the plant species also had a low antioxidant activity this may
be due to the insolubility of the dried water fraction. If polyphenolics reacted with e.g. sugars to form insoluble
complexes it would explain the results. The least active samples are the residual water fractions of C. harveyi
(310.53±12.00 µg/ml) and Maytenus undata (303.61±3.84 µg/ml).
Other fractions with notable antioxidant activity were the butanol fractions of Bauhinia bowkeri (0.64±0.05 µg/ml),
Bauhinia galpinii (1.02±0.06 µg/ml), Bauhinia variegata (1.51±0.07 µg/ml) and Commiphora harveyi (1.13±0.22
89
µg/ml); the ethyl acetate fractions of C. padoides (0.44±0.06 µg/ml), C. vendae (1.02±0.14 µg/ml), Ozoroa
paniculosa (1.22±0.44 µg/ml), Searsia leptodictya (0.91±0.04 µg/ml), C. harveyi (0.90±0.00 µg/ml); and residual
water fractions C. padoides (0.84±0.15 µg/ml), C. vendae (0.96±0.12 µg/ml) and C. woodii (1.10±0.07 µg/ml).
The ethyl acetate fractions of all the 27 plant samples exhibited good antiradical activities against DPPH radical
with EC50 ranging between 0.44±0.06 (C. padoides) – 7.92±1.63 µg/ml (Ozoroa mucronata). From the estimated
EC50 values, the order of potency is butanol fraction > ethyl acetate fraction > crude extract > dichloromethane
fraction > residual water fraction > hexane fraction.
5.4.3. Effective concentration required to reduce ABTS radical (oxidant) by half (EC50)
The ABTS˙+ radical scavenging capacity of the crude extract and the fractions expressed as EC50 are presented
in Table 5.2 with lower EC50 indicate higher antiradical activity. A wide variation in the ABTS˙+ radical scavenging
capacity of the crude extracts and the fractions which range from 0.43 ± 0.03 to 1709 ± 91.44 µg/ml.
The same trend of DPPH radical scavenging activity is also noticeable with the ABTS radical scavenging assay
though the EC50 are slightly higher. Butanol fractions of C. padoides had the highest anti DPPH radical activities
compared with crude extracts and other fractions with EC50 0.21±0.03 µg/ml followed by butanol fractions of C.
vendae and C. woodii with EC50 0.25±0.06 and 0.33±0.01 µg/ml respectively. The EC50 of these fractions are
notably lower than the EC50 of the positive controls (Trolox 1.18±0.06 – 1.31±0.07 µg/ml and ascorbic acid
1.50±0.06 – 1.68±0.07 µg/ml). Other fractions which have notable antioxidant activity were the butanol fractions
of B. bowkeri (0.88±0.18 µg/ml), B. galpinii (0.89±0.04 µg/ml), B. variegata (1.05±0.11 µg/ml), C. vendae
(0.60±0.03 µg/ml), C. woodii (0.89±0.06 µg/ml) and E. crispa (1.45±0.08 µg/ml).The ethyl acetate fractions of
C. padoides (0.79±0.01 µg/ml), C. vendae (1.20±0.30 µg/ml), C. woodii (1.30±0.13 µg/ml), S. pentheri
(1.25±0.08 µg/ml), E. crispa (1.34±0.03 µg/ml) including the crude extract of O. paniculosa (0.99±0.05 µg/ml)
also have EC50 lower or comparable to the control. The hexane fractions exhibited poor antiradical activity with
the EC50 being 6.78±0.29 and 1709.0±91.44 µg/ml for C. woodii and C. bracteosum respectively. From the
estimated EC50 values, the order of potency is ethyl acetate fraction > butanol fraction > crude extract >
dichloromethane fraction > residual water fraction > hexane fraction.
Table 5.2. ABTS radical scavenging potential of the crude extract and fractions expressed as EC50
(µg/ml)
Plant spp
Crude
Hexane
DCM
ETOAc
Butanol
Residual Water
Bab
14.50±0.50
50.63±6.37b
5.54±0.48c
1.81±0.13c
0.88±0.18c
17.99±2.34a
Bag
55.01±0.25
102.25±5.04b
11.81±0.90c
3.21±0.22d
0.89±0.04d
6.21±1.03c
Bap
17.19±0.14
116.27±0.57b
9.76±0.20a
5.01±0.07c
7.31±0.85c
40.44±7.09c
Bav
9.24±1.30a
85.84±2.55b
8.58±0.43a
2.40±0.22a
1.05±0.11a
73.91±3.68b
Erl
246.37±17.73
50.89±6.08
8.76±0.22
6.52±0.19
18.52±1.10
125.00±4.22
90
Inc
44.95±5.60
1017.73±65.41
49.89±10.43
3.19±1.16
ND
ND
Scb
4.12±0.53
276.76±54.53
ND
5.3.3±0.60
2.09±0.45
61.64±15.23
Cob
11.34±1.5
1709±91.44
59.12±2.33
7.38±0.71
22.57±1.03
190.64±16.3
Cop
4.17±0.02
22.72±1.46
4.03±0.20
0.71±0.01
0.43±0.03
1.47±0.82
Cov
6.01±0.07
23.93±0.95
2.82±0.44
1.20±0.03
0.60±0.03
4.26±0.12
Cow
9.78±0.08
6.78±0.29
1.71±0.03
1.30±0.13
0.83±0.06
5.73±1.50
Ozm
15.93±2.10
43.48±4.20
17.55±2.79
10.74±0.45
68.85±23.76
288.17±7.05
Ozp
0.99±0.05
191.47±10.94
161.60±4.16
1.60±0.61
7.74±4.57
172.67±29.37
Sle
5.43±0.07
129.17±20.20
113.80±11.07
4.63±0.56
23.43±7.72
278.77±16.25
Spd
1.94±0.20
213.73±18.31
153.00±15.48
2.19±0.24
20.39±0.24
87.85±8.60
Spt
4.70±0.24
142.80±6.62
169.33±4.38
1.25±0.08
ND
ND
Mpd
8.64±0.13
114.64±25.93
33.54±1.29
6.33±0.18
52.79±14.43
74.89±2.80
Mpr
4.03±0.18
277.80±16.13
22.26±1.33
1.71±0.13
8.99±2.86
130.70±15.05
Mse
5.34±0.39
312.73±43.83
139.90±13.65
3.59±0.06
7.78±3.13
62.86±3.90
Mun
7.89±0.30
286.30±7.78
55.30±5.09
6.66±1.53
5.74±1.37
220.27±30.15
Euc
4.18±0.86
83.39±15.89
2.50±0.39
1.34±0.03
1.45±0.08
4.51±0.13
Eun
3.53±0.55
281.77±42.97
7.41±0.33
2.10±0.23
3.25±0.16
6.54±0.40
Fic
548.43±191.60
273.36±31.81
4.63±0.92
42.09±8.04
ND
Fig
285.43±12.83
187.53±4.54
9.06±1.51
2.80±0.50
13.93±1.02
Cam
7.87±0.93
293.97±77.70
11.68±3.66
2.66±0.14
2.45±0.21
17.92±1.37
Com
19.13±0.69
51.11±2.36
6.14±0.49
2.07±0.10
3.35±
270.03±20.94
Syp
Bab (Bauhinia bowkeri), Bag (Bauhinia galpinii), Bap (Bauhinia petersiana), Bav (Bauhinia variegata), Cam (Carissa macrocarpa), Cob (Combretum
bracteosum), Cop (Combretum padoides), Cov (Combretum vendae), Cow (Combretum woodii), Com (Commiphora harveyi), Erl (Erythrina latissima), Euc,
(Euclea crispa), Eun (Euclea natalensis), Fic (Ficus craterestoma), Fig (Ficus glumosa), Inc (Indigofera cylindrica), Mpd (Maytenus peduncularis), Mpr
(Maytenus procumbens), Mse (Maytenus senegalensis), Mun (Maytenus undata), Ozm (Ozoroa mucronata), Ozp (Ozoroa paniculosa), Sle (Searsia
leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri), Scb (Schotia brachypetala), Syp (Syzygium paniculatum)
5.4.4. Ferric reducing antioxidant power (FRAP) gradient
The FRAP results are presented in Table 5.3 as the slope of the best fit linear regression analysis. Some of the
ethyl acetate and butanol fractions had moderate to good dose-dependent ferric ion reducing capability
comparable to the controls (trolox and ascorbic acid). The ethyl acetate fractions Carissa macrocarpa (45.0±3.7),
Combretumpadoides (54.15±4.87), Combretum vendae (49.87±2.91), Combretum woodii (45.89 ± 3.87),
Commiphora harveyi (50±3.5), Euclea crispa (48.0±4.8) and Euclea natalensis (42.0±3.9) have reducing power
gradients compared to the trolox and ascorbic acid. The butanol fractions of Bauhinia bowkeri (40.92±2.14), C.
padoides (44.11±4.06), C. vendae (42.59± 3.81), C. woodii (41.19 ± 1.28) and E. crispa (45.0±4.2) also
exhibited good reducing power comparable with the control. The orders of reducing capacity is ethyl acetate >
butanol > DCM > water > hexane > crude extract.
Table 5.3. Ferric reducing antioxidant power (FRAP) of the crude extracts and fractions expressed as
the gradient of the linear curve
91
Plant
Crude extract
species
Hexane
DCM fraction
fraction
ETOAc
Butanol
Water
fraction
fraction
fraction
Bab
3.30±0.17
8.15±0.76
18.75±1.18
31.84±3.36
40.92±2.14
10.26±1.21
Bag
3.63±0.19
8.36±0.41
15.20±0.59
21.48±1.55
32.70±2.10
31.75±2.08
Bap
1.51±0.05
7.57±0.34
16.42±0.83
15.50±1.69
24.19±1.22
24.63±1.14
Bav
2.63±0.27
11.74±0.62
15.22±0.75
27.75±2.58
32.06±1.11
0.47±0.05
Cam
1.70±0.10
6.1±0.57
17.0±0.87
45.0±3.7
29.1±1.7
5.2±0.59
Cob
9.4±0.32
0.27±0.037
1.6±0.10
2.7±0.40
5.2±0.25
11.0±0.37
Cop
6.36±0.26
16.96±0.91
22.68±1.04
54.15±4.87
44.11±4.06
9.59±0.58
Cov
3.310 ± 0.25
14.28±0.72
27.84±1.66
49.87±2.91
42.59±3.81
3.59±0.37
Cow
4.98±0.21
24.02±1.79
25.91±2.98
45.89±3.87
41.19±1.28
3.43±0.67
Com
1.1±0.15
14.0±0.57
26±1.7
50±3.5
36±3.0
0.26±0.08
Erl
0.29±0.07
16.0±0.60
23.0±1.3
33.0±2.9
18.0±0.84
1.8±0.13
Euc
5.5±0.17
11.0±1.2
25.0±2.1
48.0±4.8
45.0±4.2
20.0±0.91
Eun
5.3±0.27
6.1±0.18
21.0±1.2
42.0±3.9
34.0±2.2
20.0±1.9
Bab (Bauhinia bowkeri), Bag (Bauhinia galpinii), Bap (Bauhinia petersiana), Bav (Bauhinia variegata), Cam (Carissa macrocarpa), Cob (Combretum
bracteosum), Cop (Combretum padoides), Cov (Combretum vendae), Cow (Combretum woodii), Com (Commiphora harveyi), Erl (Erythrina latissima), Euc,
(Euclea crispa), Eun (Euclea natalensis)
5.4.5. Effective concentration required to reduce hydroxyl radical (oxidant) by half (EC50)
The scavenging ability of the crude extracts and fractions expressed as EC50 are presented in Table 5.4. The
EC50 ranged from 11.03±2.80 µg/ml (dichloromethane fraction of O. paniculosa) to 356.80±2.39 µg/ml
(dichloromethane fraction of M. senegalensis). The order of hydroxyl radical inhibition is butanol > ethyl acetate >
crude extract > dichloromethane > hexane.
5.4. Hydroxyl radical scavenging potential of the crude extract and fraction expressed as EC50 (µg/ml)
Plant
Crude
Hexane
DCM
ETOAc
Butanol
species
Residual
Water
Mpd
23.92±2.28
110.54±17.91
122.07±20.50
70.86±18.09
49.55±5.70
Mpr
107.69±12.32
179.70±41.17
223.96±42.04
76.70±11.56
48.79±12.42
Mse
146.30±21.60
187.40±55.56
356.80±2.39
42.06±12.90
30.81±1.78
Mun
80.68±2.90
284.36±27.04
311.90±150.33
30.81±1.78
51.19±5.30
Ozm
44.29±4.20
175.56±6.88
45.77±0.98
Ozp
33.02±6.46
35.90±3.20
11.03±2.80
33.07±0.85
17.17±3.39
Sle
43.88±8.57
128.69±8.96
27.76±0.90
41.17±7.90
64.23±9.55
Spd
83.46±10.45
130.45±5.70
39.02±0.90
74.51±15.46
Spt
74.69±2.87
73.93±4.93
22.59±6.5
38.16±
19.02±2.70
82.24±0.97
92
Mpd (Maytenus peduncularis), Mpr (Maytenus procumbens), Mse (Maytenus senegalensis), Mun (Maytenus undata), Ozm (Ozoroa
mucronata), Ozp (Ozoroa paniculosa), Sle (Searsia leptodictya), Spd (Searsia pendulina), Spt (Searsia pentheri)
5.4.6. Lipid peroxidation inhibition effective concentration (EC50)
The inhibitory effect on the lipid peroxidation expressed as EC50 values are presented in Table of 5.5. The most
active are O. mucronata and C. woodii with EC50 of 13.95±2.25 and 13.24±1.17 µg/ml respectively followed by
C. bracteosum with 17.89±1.72 µg/ml. The least active extracts were S. leptodictya and M. peduncularis with
EC50 of 40.45±13.38 and 39.84±5.52 µg/ml respectively.
Table 5.5: Linoleic acid peroxidation inhibition expressed as EC50 (µg/ml)
Plant species
EC50 (µg/ml)
Combretum bracteosum
17.89±1.72
Combretum padoides
35.62±4.37
Combretum vendae
30.91±2.53
Combretum woodii
13.24±1.17
Maytenus peduncularis
39. 84±5.52
Maytenus procumbens
34.21±1.63
Maytenus senegalensis
27.21±2.30
Maytenus undata
33.70±0.85
Ozoroa mucronata
13.95±2.25
Ozoroa paniculosa
25.20±8.10
Searsia leptodictya
40.45±13.38
Searsia pendulina
30.21±5.49
Searsia pentheri
25.53±6.20
5.5. Discussion
5.5.1. Qualitative antioxidant analyses (DPPH-TLC bioautography)
The antioxidant assay using DPPH on TLC plates to screen plant extracts is a quick method used to confirm the
potential of the extracts for further evaluations. The intensity of the yellow spot depends on the amount and
chemical characteristics of the compound present. The reaction kinetics between the DPPH radical and the
active compounds varies as some compounds react slowly while others react fast. Some antioxidant spots were
not readily visible immediately after sprayed with DPPH but appeared after incubation at room temperature for 212h. Also the mechanism of the reaction may differ as some of the compounds act as hydrogen donors and
others may act as electron donors. In the DPPH radical scavenging process hydrogen donation is the
predominant mechanism (Rekka and Kourounakis, 1991). This antioxidant assay is fast, simple and the image
can be stored for future reference. All the extracts and fractions of the 27 plants used had antioxidant properties
with varying number of yellow spots as free radical scavenging potential against the purple background of the
DPPH radical on the plate. The three different mobile phases (5, 6 and 8) used to develop the chromatogram for
93
TLC-DPPH analyses demonstrated close relationships between the compounds present in the dichloromethane
fraction of E. latissima, C. vendae, and C. woodii. These results indicate the danger of considering only chemical
markers in taxonomy because Combretum and Erythrina are not closely related based on classical taxonomic
parameters.
The antioxidant activities of the crude extracts and their fractions of varying polarities were quantified several
different antioxidant assays such as DPPH˙ and ABTS˙+ synthetic free radicals, the hydroxyl radical and their
ferric reducing capacities using the FRAP assay and lipid peroxidation inhibition. More than one type of
antioxidant capacity measurement usually performed to take into account the various modes of antioxidant
mechanism. These methods were not specific to any particular antioxidant component rather to the overall
capacity of the extract. Of these methods only hydroxyl radical scavenging, FRAP and LPO assays have direct
physiological importance as a measure of plant extracts protective performance against free radical chain
reactions in cellular membranes.
For the DPPH and ABTS assay which involves hydrogen atom transfer and electron transfer processes
respectively, all the extracts and fractions have a dose-dependent radical activity with butanol fraction being the
most active with EC50 ranges from 0.21 ± 0.03 µg/ml for Combretum padoides to 41.8 ± 3.37 µg/ml for Searsia
pendulina (DPPH) and EC50 ranges from 0.43 ± 0.03 µg/ml for C. padoides to 68.85 ± 23.76 µg/ml for Ozoroa
mucronata (ABTS). These results suggested that the strong DPPH radical scavenging ability of ethyl acetate
fraction was closely related to the high levels of phenolic compounds and due to the scavenging of the radical by
hydrogen donation. Ethyl acetate fractions with EC50 ranges from 0.44 ± 0.06 µg/ml for C. padoides to 7.92 ±
1.63 µg/ml for Ozoroa mucronata (DPPH) and EC50 ranges from 0.71 ± 0.01 µg/ml for C. padoides to 10.74 ±
0.45 µg/ml for O. mucronata (ABTS) and the crude extracts with EC50 ranges from 1.91 ± 0.15 µg/ml for S.
pendulina to 123.60 ± 11.05 µg/ml Bauhinia variegata (DPPH) and EC50 ranges from 0.99 ± 0.05 µg/ml for
Ozoroa paniculosa to 246.37 ± 17.73 µg/ml for Erythrina latissima (ABTS).
In the results presented in Table 5.3, the higher the slope value the stronger the total antioxidant capacity
(reduction power) of the tested extracts or fractions. The ferric reducing antioxidant power (FRAP) serve as a
significant indicator of antioxidant potential of medicinal plant preparations and the activity are potentiated in the
butanol and ethyl acetate fractions as observed in the DPPH and ABTS assays results. Antioxidant capacity of
plant extracts and fractions depend on factors such as the compositions, chemical structures of the constituents
and conditions of the test used. These results indicates that the phytochemical present in the plants performed
as good electron or hydrogen donors and therefore should be able to terminate radical chain reaction by
converting free radicals and reactive oxygen species to more stable products.
Ferric ions generate hydroxyl radical in vivo through Haber-Weiss and Fenton reaction mechanisms to hydroxyl.
Hydroxyl radicals are highly strong reactive oxygen species, and there is no specific enzyme to defend against
them in living organisms (Liu et al., 2005). Hydroxyl radicals and other reactive species are also produced by
94
activated neutrophils, eosinophils monocytes and macrophages during inflammatory responses of the immune
process. Hydroxyl radicals-mediated and propagated lipid peroxidation of the gastrointestinal tract mucosa
phospholipids are considered to play a crucial role in the pathophysiology of numerous chronic diseases. The
major toxic products of LPO are 4-hydroxyl-2-nonenal and malondialdehyde (MDA) which can react with
intestinal epithelium mucosa resulting in altered transport process (fluid and ions). These mechanisms are
involved in varieties of diarrhoea aetiology such as infection, toxin, and inflammations.
ROS are important mediators that initiate and propagate inflammatory responses by inducing the formation of
pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumour necrosis factors (TNF-α). ROS/RNS are
generated directly by COX at the site of inflammation and have regulatory role in the expression of COX and
subsequent synthesis of PGE, therefore amplifying the acute phase of the inflammatory responses. These
inflammatory mediators contribute to diarrhoea aetiology as direct secretagogue (pro-secretory), reduced fluid
absorption capacity due to damaged to mucosa epithelial tissue and/or modulation of the intestinal contractility
through enteric nervous system (ENS).
Oxidative damage to cellular components such as cell membrane by free radicals is believed to be associated
with immune system decline and hyperactivation. Immune activation of PMNs and monocytes result in formation
of potent hypochlorous acid (HOCl) from myeloperoxidase (MPO)-catalyzed oxidation of Cl- by H2O2. In addition,
the HOCl react with primary amine groups (RNH2) to produce N-chloramines (RNHCl). Both HOCl and RNHCl
are cytotoxic (Pavlick et al., 2002). Some of the ROS/RSN and their products enhances intestinal and colonic Clacts directly or indirectly to initiate diarrhoea as secretagogue (Gaginella et al., 1995).
Considerable interest is focused in finding natural antioxidants which can help on the management of numerous
diseases with oxidative stress aetiology and maintenance of good health. Oxidative stress and the associated
diseases resulting from an imbalance between the endogenous antioxidant defence mechanisms and prooxidative forces in favour can be alleviate by increased expression of antioxidant (Pavlick et al., 2002).
Antioxidants have the capacity of stopping the chain reaction of oxidative species and the deleterious health
hazard to the body. The crude extract of these medicinal plants inhibit Fenton-generated hydroxyl radicalmediated peroxidation of a heterogeneous phospholipid-aqueous phosphate buffered system and scavenged
hydroxyl radical which are important characteristic of phenolic compounds. Free radical scavenging and
antioxidant activity of these medicinal plants contribute their therapeutic effect against diarrhoea diseases and
other GIT disorders for which they are being use ethnopharmacological in South African traditional medicine to
treat.
5.6. Conclusion
In this study, the extracts and their fractions were found to have various forms of antioxidant activities that could
possibly be attributed to the phenolic constituents. The extraction and fractionation protocols potentiate the
95
antioxidant components in the polar fractions while the non-polar hexane fractions demonstrated little or no
antioxidant activity except one prominent spot from the Erythrina latissima extract in TLC-DPPH analyses.
Linoleic acid and arachidonic acid are indigenous compounds of the cell membrane with a task to protect the
cell. The two membrane lipids are prone to attack during induce inflammatory and oxidative stress. However, the
increases of intracellular ROS level, due to increased production or impaired removal, can also cause cell
damage ranging from cytoplasmic swelling to cell death. In view of the involvement of the many oxidative
mechanisms in the pathogenesis of various diseases, free radicals scavenging and removal of excessive ROS
are important for restoring normal conditions, which might be the possible reasons of the correlation between
antioxidant activity and other therapeutic activities. This investigation provided data clarifying the potentials of
some of the plants as promising sources of natural antioxidants.
Further work on in vivo verification of the antioxidant therapeutic effectiveness, bioavailability, absorption and
metabolism of the active component is needed. Finally identification, isolation, characterization and absence of
possible toxicity of the bioactive compounds also required further investigation. Free radicals and oxidative
species play some critical roles in diseases with inflammatory aetiologies including the GIT disorders and
immunosuppression mechanism. The crude extracts and the polar fractions (ethyl acetate and butanol) of many
of the plant species have strong antioxidant activities, consequently may reduce inflammation or stimulate the
immune system of host. This could be one of the anti-diarrhoeal mechanisms and therefore explaining the
traditional use of these medicinal plants. The anti-inflammatory potentials of the crude extracts will be
investigated in the next chapter
96
CHAPTER SIX
Anti-inflammatory activities of the crude extracts as antidiarrhoeal mechanisms
6.0. Introduction
Inflammation is an important component of immune response to pathogens and damaged cell characterized by
heat, redness, pains, swelling and sometimes loss of tissue functionality in chronic situation (MacNaughton,
2006). Although inflammatory response provides an important defence mechanism to injurious agents, injury to
some healthy cells at the inflammatory site could also occur (Sprague and Khalil, 2009).
The cellular immune systems including that of the gastrointestinal tract (GIT) act as defence mechanisms by
mobilizing white blood cells (leukocytes and other chemicals) to fight infections and harmful stimuli. The body’s
reaction to this phenomenon may trigger inflammatory responses through the release of pro-inflammatory
eicosanoids such as prostaglandins, prostacyclins and leukotrienes, and pro-inflammatory cytokines (Nardi et al,
2007) such as interleukins (IL-1B, IL-3,IL-6), interferons (IFN), tumor nuclear factor (TNF-α) and plateletactivating factor (PAF) (Conforti et al, 2008; Kunkel et al, 1996).
Cyclooxygenase (COX) and lipoxygenase (LOX) oxidation of polyunsaturated fatty acid (PUFA) such arachidonic
acid or linoleic acid forming bioactive eicosanoids are the major features of inflammatory response (Haeggstrom
et al., 2010) (Fig 6.1). The generation and release of reactive species (ROS/RNS) by inflammatory cells in
response to pathogens and stimuli is considered the major microbicidal mechanism in the body. However,
excessive generation of ROS/RNS exacerbate inflammatory responses that may lead to development of disease
state.
In the GIT, inflammation affects epithelial cells as well as the more specialised mucus secreting and
enteroendocrine cells of the gut mucosa (Spiller, 2004). Inflamed intestinal epithelial mucosa usually results in
increased permeability, increased bowel movement or contractility, inadequate digestion of food materials and
impaired absorption of essential food components (Spiller, 2004). Some inflammatory mediators are ion or fluid
secretagogue and prokinetic of enteric nervous system (ENS) causing diarrhoea, and malnutrition (See section
2.10.2 for detailed discussion).
Intestinal inflammation causes damage to mucosal barrier function comprised of physical diffusion barriers,
physiologic and enzymatic barriers, and immunologic barriers (Soderholm and perdue, 2006). The continuous
layer of epithelial cells interconnected by tight junction, restricts both transcellular and paracellular permeation,
therefore, constituting the major part of the mucosal barrier. Active secretion of fluid and mucus containing
secretory immunoglobulin isotype A (IgA) also serve to bind, dilute, and cleaning mechanisms of the intestine.
The intestinal propulsive movement is also an important protective process against noxious substances
(Soderholm and perdue, 2006).
6.1. Effect of cyclooxygenases (COX) on GIT
97
Cyclooxygenases (COXs) are oxidizing enzymes which metabolize polyunsaturated fatty acid (PUFA) such as
arachidonic acid liberated from membrane phospholipid by phospholipases A to various eicosanoids such as
prostaglandin D (PGD), prostaglandin E (PGE), prostacyclin (PC), thromboxane (TXA2) (Xu et al, 2007; Simon et
al, 2004). The physiological activities of these inflammatory mediators are mediated by G-protein-coupled
prostanoid receptors such as DP, EP1-4, FP, IP and TP which preferentially respond to PGD, PGE, PGF2α, PGI
and TXA respectively. There are two isoforms of COX namely COX-1 and 2. COX-1 is constitutively expressed to
produce PG series which are involve in the regulation of physiological housekeeping such as platelet
aggregation, homeostasis of the GIT and the kidney. The COX-2 is an inducible enzyme expressed to produce
PG series which are responsible for pro-inflammatory stimuli such as cytokines, growth factors, tumor promoting
agents and bacterial endotoxins.
Prostaglandins are widely distributed along the GIT and are involved in a number of physiological and
pathological processes including motility, blood flow, water and electrolyte absorption, and mucus secretion.
PGE2 is cytoprotective to the intestinal epithelium by decreasing gastric acid secretion, thus prevents ulceration.
However, in pathological situation, it also increases intestinal motility and intestinal secretion causing secretory
diarrhoea.
6.2. Effects of Lipoxygenase in GIT
Lipoxygenases (LOXs) comprise a family of non-heme iron-containing dioxygenases, representing the key
enzymes in the biosynthesis of leukotrienes from PUFA. Leukotrienes have been postulated to play essential
role in the pathophysiology of several inflammatory and allergic diseases. The LOXs are classified with respect
to their positional specificity of arachidonic acid oxygenation as 5-LOX, 9-LOX, 12- LOX, 15-LOX. The products
of LOXs catalysed oxygenation include leukotrienes, lipoxins, hydroperoxyeicosatetraenoic acids (HPETE), and
hydroxyeicosatetraenoic acids (HETE).
LTB4 is synthesized by 5-LOX from arachidonic acid. It is a potent chemotactic agent for inflammatory cells such
as neutrophils and macrophages. It elicits leukocytes migration towards inflammatory sites and activates
neutrophils, causing their degranulation associated with enzyme release as well as superoxide radicals. It also
plays an important role in immune systems by enhancing the release of pro-inflammatory cytokines by
macrophages and lymphocytes.
6.3. Effects of cytokines on GIT
Secretion of cytokines by the intestinal immune system is one of the main factors in maintaining the gut integrity
in quiescent homeostasis. Cytokines are classified as pro-inflammatory (TNF-a, IL-1, -6, -12, -15, -18, and -32,
as well as the anti-inflammatory cytokines IL-10 and TGF-β produced predominantly by activated macrophages,
involved in the up-regulation of inflammatory reactions and IFN-γ and IL-4 from T-cells (MacNaughton, 2006;
Sprague and Khalil, 2009). Anti inflammatory cytokines such as IL-4, IL-10, IL-13, IFN-α, and TGF-β are involved
98
in the down regulation of inflammatory reactions (Sprague and Khalil, 2009). Of major importance is the balance
between pro-inflammatory cytokines such as TNF-α, IL-1 and IFN-γ and regulatory cytokines like IL-10 and
transforming growth factor-β. The features ultimately determine the capacity of an immune response as either
detrimental or innocuous to the gut. TNF-α is a critical cytokine that elaborate inflammatory responses by
activating a number of inflammatory cells including neutrophils, macrophages and NK cells which induces the
production of inflammatory cytokine such as IL-1β, IL-6 and IL-8 and upregulation of adhesion molecules on cell
surface. In addition TNF-α also directly potentiate the immune response of other pro-inflammatory cytokines such
as IL-1, IL-6, IL-12 and IFN-γ consequently enhancing the anti-inflammatory and anti-apoptotic effect. IFN-γ can
stimulate the production of IL-1β, platelet-activating factor, H2O2, NO and downregulate IL-8. As a proinflammatory cytokine, IFN-γ sensitizes intestinal epithelial cells to physiological and therapeutic inducers of
apoptosis.
6.4. Oxidative species as inflammatory mediator
Production of reactive species (H2O2, O2˙–, and OH˙), nitric oxide (NO) and per-oxy-nitrite occurs at the site of
inflammation and contributes to the exacerbation of inflammatory disease and tissue damage. Oxidative species
stimulates the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor
(TNF-α). In addition, ROS induced by activated neutrophils, eosinophils, monocytes and macrophages during the
inflammation process leads to tissue injury by damaging macromolecules and effecting the lipid peroxidation of
membranes. In acute or chronic inflammations, the production of O2˙– is increased at a rate that overwhelms the
capacity of the endogenous SOD enzyme defence to dissipate. Reduction in the O2˙– generation can decrease
side-effects of the radical in inflammatory conditions.
Nitric oxide (NO) is a free radical gas synthesized by nitric oxide synthase (NOS) from L-arginine and initiates
diverse physiological and pathological processes (Lee et al,, 2007b). Three iso-forms of NOS had been
identified; they are neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). The first two isoforms (nNOS and eNOS) are constitutive NOS (cNOS) while iNOS is produced only by specific stimulants in
some cells. The iNOS stimulants include cytokines or bacterial lipopolysaccharides or endotoxin. Inflammatory
responses are associated with the production of large quantity of NO (Cuzzocrea et al, 2001). The deleterious
effects of NO include mitochondrial enzymes inhibition (Nathan, 1992) and activation of COXs to produce
inflammatory PGs (Salvemini and Masferrer, 1996) and interaction with superoxide to generate cytotoxic
peroxynitrite. NO is an important mediator in the inflammatory process and is produced at inflamed sites by
iNOS. High levels of NO have been linked to a number of pathological processes including various forms of
inflammation, circulatory shock, and carcinogenesis. Therefore, an inhibitor of NOS might be effective as a
therapeutic agent for inflammatory diseases.
6.5. Allopathic anti-inflammatory therapies and adverse effects on GIT
99
In view of the importance of PGs in inflammatory response, the rate-limiting enzymes for PGs synthesis are the
therapeutic targets in controlling inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs) exert their action
by inhibiting the activity of COX enzymes, thereby reducing the production of pro-inflammatory prostaglandins.
NSAIDs are structurally diverse, including compounds in the salicylic acid, arylalkanoic acid, propionic acid
(profens), N-arylanthranilic acid (fenamic acid), pyrozolidine derivatives, oxicam and sulphonanilide families.
Classical NSAIDs exhibited non-selective inhibition of both COX-1 and COX-2 while some other NSAIDs
however show preferential inhibitory activity toward one isoform or the other.
Although NSAIDs provides good therapeutic relief against inflammation, some of these drugs currently in use
have various side effects, particularly in the gastrointestinal tract ulceration and kidney (Charlier and Michaux,
2003). Prolonged use of nonselective NSAIDs has adverse effects such as nausea, dyspepsia, gastritis,
abdominal pain, peptic ulceration, gastrointestinal bleeding, and/or perforation of gastroduodenal ulcers. In
addition, NSAIDs are postulated to shift the metabolite profile from COX derivatives to lipoxygenase (LOX)
derivatives resulting in the accumulation of substrate for the LOX-derived metabolites. LOX products stimulate
neutrophil migration, increase adhesion of leukocytes to endothelial cells, cause smooth muscle contraction,
increase vascular permeability, and increase ion and mucus secretion. Inhibition of leukotriene biosynthesis
decreases inflammation and accelerates gastrointestinal healing. However, there are controversies in the recent
findings that non selective NSAID (indomethacin) causes GIT damage and neither selective COX-1 inhibitors nor
selective COX-2 inhibitor causes any intestinal damage. The combine use of selective COX-1 and selective
COX-2 inhibitors produces intestinal haemorrhage (Takeuchi et al., 2010) (Fig 6.2). Aspirin a known non
selective NSAID as its metabolite such as salicylic acid causes no intestinal damage but instead provided
protection against ulcerogenic response induced by other classical NSAID. This also indicates that some
complex mechanisms are responsible for the intestinal damage by a number of non selective NSAIDs other than
COX-1 inhibition only (Takeuchi et al., 2010). Some of the factors involved in the pathogenesis of NSAIDs
toxicity include
•
Bile acids secretion: NSAIDs increase the secretion of bile acids in the GIT causing complications such
as colonic mucosa damage and diarrhoea. Bile acids induce the liberation of arachidonic acid from
epithelial membrane, and the generation of COX and LOX metabolites along with the secondary active
oxygen radicals.
•
Intestinal motility: NSAIDs such as COX-1inhibitors causes marked enhancement of intestinal motility
with regard to both the amplitude and frequency of contractility. Intestinal hypermotility caused mucosal
hypoxia and microvascular injury due to smooth muscle contraction (Takeuchi et al., 2002).
•
Neutrophil infiltration: NSAIDs cause severe damage to the GIT resulting in loss of surface epithelium,
mucosal necrosis and massive neutrophil infiltration.
100
•
Bacterial flora: Non selective and COX-1 selective NSAIDs increased number of enterobacteria in the
intestinal mucosa homogenates and luminal bacterial adherence to the mucosa induced severe
intestinal injury (Takeuchi et al., 2011).
•
Nitric oxide (NO): Non selective and COX-1 selective NSAIDs cause an up-regulation of inducible NO
synthases (iNOS) in the GIT. This is due to bacterial endotoxin increased intestinal permeability which
induces the expression of iNOS and enhanced the generation of NO in the mucosa (Takeuchi et al.,
2011).
•
Prostaglandins (PGs) deficiency: COX-1 isoform is expressed in most tissues, producing prostaglandins
that play an important protective role in the gut by stimulating the synthesis and secretion of mucus and
bicarbonate, increasing mucosal blood flow and promoting epithelial proliferation. The inhibitions of this
enzyme by NSAIDs create PGs deficiency. In addition the inhibition of the COX-1 blocks platelet
production of thromboxane, which increases bleeding when an active GI bleeding site is present. COX-2
isoform is induced in most tissues in response to inflammatory stimuli. Prostaglandins derived from
COX-2 can be generated at the ulcer margin and appear to play an important role in ulcer healing
through triggering the cell proliferation, promotion of angiogenesis and restoration of mucosal integrity
(Takeuchi et al., 2011). Effects of NSAIDs on GIT are presented in Fig 6.1 and Fig 6.2.
Fig. 6.1. Roles of COX-1 and COX-2 in the pathogenic mechanism of NSAID-induced intestinal damage
(Takeuchi et al, 2010)
•
Effect COX-2 inhibition: The NSAIDs selective inhibition of COX-2 has adverse effect on cardiovascular
function (Grosser et al, 2006) due to suppression of PGI (anti-thrombotic) promoting hypertension and
blood coagulation while the synthesis of TXA2 (pro-thrombotic) by COX-1 remain unchanged
(Fitzgerald, 2004).
101
Fig. 6.2. Various factors involved in the pathogenesis of indomethacin-induced small intestinal lesions (Takeuchi
et al, 2010)
6.6. Plant phytochemicals as anti-inflammatory agents
Plant extracts are suspected to contain potential bioactive component that can strongly inhibit the expression of
LOX and COX. Therefore, there is continuous need to search for new drugs from natural products with antiinflammatory properties and minimum side effects. Modulation of the activities of the enzymes implies that the
inflammation process can be modified. Anti-inflammatory agent may also have an antioxidant and radicalscavenging mechanisms as part of it activity.
Phenolics, alkaloids and triterpenoids have been demonstrated to exhibit anti-inflammatory activity by exerting
anti-oxidative properties in reducing O2˙– and malondialdehyde (MDA) production, plasma extravasations and cell
migration mainly of leukocytes and potentiates the activity of SOD in radical scavenging (Nardi et al, 2007).
Reactive species are one of the most important mediators that provokes or sustain inflammatory processes and
consequently, their annihilation by antioxidants and radical scavenger such as phenolic compounds can alleviate
inflammation (Delaporte et al, 2002; Geronikaki and Garalas, 2006).
6.7. Mechanisms of anti-inflammatory assay models
Anti-inflammatory potential were determined by in vitro assays based on the inhibitory effect on the biosynthesis
of 12(S)-hydroxy-(5Z, 8E, 10E)-heptadecatrienoic acid (12-HHT), and 12(S)-hydroxy-(5Z, 8Z, 10E, 14Z)eicosatetraenoicacid (12-HETE). 12-HHT and 12-HETE are inflammation mediators derived from arachidonic
acid metabolism, which is catalysed by enzymes of inflammatory response, cyclooxygenase (COX-1) and
lipoxygenase (12-LOX), respectively. The advantage of this type of experiment is avoidance of the undesirable in
vivo tests on experimental animals, since the tests commonly used to detect the anti-inflammatory activity is
carrageenan induced paw edema in rats.
6.8. Materials and Methods
102
6.8.1. Lipoxygenase inhibition assay
Lipoxygenase activity was determined spectrophotometrically according to Taraporewala and Kauffman, (1990);
Lyckander and Malterud, (1992) which is based on the enzymatic oxidation of linoleic acid to the corresponding
hydroperoxide. To determine hydroperoxide, soy lipoxygenase-1 (200 U) was incubated with linoleic acid (50
µM) in sodium borate buffer (200 mM, pH 9.0) for 4 min at 25oC. The absorbance at 234 nm was measured on a
Heλios β (Thermo Electron Corporation) spectrophotometer using a quartz cuvette. The inhibitory assays were
performed in presence of extracts in different concentrations ranging from 0.15 - 25 µg/ml. The anti-inflammatory
effect was evaluated by calculating percentage inhibition of hydroperoxide production from the changes in optical
density values at 234 nm for 5 min. The test compound concentration causing 50% inhibition of hydroperoxiderelease (IC50) was calculated from the concentration–inhibition response curve by best fit non-linear regression
analysis. The extinction coefficient of 25 mM−1 cm−1 was used for quantification of lipid hydroperoxides. DMSO
was used as negative control.
6.8.2. Cyclooxygenase enzymes inhibition (COX 1 and 2) assay
The experiments were performed using an assay originally described by Noreen et al, (1998), with some
modification by du Toit et al, (2005). For COX-1 assay, commercial COX-1 (from ram seminal vesicles, SigmaAldrich) (10 µl/sample) enzymes and Hematin (co-factor) (50 µl/sample) was pre-incubated for 5 min on ice. The
mixture (enzyme and cofactor) was added to the test sample (2.5 µl of test sample and 17.5 µl of water) to make
a concentration of 0.25 µg/µl in the final assay volume and pre-incubated for 5 min at room temperature. I -14Carachidonic acid (20 µl) was added to the enzyme-test sample mixture and incubated for 10 min in a water bath
at 370C. The reaction was terminated by adding 10 µl of 2M HCl. The amount of [14C]-labelled PG synthesized
was measured using a Packard scintillation counter after removing the unmetabolized [14C]-arachidonic acid
substrate by column chromatography using Pasteur pipette as column. Unmetabolized [14C]-arachidonic acid
substrate was eluted with n-hexane-dioxane-glacial acetic acid (70:30:1) while [14C]-labelled PG synthesized in
the reaction was eluted with ethyl acetate: methanol (85:15).
The same procedure was adopted for COX-2 assay using three units of COX-2 enzymes (human recombinant,
Sigma-Aldrich). Indomethacin at 12.5 µM and 200 µM used as positive controls for the COX-1 assay and COX-2
assays respectively. Two background controls in which the enzymes were inactivated with HCl before the
addition of [14C]-arachidonic acid and two solvent blanks were prepared for experiment. The results were
expressed as percentage inhibition (% I) using equation described by Lin et al, 1999.
% I = [(1-DPM of sample) – (DPM of background/DPM of blank - DPM of background)] X100
Where DPM is disintegration min-1
6.9. Results
6.9.1. Cyclooxygenase inhibition assay
103
Results from cyclooxygenase assay against COX-1 are presented in Fig. 6.3. All the extracts exhibited moderate
to good activity with the inhibitory effects ranging between 41.70 to 84.61%. The most active extract against
COX-1 was Carissa macrocarpa with 82.98±1.62% inhibition at 250 µg/ml and 69.72±1.91% at 62.5 µg/ml. The
inhibition of COX-1 enzyme was concentration dependent as Bauhinia petersiana inhibited 50% of the enzyme at
a concentration of 167 µg/ml (R2=0.989). Bauhinia bowkeri and Bauhinia galpinii inhibited 50% of the enzyme at
the concentrations of 241 µg/ml (R2=0.9645) and 377.66 µg/ml (R2=0.9216) respectively. Commiphora harveyi
was only active at concentration of 250 µg/ml by inhibiting 45.45±2.96% of COX-1 enzyme.
Percentage inhibition of COX-1 against concentration (µ
µ g/ml)
%Inhibition of COX-1
100
BAB
BAG
BAP
Cam
Com
80
60
40
20
65
12
5
25
0
0
Concentration (µg/ml)
Fig. 6.3. COX-1 inhibitory activity of some selected phenolic-enriched crude extracts
The phenolic-rich crude extracts of these plants had no inhibitory activity against COX-2 at the maximum
concentration tested (250 µg/ml) indicating that the extracts were COX-1 selective inhibitor. The phenolic enrich
crude extracts of C. padoides, C. vendae and C. woodii exhibit no cyclooxygenase inhibitory activity against
COX-1 and 2 enzymes. Indomethacin used as reference compound had IC50 of 3.30±0.006 and 122.5 µM
against COX-1and COX-2 respectively.
6.9.2 Lipoxygenase inhibitory assay
The lipoxygenase inhibitory capacity expressed as LC50 and percentage inhibition are presented in Table 6.1.
The activity of the extract varied widely ranging between 0.86±0.27 and 111.44±37.28 µg/ml. The most active
extract was obtained from Syzygium paniculatum with LC50 of 0.86±0.27 µg/ml and percentage inhibition of
66.74±2.07 followed by Euclea crispa with LC50 of 2.55±0.13 µg/ml and percentage inhibition of 63.06±1.75.
The least active extract was from Commiphora harveyi with LC50 of 111.44±37.28 µg/ml and percentage
inhibition of 39.15±1.92 (extract concentration equals 25.6 µg/ml).
Table 6.1: Lipoxygenase inhibitory activity of the crude extracts
Plant species
LC50 (µg/ml)
% inhibition (25 µg/ml)
104
Bag
4.10±0.62
56.31±4.36
Bap
10.18±2.25
52.15±2.1
Bav
5.07±1.11
56.00±0.97
Cam
30.22±0.83
46.29±1.69
Cop
25.12±2.05
44.97±0.53
Cov
33.48±6.01
45.30±0.70
Cow
15.70±5.57
49.21±1.25
Com
111.44±37.28
39.15±1.92
Euc
2.55±0.13
63.06±1.75
Eun
17.23±0.13
50.46±1.49
Erl
7.25±1.84
55.09±1.85
Fic
8.48±4.35
53.87±2.25
Fig
5.02±1.46
66.66±3.64
Inc
7.90±1.87
55.37±4.25
Mpd
4.08±0.51
61.54±4.19
Mpr
11.08±3.50
55.80±3.61
Mse
10.88±1.92
53.18±2.31
Mun
4.68±2.44
56.50±1.81
Ozm
2.88±1.10
57.90±1.62
Ozp
27.33±9.16
46.80±2.20
Sle
11.60±2.61
54.80±2.21
Spt
9.16±2.07
54.76±2.79
Scb
4.09±2.37
60.19
Syp
0.86±0.27
66.74±2.07
Spd
6.9. Discussion
6.9.1. Cyclooxygenase assay
COX enzymes are the rate-determining enzymes in the prostaglandin biosynthetic pathways. The modulation of
the enzymes can help in anti-inflammatory treatments due to the key role of PG especially PGE2 in the
inflammatory response (Gale et al, 2007). ROS have been reported to have a regulatory role in the expression of
COX, particularly COX-2 and subsequent synthesis of PGE2 which is responsible for inflammation. Classification
of inflammatory activity of extract based on extractants as 59% (minimum inhibition) by aqueous extracts tested
at a final concentration of 250 µg per test solution and for organic extracts is 70%, when tested at a final
concentration of 250 µg per test solution (Fennell et al., 2004). The polyphenolic-rich extracts of the plant
species tested exhibited selective inhibition of COX-1. The results confirmed the postulation that most phenolic
compounds like flavonoids exhibit COX-1 selective inhibitory activity and have no effect on COX-2 isoform (Kim
et al, 2004). COX-1 is also reported to be involved in the inflammatory response and compensatory mechanisms
105
between COX-1 and COX-2 have been demonstrated (Gale et al, 2007). COX-1 is the predominant isoenzyme in
the normal gastrointestinal tract (Radi and Khan, 2006) and modulates neurogenic contraction (Smid and
Svensson, 2009), while COX-2 expression is up-regulated during inflammation, where it modulates cholinergic
contraction and small bowel motility. COX-2 mediated PGs from inflamed gastrointestinal mucosa may play a
role in the chloride and fluid flux that helps flush GI bacteria.
However, the inhibition of COX-2 in the inflamed GI mucosa has been hypothesized to delay the resolution of GI
injury. Since the polyphenolic-rich crude extracts of C. macrocarpa, B. bowkeri, B. galpinii and B.petersiana
exhibited selective inhibition of COX-1, the use of these plants in traditional medicine as antidiarrhoea agents
need to be monitored critically especially in term of dosage. The phenolic-enriched extracts of C. padoides, C.
vendae, C. woodii and Syzygium paniculatum did not exhibit activities against COX-1 and COX-2. However, the
anti-inflammatory activities of these extracts cannot be ruled out as it may involve in other inflammatory
mediators. In diarrhoea disease, ROS and RNS are known to activate many pro-inflammatory cytokines
(interleukins and TNFα), cell adhesions and COX enzymes. Oxidative damage exacerbates intestinal
inflammatory response and causes a virulent cycle of oxidative stress, inflammation and increased mucosal
permeability (Chen et al., 2007). Though, the phenolic-enriched extracts of the three Combretum species may
not have a direct effect on COX enzymes, the significant free radical, ferric reducing properties and inhibition of
lipid peroxidation may probably influence inflammation process.
6.9.2. Lipoxygenase assay
The anti-inflammatory activities of phenolic-enriched crude extracts on inflammatory mediators were measured
against soybean lipoxygenase enzyme (Table 6.1). All medicinal extracts inhibited the lipoxygenase enzyme and
these inhibitory effects are concentration dependent. Lipoxygenases inhibition correlate to antioxidants because
lipid hydroperoxide formations are usually inhibited as a result of the scavenging of lipid-oxy- or lipid-peroxyradicals formed in the course of enzymatic peroxidation. Consequently, limiting the availability of lipid
hydroperoxide substrates required for the catalytic cycle of lipoxygenase oxidative process (Cuello et al., 2011)
6.10. Conclusion
Some of the crude extract exhibited selective COX-1 and LOX inhibitory activities in the in vitro enzymatic assays
conducted in this study. The release of arachidonic acid is closely related to the cyclooxygenase (COX) and 5lipoxygenase (LOX) enzyme systems. The ability of plant extracts, fractions and isolated pure compounds to
inhibit both COX and LOX pathways of the arachidonate metabolism have been suggested to contribute to antiinflammatory action (Middleton et al., 2000). The inhibition of COX enzymes result in the shifting of arachidonic
acid to the LOX pathway, which promotes gastrointestinal damage by recruiting leukocytes to the mucosal and
stimulating gastric acid secretion. It is proposed that drugs that are capable of block both COX and LOX
metabolic pathways (dual inhibitors) are best option in terms of NSAIDs. The dual inhibition of COX and LOX
enhances their individual anti-inflammatory effects and reduce the undesirable side effects associated with
106
NSAIDs, especially of the gastrointestinal tract (Fiorucci et al., 2001). Further work on in vivo anti-inflammatory
evaluation of the extracts in an animal model is needed to confirm the therapeutic potentials of these plant
extracts. The crude B. galpinii had COX-1 and LOX inhibitory activity above 50% at concentration of 250 and 25
µg/ml respectively. Considering the GIT injury potential of some of the plants due to selective COX-1 inhibition,
cellular toxicity will be evaluated in the next chapter.
107
Chapter Seven
Cytotoxicity evaluation of the crude extracts against Vero African green monkey kidney cell lines
7.0. Introduction
Medicinal plants are assumed to be non-toxic and regarded safe due to their natural origin and long use in
traditional medicine to treat various forms of diseases (Chen et al., 2011; Fennell et al., 2004). Medicinal plant
preparations are administered with the hope of promoting health and treating various diseases such as
infections, colds, inflammation, GIT disorders, insomnia, depression, heart diseases, diabetes, cancer, acquired
immunodeficiency syndrome, and liver diseases has increased in recent times (Chen et al., 2011). However,
scientific studies on efficacy and safety of some medicinal plants indicated that there are many phytochemicals
that have cytotoxic, genotoxic, and carcinogenic effects when used chronically (Ernst, 2004; Rietjens et al.,
2005). It should also be kept in mind that if a different extractant is used, the safety ascribed to traditional use
based mainly on aqueous extracts may not be relevant at all.
The adverse effects of medicinal plant use arise due to organ toxicity, adulteration, contamination, contents of
heavy metals, herb–drug interactions, poor quality control and inherent poisonous phytochemical (Jordan et al.,
2010). Some medicinal plant phytochemicals are associated with toxicities of the heart, liver, blood, kidney,
central nervous system, gastrointestinal disorder such as diarrhoea, and less frequently carcinogenesis (Jordan
et al., 2010). In the formal herbal industry the toxicity problems of medicinal plants are attributable to insufficient
quality assurance and non compliance to the standards of Good Manufacturing Practise (Palombo, 2006).
Furthermore, the problem is complicated by adulteration of herbal remedies by surreptitious addition of synthetic
drugs and other potentially toxic compounds such as other botanicals, microorganisms, toxins, pesticides, and
fumigants (Palombo, 2006).
More importantly, if herbal medicines are used with prescription drugs especially those with narrow therapeutic
indices it can result in potential harmful herb–drug interactions that cause altered drug response and toxicity
(Chen et al., 2011). The fact that herbal medicines contain many compounds (active and non active), the large
number of pharmacologically active compounds also increases the chance of herb-drug interaction (Palombo,
2006). Like synthetic drugs, herbal bioactive compounds can also undergo Phase I and Phase II enzymatic
transformations to form nontoxic metabolites which are excreted through the faeces and urine. However, the
production of reactive and potentially toxic metabolites is feasible with associated toxicity implications (Chen et
al., 2011).
With the current emphasis on research and development of medicinal plant worldwide, it is important to have
some information regarding the toxicity potential and efficacy of plants utilized ethnobotanically to treat ailments.
As part of ethnopharmacological studies of medicinal plant available literature should be searched for known
toxic properties of plants of interest before embarking on biological activity studies. However, where toxic effects
are unavailable, the inclusion of cytotoxicity and other toxicity protocols in the study are useful in detecting
108
potential toxicity. This strategy is applicable when screening plant extracts or isolated natural products for some
other biological activities such as antiinfectious, anti-inflammatory, antioxidant, antidiarrhoea and antiparasitic
property. The aim of this work was to determine the potential risk of the crude phenolic-enriched extracts by
evaluating the cytotoxicity using Vero cell lines.
7.1. Materials and Methods
7.1.1. Preparation of plant extract
The plant extracts were prepared as described in section 3.6.3. The dried sample were reconstituted in 70%
acetone at the concentration of 1.0 mg/ml (3 ml) and from it a serial dilution of the concentration range of 1.0 to
0.001 mg/ml were made on the 96 well tissue culture plate.
7.1.2. Cytotoxicity assay against Vero cell
Cytotoxicity of the extract was determined by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide]
assay (Mosmann, 1983) using Vero African green monkey kidney cell lines. The cells were cultured in Minimal
Essential Medium (MEM) Earle’s Base, supplemented with 20 mM L-glutamine, 16.5 mM NaHCO3 supplemented
with 0.1% gentamicin and 5% foetal calf serum. Confluent monolayer culture suspensions of the cells were
seeded into 96-well tissue culture microtitre plate at a density of 0.5×103 cells per well and incubated for 24 h.at
37oC in a 5.0% CO2 incubator. The cells were washed with cultured media and extract (1.0, 0.1, 0.001 mg/ml),
positive control (berberine at concentrations of 100, 10, 1.0, 0.1 µg/ml) were added and incubated for 5 days.
The cells were observed using inverted microscope to check for cytopathic effect from the extract. The cells
proliferation and viability was examined by addition of 30 µl of a 5 mg/ml solution of MTT in PBS to each well and
incubated for another 4 h.at 37oC. The medium was carefully removed from the wells without disturbing the MTT
concentrate and washed twice with PBS. The liquid was aspirated from the cells and 50 µl of DMSO was added
to each well to dissolve the crystallized MTT formazan. The amount of reduced MTT was measured as
absorbance at 570 nm using microtitre plate reader. The result expressed as a percentage of the control cells
and IC50 was calculated
Dose response curves were obtained by plotting the percentage growth of cells versus log concentration of the
compound. The LC50 (50% inhibitory concentration) values were calculated from a non-linear regression model
(best fit curve) of sigmoidal dose-response curve (variable) and computed using GraphPad Prism 5.04
(Graphpad, USA).
7.2. Results
The cytotoxicity of phenolic-enriched crude leaf extracts of the 19 medicinal plants used ethnopharmacologically
in treating diarrhoea and other GIT disorders is presented in Table 7.1. The results indicate that the extracts had
varying degrees of toxicity to Vero cell lines with LC50 ranging from 3.51±2.03 to 741.90±44.22 µg/ml. The most
cytotoxic extract was Combretum woodii (3.51±2.03µg/ml) followed by Combretum vendae (5.70±1.25 µg/ml)
109
while the least cytotoxic extract was O. mucronata (741.90±44.22 µg/ml) followed by Maytenus procumbens
(187.71±19.92 µg/ml).
Table 7.1 The LD50 of the cytotoxicity assay of some medicinal plants used in South African traditional medicine
to treat diarrhoea and related ailments
Plant species
LC50 (µg/ml)
Bab
17.90±2.56
Bag
35.68±2.15
Bap
40.68±18.13
Bav
76.37±7.50
Cam
ND
Cob
48.81±6.15
Cop
9.03±0.20
Cop
5.70±1.25
Cow
3.51±2.03
Com
-ND
Euc
31.61±4.04
Eun
26.99±4.48
Erl
ND
Fic
ND
Fig
ND
Inc
ND
Mpd
89.41±16.37
Mpr
187.71±19.92
Mse
87.62±3.03
Mun
99.17±11.88
Ozm
741.90±44.22
Ozp
16.58±1.85
Sle
25.09±2.40
Spd
22.30±2.42
Spt
50.62±4.30
Scb
ND
Syp
ND
ND = not determined
7.3. Discussion
For medicinal plant extracts to be useful in clinical application, the preparation must be selectively toxic to the
targeted organism or interfere directly with specific reaction pathway without a major effect on the host cell or
interference with normal physiological pathways. In categorization of crude extract safety, IC50 value of 20 µg/ml
and below were considered to be cytotoxic in an in vitro assay according to US National Cancer Institute (NCI)
plant screening program (Kuete et al., 2011) following incubation for more than 48 h. Some of the phenolic-rich
crude leaf extract of the medicinal plants tested in this study are relatively toxic compared to the positive
berberine control.
110
The cellular toxicity effects of the crude extracts were evaluated by MTT-formazan viability assay. Cellular
viability and proliferation are considered to be an important functional characteristic of healthy growing cells.
Increase in cell viability indicate cell proliferation, while decrease in cell viability indicate cell death as a result of
either toxic effects of the test extracts or sub optimal culture conditions. With the cell viability of the negative
control (DMSO) at the highest concentration of 1000 µg/ml under the same experimental condition, the latter
postulate is eliminated. Therefore, all the phenolic-enriched extracts of the medicinal plants tested may be
suggested to be safe for use in treating diarrhoea if the dosage is below the cytotoxic level. Although, Ozoroa
paniculosa (16.58±1.85 µg/ml), Searsia pendulina (22.30±2.42 µg/ml), Searsia leptodictya (25.09±2.40 µg/ml)
and Euclea natalensis (26.99±4.48 µg/ml) are within the defined cytotoxicity range, therefore the use of these
extracts in traditional medicine need to be monitored carefully. It is also important to note that no report of toxicity
has been recorded for the traditional use of these plant extracts. One should however remember that cellular
toxicity does not necessarily equate to whole animal toxicity due to possibly interactions in the gut and
bioavailability issues.
C. woodii acetone extracts have however been reported to be toxic in an in vivo test as anticoccidial in poultry at
concentration of 160 mg/kg (Naidoo et al, 2008). Furthermore, several cytotoxic and anti-tumour derivative of
stilbenoids such as Combretastin A and Combretastatin B5 (IC50 value of 10 µg/ml) have been isolated from the
genus Combretum.
Toxicity is usually encountered due to irrational use causing accumulation of potentially toxic constituents or
interactions between herbal medicinal products and conventional therapies. Indicative observations of toxicity is
alterations of one of the clinical signs such as diarrhoea, weight loss, agitation, hispid hair, convulsions, tremors,
dyspnoea among other) and mortality (Caparroz-Assef et al., 2005).
Some medicinal plant metabolites can cause GIT toxicity. The mechanism of action can be primarily irritative or
cytotoxic in nature resulting in an initial release of mucus from goblet cells, hypersecretion from crypt cells, and
maladsorption causing diarrhoea and emesis. Administration of high dose of some phytochemicals can cause
effects such as necrosis, haemorrhage, and even ulceration on the GIT. Medicinal plant toxins can have
additional toxicity or more directly life-threatening effects on other organ system.
7.4. Conclusion
These results are important because they show that there are risks of toxicity with an inappropriate use of some
of these extracts as therapeutics for any ailments. In vivo acute toxicity studies may be necessary to establish
the safety level of the extracts as in vitro assay results not necessarily translate to in vivo activity. Long term
effect of the use of the extracts such as mutagenicity and genotoxicity also need to be determined.
In vivo animal studies are frequently very expensive and requires much work to establish changes in enzyme
concentrations or histological evaluation of toxicity. It is also possible to do ex vivo studies using isolated organs.
111
In the next chapter some ex vivo studies will be described to investigate the possible mechanism of activity of
two selected species.
112
CHAPTER EIGHT
Motility modulation potential of Bauhinia galpinii and Combretum vendae phenolic-enriched leaf extracts
on isolated rat ileum
8.0. Introduction
Gastrointestinal tract (GIT) used the smooth muscle of the mucosal lining enriched with an enteric neural network
to regulate propulsive transport and mixing of food material directionally through the digestive systems (Wood,
2004). The neural network initiates and coordinates secretion and absorption across the intestinal lumen as well
(Bohn and Raehal, 2006).The enteric neurons function independent of the central nervous system (CNS),
therefore referred to as enteric nervous system (ENS). Enteric nervous system controls the motility and
contractility of the GIT as its rate and intensity of contraction regulates the absorption of fluid, and expulsion of
solid material. Therefore ENS exhibit significant role in GIT disorders such as diarrhoea and constipation through
these means. Neurotransmitters such as acetylcholine (ACh), serotonin (5-hydroxytryptamine (5-HT)), substance
P, histamine and opioids are the important chemical mediators in contractile regulatory actions of ENS (Farthing,
2002). The activities of the neurotransmitters in the intestine are coordinated by a large number of receptors and
sub-receptors. Some of the receptors have been proved to play essential roles in GIT disorders such as
peristaltic colonic motility, diarrhoeal and constipation diseases.
Some of the diarrhoea aetiologies such as infectious pathogens or their toxins, inflammatory mediators and
oxidation by-products targets to control the peristaltic colonic movement by manipulating the ENS, and also
control fluid and electrolyte movement across the intestinal mucosa (Guttman and Finlay, 2008). The
modulations in the quantity of the neurotransmitters or the activity of the receptors can have enormous effects on
intestinal motility and contraction. The process may help in regulating absorption or secretion of fluid and
electrolyte by the intestine; hence provide relief against GIT disorders including diarrhoea and constipation
diseases (Sikander et al., 2009).
Enteric nervous system presents an attractive potential target for pharmacological intervention in diarrhoea. The
use of agonists and antagonist that target these ENS hormone receptors are routinely used clinically to modulate
intestinal motility, absorption and secretion. Antispasmodic or antimotility (atropine, clonidine and deodorized
tincture opium), and antisecretory agents (racecadotril, octreotide) are used to treat or prevent smooth muscle
contraction and control intestinal secretion, thus alleviating many symptoms of GIT disorder including diarrhoea.
However, prolonged uses of these drugs are often associated with some side effects such as dry mouth and
urinary retention for antimuscarinic drugs, headache, nausea, vomiting and constipation for calcium blockers.
Several medicinal plants are used by different traditional cultures across the world in alleviating GIT disorders
clinically manifesting as diarrhoea without reported cases of adverse effects. These provide the rationale in
continuous search for safer and efficient drugs from plant phytochemicals that might target a specific receptor.
113
In South Africa and other developing countries treatment of gastrointestinal disorders such as diarrhoea with
medicinal plants are particularly common in rural areas. The antidiarrhoea activities of medicinal plant extracts
can be exhibited through spasmolytic effects (intestinal smooth muscle relaxation), delay gastrointestinal transit,
suppress gut motility, stimulate water absorption or reduce electrolyte secretion. In contrast, the mechanism of
actions of medicinal plants used in constipation include spasmogenic effects (intestinal smooth muscle
contraction), rapid gastrointestinal transit, activated gut motility, suppressed water absorption or increase
electrolyte secretion (Gilani et al, 2005a). All these effects are related to the regulation of ENS motility and
contractility. However, scientific evaluations of the therapeutic claims as well as mechanisms of action are still
unreported for many of the antidiarrhoeal plants used in traditional medicine. The aim of this study therefore is to
evaluated motility regulatory potentials and determined possible mechanism of action of phenolic-enriched leaf of
Bauhinia galpinii and Combretum vendae as antidiarrhoea medicinal plants on isolated rat ileum.
8.1. Drugs and reagents
Acetylcholine hydrochloride (Ach), serotonin (5-HT), nicotine, Histamine, Prostaglandin E2 (PGE2), Prostaglandin
F2α (PGF2α), NG-nitro-l-arginine methyl ester (L-NAME), Carbachol, Pilocarpine, , Cyclopiazonic acid,
Dimethylsulphoxide (DMSO), Sodium chloride (NaCl), Potassium chloride (KCl), Calcium chloride (CaCl2),
Sodium bicarbonate (NaHCO3), Magnesium sulphate (MgSO4), Potassium hydrogen phosphate (KH2PO4),
Glucose, and carbogen
8.2. Animals
Male Wistar rats (250-300 g) obtained from University of Pretoria Biological Research Centre (UPBRC), Faculty
of Veterinary Science, Onderstepoort, Pretoria were used. All animals were housed under standard
environmental conditions and provided with food and water ad libitum. All the procedures were in accordance
with the guidelines for use of experimental animals established by the Animal Use and Care Committee (AUCC),
University of Pretoria based on specification in the South African National Standard (SAN 10386-2008). The
approval of ethical committee at Faculty of Veterinary Science, University of Pretoria was obtained before the
start of the work. The project was also approved by Faculty of Veterinary Science, University of Pretoria research
committee (UP-RESCOM) with approval number of V027-10.
8.2.1. Isolated ileum preparation
The animal was humanely sacrificed with inhalation of isoflurane and dissected immediately. The ileum was
removed and placed in carbogenated (95% O2 and 5% CO2) Krebs solution with the following composition (g/l):
NaCl, 6.94; KCl, 0.354; KH2PO4, 0.163; NaHCO3, 2.1; MgSO4, 0.370; CaCl2, 0.367; glucose, 2.07 and pH 7.4.
114
The intestinal content was removed by washing with Kreb’s solution and the mesenteric constituents were
eliminated. Longitudinal segments (1.5–2.0 cm) obtained from the distal ileum were placed in a 50 mL
thermostatically controlled (37oC) organ bath containing Krebs solution gassed with carbogen. The preparations
were connected to an isotonic transducer (load 0.5 g) in such a way as to record contractions mainly from the
longitudinal axis and allowed to equilibrate for 60 min before the start of experiment: contractions were recorded
using Bioscience transducers.
8.3. Contractility test
8.3.1. Spasmogenic assays
The crude extracts was prepared in stock solution of 20 mg/ml in DMSO and cumulatively added to the organ
bath from concentration of 10, 25, 50, 100, 250, 500, 750 and 1000 µg/ml. The effective concentration of DMSO
in the waterbath was less than 5%in all the experiments. Effect of the extracts on spontaneous motility of the ileal
preparations were monitored at 20 min contact time for each concentration and cumulative dose-dependent
curves for the extracts were determined to measure stimulatory effects.
8.3.2. Spasmolytic assays
8.3.2.1. Effects on acetylcholine-induced contraction
Acetylcholine hydrogen chloride was added to the organ bath cumulatively in the absence of test extracts at
concentration ranging between 0.01-1.00 µg/ml in water. The process was repeated with addition of ACH (0.011.00 µg/ml) after 20 min pre-incubation of the isolated ileum with the extracts (10, 25, 50, 100, 250, 500, 750 and
1000 µg/ml).
8.3.2.2. Effects on Serotonin-induced contraction
Serotonin was added to the organ bath cumulatively in the absence of test extracts at concentration ranging
between 0.001 - 0.1 µg/ml. The process was repeated with addition of 5-HT after 20 min pre-incubation of the
isolated ileum with the extracts (10, 25, 50, 100, 250, 500, 750 and 1000 µg/ml).
8.3.2.3. Effects on K-induced contraction
The isolated ileum preparation was washed with K+ free Kreb’s solution (composition (g/l): NaCl, 6.94; KCl,
0.354; KH2PO4, 0.163; NaHCO3, 2.1; MgSO4, 0.370; CaCl2, 0.367; glucose, 2.07 and pH 7.4) for 20 min after
equilibration and incubated with the extracts for 20 min. Thereafter, KCl solution (100 µl) was added
cumulatively.
Rat
115
Isolation of intestine
Preparation of ileum segment
Agonist, antagonist, ionic solution
Spasmogenic assay
Spasmolytic assay
Ionic channel modulation (80mM KCl)
Fig. 8.1. Schematic presentation of the contractility assay
8.4 Data analysis
The inhibition of ileum contraction by test sample was normalized and expressed as a percentage of mean±
SEM from 3-4 experiments of the references responses induced by acetylcholine (10 µg/ml), other spasmogens,
receptor agonists and antagonists using the following formula:
% Inhibition = [AC - AT/AC] × 100
Where AC is the amplitude (cm) of the ileum contraction induced by the agonists and antagonists in the absence
of the test sample; AT is the amplitude (cm) of the ileum contraction by the agonists and antagonists in the
presence of the test sample. The changes in EC50 will be used to compare the effect of the extracts using an
ANOVA.
8.5. Results.
8.5.1. Effect of B. galpinii crude extract on isolated rat ileum
The 70% acetone extract which should have high concentration of phenolics of B. galpinii (10 - 1000 µg/ml)
stimulate spontaneous contraction of the rat ileum as shown in Fig 8.2 with EC50 value of 27.85 µg/ml. Maximum
contraction (Emax) of 44 mm was obtained at 200 µg/ml and additional doses causes suboptimal response but
increase duration of response caused an irreversible spasm at the maximum dosage of 1000 µg/ml. Repeated
administration of the extract at maximum dosage (1000 µg/ml) caused exhaustion of the ileum.
Effects of the extract on acetylcholine, serotonin, K+ induced contractions and acetylcholine in the presence of
atropine (acetylcholine non-specific muscarinic receptors antagonist) indicated dual mechanisms of being an
agonist (prokinetic) and an antagonist (relaxant) agent. The extract also exhibited additive contractility activity
with acetylcholine and agonistic tendency to serotonin-induced contraction of the isolated rat ileum (Fig 8.3 and
8.4).
116
Effect of Bauhinia galpinii (Bag) on spontaneous contractility
% contraction of B. galpinii
100
% Contraction ofB. galpinii+atropine
% Contraction
80
60
40
20
0
1.0
1.5
2.0
2.5
3.0
Log Concentration (µg/ml)
Fig.8.2: Stimulatory effect of 70% acetone leaf extract of B. galpinii on spontaneous contractility of isolated rat
ileum and the antagonised effect of atropine.
T ransform of Acetylcholinestrase contractility
Ach
100
Ach+ Bag
Ach+ Bag +atropine
% Contractility
80
60
40
20
0
-0.5
0.0
0.5
Log [Ach] (µg/ml)
Fig. 8.3. Effect of 70% acetone leaf extract of B. galpinii (200 µg/ml) on the acetylcholine cumulative
concentration-effect curves in the presence and absence of atropine
From the concentration-response curve (CRC) for acetylcholine-induced contraction, the EC50 value in the
absence of B. galpinii was 0.033 µg/ml and the EC50 in the presence of B. galpinii was 0.049 µg/ml. The
stimulation of spontaneous contraction and agonistic effects on acetylcholine-induced contraction were partially
abolished by atropine (Fig 8.3). In the CRC for serotonin-induced contraction, the EC50 value in the absence of B.
galpinii was 0.0025 µg/ml and the EC50 in the presence of B. galpinii was 0.0014 µg/ml. In contrast, the B. galpinii
extract resulted in a concentration-dependent spasmolytic effect (antagonist) on K+-induced contraction of the
isolated rat ileum (Fig. 8.5) with maximum effect (Emax) of 40.66±5.13 mm at concentration of 200 µg/ml.
117
Agonist effect of B. galpinii on serotonin-induced ileum contraction
Serotonin
100
Serotonin + B. galpinii
% Contractilty
80
60
40
20
0
-3.0
-2.5
-2.0
Log Concnetration (Serotonin (ug/ml))
-1.5
Fig. 8.4. Agonised effect of 70% acetone leaf extract of B. galpinii (200 µg/ml) on serotonin induced-contraction
on rat isolated ileum.
Spasmolytic effect of B. galpinii on KCl-induced ileum contraction
KCl contractility (mm)
60
40
20
40
0
20
0
10
0
50
25
10
0
0
Concentration of B. galpinii (µg/ml)
Fig. 8.5. Relaxant effect of 70% acetone leaf extract of B. galpinii on KCl induced contractility of isolated rat ileum
8.5.2.
Effect of C. vendae crude extract on isolated rat ileum
The phenolic-enriched extract leaf extract of C. vendae do not stimulate spontaneous contraction (spasmogenic)
of the isolated rat ileum, we therefore conclude that the extract have spasmolytic potential. The crude extract of
C. vendae exhibited concentration-dependent spasmolytic effect on acetylcholine-induced contraction with EC50
values of 0.037, 0.027, 0.117, 0.365, and 0.396 µg/ml at the concentration of 0, 100, 200, 400, and 600 µg/ml of
C. vendae in the organ bath (Fig 8.6) and concentration-dependent spasmolytic effect on serotonin-induced
contraction of isolated rat ileum with EC50 value of 0.0017, 0.0044 and 0.012 µg/ml at the concentration of 0, 100,
200 µg/ml of C. vendae in the organ bath respectively (Fig 8.7). Equivalent volume of the solvent (5% DMSO)
used in dissolving the extract had no effect on the spontaneous contraction or on 5-HT-induced contraction.
118
Effects of C. vendae on acetylcholine-induced contraction
0
100
100 µg/ml
% Contractilty
80
200 µg/ml
400 µg/ml
60
600 µg/ml
40
20
0
-2.0
-1.5
-1.0
Log Concentration (acetylcholine (µg/ml))
-0.5
Fig.8.6. Spasmolytic effect of 70% acetone leaf extract of C. vendae on Ach-induced contractility of isolated rat
ileum
Effects of C. vendae on serotonin-induced contraction
100
0
100 µg/ml
200 µg/ml
80
% Contractilty
400 µg/ml
60
40
20
0
-3.0
-2.5
-2.0
-1.5
Log Concentration (serotonin (µg/ml))
Fig.8.7. Relaxant effect of 70% acetone leaf extract of C. vendae on 5-HT-induced contractility of isolated rat
ileum
Addition of depolarised KCl solution (80mM) caused sustained contractions which were inhibited by C.
vendae phenolic enriched leaf extracts in concentration-dependent response (Fig. 8.8). Therefore, agent that
inhibits contraction induced by depolarised KCl solution is considered to be a calcium channel blocker (Godfraind
et al., 1986). The spasmolytic effects were reversible and the spontaneous contraction returned to normal after
washing three times with kerbs’ solution.
119
Spasmolytic effects of the crude extracts on 80 mM KCl-induced ileum contraction
50.00
KCl contractility (mm)
40.00
30.00
20.00
10.00
40
0
20
0
10
0
50
25
10
0
0.00
Concentration of C. vendae (µg/ml)
Fig. 8.8. Spasmolytic effect of the C. vendae on the depolarised KCl-induced isolated rat ileum contractions
8.6. Discussion
Gastrointestinal motor tone is modulated through multiple physiological mediators which include
neurotransmitters, inflammatory mediators and oxidative metabolites (Hoogerwerf and Pasricha, 2006). The
release of these chemical modulators in GIT causes stimulatory effect mediated through an ultimate increase in
cytosolic Ca2+ (Burks, 1987). Drug substances with ability to block or alter any of the above pathways or with
non-receptor specific inhibitory action such as Ca2+ antagonists could be considered to be effective as
therapeutic agent in hyperactive or hypoactive GIT disorders. These are important in control or alleviating
diseases such as diarrhoea, constipation, emesis and dyspepsia. To study the pharmacology and possible
mechanism of smooth muscle excitatory or inhibitory effect of drugs and medicinal plant extracts isolated tissue
preparations of laboratory animal are usually used for in vitro assays.
Acetylcholine (ACh) is a neurotransmitter released by the parasympathetic nervous system mediating its action
in the GIT by stimulation of nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors
(mAChR). In the GIT, five subtypes of the muscarinic receptors, namely M1, M2, M3, M4 and M5 have been
identified (Tobin et al. 2009). However, M2 and M3 receptors play some essential roles in the smooth muscle
contraction/relaxation of GIT (Matsui et al., 2002; Takeuchi et al., 2005; Unno et al., 2005). Through this
mechanism, acetylcholine plays a critical physiological role in regulating the peristaltic movements of the GIT
(Brown and Taylor, 1996). The possible mechanisms responsible for contractility mediating action of drugs
including medicinal plant extracts may include one or combinations of:
•
Stimulation/inhibition of ACh release from the cholinergic nerve endings.
•
Stimulation/inhibition of acetylcholinesterase (AChE) enzyme at the neuro-effector junction.
120
•
Direct activation/inactivation of the muscarinic receptors of all smooth muscles, including those of GIT.
The effects of serotonin in the ENS are complex and diverse including modulation of smooth muscle function
(promoting both contraction and relaxation), potent intestinal secretagogue (predominantly pro-secretory) and
responses to visceral pain. Serotonin (5-HT) receptors found within the ENS and motor neurones of the GIT
include 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7. However, only 5-HT1, 5-HT2, 5-HT3, 5-HT4 and 5HT7 receptor subtypes are known to affect GIT motor functionality.
The 5-HT3 and 5-HT4 receptors are the most studied subtypes with the regards to physiological function and
histological distribution in GIT (Chetty et al., 2006; Cellek et al., 2006). The 5-HT3 receptor induces a rapid
depolarization of the myenteric neuron through enhancing ACh release (Kim, 2009), while 5-HT4 receptor
expressed in the nerve terminal facilitates the releases of neurotransmitters including ACh, substance P and
vasoactive intestinal peptides (Kim, 2009; Wouters et al. 2007). These cellular events of 5-HT lead to an upstream regulation enhancing the excitatory activity of GIT smooth muscles through mediating the ACh release.
Serotonin is involved in cholera toxin-and bile salt-induced fluid and electrolyte secretion by activating the ENS.
Contractions of all smooth muscles, including those of GIT depend on the presence of Ca2+. Increase and
decrease in intracellular free Ca2+ are the principal mechanisms that initiate contraction and relaxation
respectively in smooth muscle (Sanders, 2001). Agonists-induced contractions are related to the release of
intracellular Ca2+ from sarcoplasmic stores and extracellular Ca2+ influx through L-type channels (Makhlouf,
1994). Therefore smooth muscle relaxations can be effected by antispasmodic drugs through the inhibition of
Ca2+ entry or release into the cells. Exposure of smooth muscle cells to high concentration of K+ (>30 mM)
stimulate contractions through opening of voltage-dependent L-type Ca2+ channels and influx of extracellular
Ca2+ (Bolton, 1979; Godfraind et al., 1986).
The results obtained in this work indicated that phenolic-enriched crude leaf extract of B. galpinii contracted the
rat ileum dose-dependently and its initial contractile phase was partially blocked by atropine, a naturally occurring
alkaloid and a well known non-selective muscarinic receptor antagonists. Atropine competes with ACh and other
muscarinic agonists for a common binding site on the muscarinic receptor. This result shows the involvement of
cholinergic muscarinic receptors alongside with other stimulatory receptors exhibiting initial contraction by B.
galpinii on isolated rat ileum.
The phenolic-enriched crude leaf extract of B. galpinii also exhibited dose-dependent stimulating activity on
serotonin-induced contraction of isolated rat ileum. The spasmogenic effects of the extract on ileum longitudinal
muscle may be direct serotogenic activation of 5-HT receptor pathways or through the enhanced release of other
neurotransmitters without serotogenic potential.
Addition of KCl (80 mM) caused sustained contractions which were inhibited by B. galpinii. Therefore the
inhibitory effect of the crude extract of B. galpinii against K+-induced contractions can be as result of the blockade
121
of Ca2+ channels. Thus it can be concluded that B. galpinii has a dual-mechanism of action (prokinetic and
relaxant) on gastro-intestinal motility, depending on the prevalent patho-physiological condition. The B. galpinii
70% acetone leaf extract can therefore be clinically relevant as therapeutic agent in diarrhoea and constipation
which are both diseases with aetiology based on motility disturbances to a large extent.
Fumaria indica crude extract also has dual-spasmogenic and spasmolytic effects on isolated organs (Gilani et
al., 2005a). The aqueous-ethanolic extract (80% ethanol) of the aerial parts of Hibiscus rosasinensis Linn
(Malvaceae) contains spasmogenic and spasmolytic constituents mediating their effect through cholinergic
receptors activation and blockade of Ca2+ influx, respectively (Gilani et al., 2005b). Crude aqueous leaf extracts
of Morinda morindoides (Baker) Milne-Redh (Rubiaceae) agonise spontaneous contractility of isolated rat ileum
(Cimanga et al., 2010). The petroleum ether soluble fraction and the crude saponin constituents of the extract
are responsible for the spasmogenic activities. The spasmogenic and spasmolytic effect of a particular medicinal
plant extract on the isolated ileum depends on predominant phytochemical constituents. Phenolic compounds
exhibit spasmolytic activity while saponins are responsible for the spasmogenic activities of many plant extract
preparation. From the phytochemical analysis of the extract of B. galpinii, the extract contains high content of
phenolics. However, the result obtained in this study indicated that the crude extract of B. galpinii also contains
other active ingredients with spasmodic effect higher than the anticholinergic effect of the phenolics.
C. vendae extract did not stimulate spontaneous contractility of the rat ileum. Further investigation of its effects
on ACh-induced contraction led to a concentration-dependent inhibitory activity against ACh contraction of the rat
ileum. Anti-contractility effects of C. vendae against ACh-induced contraction are similar to atropine indicating
that the extract may be acting via nAChR or mAChR.
Addition of KCl (80 mM) caused sustained contractions which were inhibited by both B. galpinii and C. vendae
phenolic enriched leaf extracts in concentration-dependent response. Agents that inhibit contraction induced by
KCl are considered to be a calcium channel blocker (Godfraind et al., 1986). The spasmolytic effects were
reversible and the spontaneous contraction returned to normal after washing three times with Ca2+ free-krebs
solution.
The results indicate that C. vendae extract is capable of mediating spasmolytic effects on isolated rat ileum
through multiple inhibitions of a wide range of contractile stimuli, such as neurotransmitters (acetylcholine and
serotonin) and high potassium (depolarizing stimulus). This suggests that the ileum relaxant effects of the extract
are not specific to a type of receptor but rather due to either general receptor inactivation or membrane
depolarization. Muscarinic receptor antagonists, 5-HT receptor antagonist and Calcium channel blockers of the
L-type are known to be effective as antispasmodic, anti-motility and antidiarrhoeal agents (Lee et al., 1997;
Brown and Taylor, 2006; Pasricha, 2006). Hence, the presence of multiple acting spasmolytic activities in the
plant extract might be contributing towards its effectiveness in diarrhoea and abdominal spasm. The isolated
triterpenoids such as ursolic acid, maslinic acid, corosolic acid, asiatic acid and arjunolic acid from the plant also
122
have good antimicrobial activity and the stilbenoid glycosides such as combretastatin B5-O-2’-β-Dglucopyranoside and combretastatin B1-O-2’-β-D-glucopyranoside has good antioxidant activity. Such activities
of the plant could account for additional benefits providing a wider cover for its use in diarrhoea of different
aetiologies. This is also in accordance with the general understanding that plants contain multiple active
constituents with effect enhancing activities (Gilani and Rahman, 2005).
8.7. Conclusion
The result indicated the B. galpinii have dual activities with the capacity of acting as prokinetic and spasmolytic
agent while C. vendae acts as spasmolytes against the three spasmogens used to induce contraction of the
ileum.Further studies aiming to identify the targeted receptor subtype and the type of interaction with muscarinic
receptors as well as the identification of the main active principle are needed.
The results indicate that there is a scientific rationale for using extracts of these plant species to treat diarrhoea
in humans or animals. In the next section some of the antimicrobial and anti-oxidant compounds present in these
extracts will be isolated and characterized.
123
CHAPTER NINE
Isolation and characterization of antimicrobial and antioxidant compounds from Bauhinia galpinii and
Combretum vendae
9.0. Introduction
One of the cardinal objectives in medicinal plant research and development is identification, isolation and
characterization of the bioactive components present in an extract. Medicinal plant extracts are inherently
complex mixture of diverse chemical components. Separation of the active components of plant phytochemicals
from the inactive components are categorized into three parts: extraction, purification and chromatography.
Extraction and purification involved sample preparations schematically represented in Fig. 9.1 and Fig. 9.2.
Various chromatographic methods are available for qualitative and quantitative (TLC fingerprint, high
performance liquid chromatography (HPLC) fingerprints) as well as for isolations (open column chromatography
(OCC), vacuum liquid chromatography (VLC), HPLC, high-speed counter-current chromatography (HSCCC),
gas-liquid chromatography (GLC) and/or gel permeation chromatography (GPC)). The principles of separation
are based on molecular size, adsorption to the stationary phase, polarity and solubility in the mobile phase.
Structural information on isolated compounds are usually obtained from different spectroscopic techniques
namely: nuclear magnetic resonance spectroscopy (NMR), mass spectroscopy (MS), and to a lesser extent
infrared spectroscopy (IR) and ultraviolet-visible spectroscopy (UV-visible).The characteristic features of each
NMR experiments are summarized in Table 9.1.
Table 9.1 NMR experiments commonly applied for natural product structural elucidation (Simpson et al., 2011)
NMR experiment
Information/interpretation
Proton NMR (1D 1H NMR)
Quantitative overview of the distribution of protons in a
sample.
Can provide a quantitative overview of the carbon
distribution.
Separate the carbon of a compound into primary (CH3),
secondary (CH2), Tertiary (CH) and quaternary (C) spectra.
Connectivity information of protons on adjacent carbons.
Cross-peaks connect the chemical shifts of protons that are
coupled.
Symmetrical cross peaks appear around a central diagonal.
1H–13C 1 bond correlation. Cross peaks represent carbon
chemical shifts in one dimension and proton chemical shifts
in the other dimension.
1H–13C 2–4 bond correlations. Quaternary carbons are
observed. Connectivity information is read as vertical lines.
Carbon-13 NMR (1D 13C NMR)
Distortionless enhancement through polarization
transfer (DEPT) (1D 13C NMR)
1H-1H Correlation spectroscopy (COSY) (2D 1H NMR)
1H-13C
Heteronuclear single quantum correlation
(HSQC) and heteronuclear multiple quantum
correlation (HMQC) (2D 1H- 13C NMR)
1H-13C Heteronuclear bond multiple correlation (HMBC)
(2D 1H- 13C NMR)
9.1.1. Column chromatography
124
Isolation of such compounds is usually carried out by open column chromatography under gravitation force using
silica gel, sephadex, polyamides or reverse phase (RP) mode on C8 or C18-bonded silica gel stationary phase.
The separation of individual compounds from the complex extract mixture is based on the compound
characteristic ability for the stationary phase in the column relative to the polarity of the mobile phase. Changing
the polarity (gradient elution) of the mobile phase will allowed all target compounds to elute in a sequential
manner. The chromatographic process should be rapid, do not lead to decomposition of compounds, material
loss, or formation of artefacts.
Open column chromatography is simple, cheap and universally practiced despite some obvious disadvantage of
method being slow and often produces irreversible adsorption of sample onto the stationary phase. The method
is also encumbered with large sample and solvent requirement. The bioactive compounds of interests in this
project are non volatile.
9.1.2. Mass spectrometry
Mass spectrometry (MS) is an important physico-chemical tool applied for structural elucidation of compounds
from natural products including medicinal plants. The fundamental principle of MS is the use of different physical
means for sample ionization and separation of the ions generated based on their mass (m) to charge (z) ratio
(m/z) (Rijke et al., 2006). The ionization techniques available include electrospray ionization (ESI), atmospheric
pressure chemical ionization (APCI), electron ionization (EI), chemical ionization (CI), fast atom bombardment
(FAB), and matrix-assisted laser desorption ionization (MALDI) (Rijke et al., 2006). Mass spectrometry has high
sensitivity with detection limit of fentogram compared to NMR with sensitivity limit of nanogram range and above
(Simpson et al., 2011). The high sensitivity and the flexibility for hyphenation with other chromatographic
technique made MS a versatile analytical instrument.
9.2. Materials and Methods
9.2.1. Preparation of plant extracts
The extraction and fractionation protocol was followed as described in Chapter 3. The schematic diagrams of the
extraction, fractionation and isolation processes for Combretum vendae and Bauhinia galpinii are presented in
Fig 9.1 and 9.2.
9.2.2. Bioautography
The bioautography against bacteria (E. coli and S. aureus) protocol were followed as described in chapter 4
while the TLC-DPPH antioxidant assay were carried out as described in chapter 5.
9.2.3. Isolation of bioactive terpenoids from Combretum vendae
The n-Hexane and ethyl acetate fractions showed one and two clear zone(s) of microbial growth inhibition
respectively. The two fractions were subjected to gravitational column chromatography on silica gel
125
(2.5cm×73cm using 150 g silica, particle size 0.063–0.200 nm, Merck 70–230 mesh ASTM) using the solvent
mixture of hexane: ethyl acetate starting with 100% n-Hexane, 99: 1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7 and finally
90:10 as mobile phase. Schematic representation of the isolation procedure is presented in Fig 9.1.
Combretum vendae leaf powder (232.75 g)
Extraction with mixture of acidified 70%
acetone and n-hexane (73.09 g)
Decanted
Phenolic-enriched crude extracts (63.88 g)
Chlorophyll and terpenoid-enriched hexane
fraction (9.21 g)
Open column chromatography (Silica gel:
Stationary phase [100 g].
Liquid-liquid fractionation
Dichloromethane
fraction (4.3 g)
Ethyl acetate
fraction (14.39 g)
Gradient solvent starting with 100% hexane
and 5% increasing amount of ethyl acetate
Butanol
fraction
(16.27 g)
Water fraction
(28.9 g)
Open column chromatography (Silica gel:
Stationary phase [150 g].
Re-chromatographed
Compd 1 (eluted
with hexane:ethyl
acetate [90:10]
(101.3 mg
Fraction 1
(eluted with
96:4) (40
mg)
Repeated column
chromatography with
100:0, 98:2, 96:4, 94:6
DCM: methanol) to
yield compound 4 [5.3
mg]
Compd 2 (eluted
with hexane:ethyl
acetate [70:30]
(56.8 mg)
Fraction 2
(eluted with
90:10 (310
mg)
Fraction 3
(eluted with
85:15) (450
mg)
Repeated column
chromatography with
96:4, 94:6, 92:8, 90:10
DCM: methanol) to yield
compound 5 [185.0 mg]
Compd 3 (eluted
with hexane:ethyl
acetate [30:70]
(67.0 mg)
Gradient solvent starting with 100%
dichloromethane and 2% increasing amount
of methanol
Fraction 4
(eluted with
80:20) (331
mg)
Fraction 5
(eluted with
70:30) (279
mg)
Fraction 6
(eluted with
50:50) (191
mg)
Fraction 7
(eluted with
70:30) (288
mg)
Repeated column
chromatography with
96:4, 94:6, 92:8, 90:10
DCM: methanol) to yield
compound 5 [160.43 mg]
and 6 [89.71 mg]
Fig. 9.1. Extraction, fractionation and isolation of bioactive compounds from the leaf extract of Combretum
vendae
The dried fractions were reconstituted (10 mg/ml) and 10 µl of the aliquot spotted on TLC. Three mixtures of nHexane: ethyl acetate (90:10; 95:5 and 98:2) was used to develop the plates. The fractions with Rf corresponding
to the Rf values of bioautography assay were combined. The purification of the compounds was achieved by
repeated column chromatography until single spot was obtained for each compound using three different mobile
phases to develop the TLC.
9.2 4. Isolation of bioactive phenolics from Combretum vendae
126
The ethyl acetate, n-Butanol and residual water fractions exhibited good antimicrobial and antioxidant activities.
In TLC-DPPH assay four clear zones of antioxidant components were observed in ethyl acetate fraction. The
fraction was subjected to open column chromatography under gravitational force (2.5cm×73cm using 150 g
silica, particle size 0.063–0.200 nm, Merck 70–230 mesh ASTM) using the solvent mixture of dichloromethane:
methanol starting with 100% dichloromethane, 98:2, 96:4, 94:6, 92:8, 90:10, 85:15, 80:20, 70:30, 60:40, 50:50 as
mobile phase. The eluents were monitored with DPPH-TLC antioxidant assay and vanillin/H2SO4 spray reagent.
The fractions with the same compounds were combined and subjected to further cleaning by re-chromatography
until single spots were obtained on TLC chromatogram using three different mobile phases. Schematic
representation of the isolation procedure is presented in Fig 9.1.
9.3. Isolation of compounds from B. galpinii
9.3.1. Isolation of bioactive terpenoid from Bauhinia galpinii
The n-Hexane fractions showed one clear zone of microbial growth inhibition and was subjected to open column
chromatography under gravity on silica gel (2.5cm×73cm using 150 g silica, particle size 0.063–0.200 nm, Merck
70–230 mesh ASTM) using the solvent mixture of hexane: ethyl acetate starting with 100% n-Hexane, 98:2,
96:4, 94:6, 92:8 and finally 90:10 as mobile phase. The eluent were monitored using TLC and vanillin/H2SO4
spray. The fractions containing the target compound was combined and the chromatography process was
repeated until a single spot using three mixtures of n-Hexane: ethyl acetate (90:10; 95:5 and 98:2) as mobile
phases for TLC chromatogram was obtained. Schematic representation of the isolation procedure is presented in
Fig 9.2.
9.3.2. Isolation of bioactive phenolics from Bauhinia galpinii
The ethyl acetate, n-Butanol and residual water fractions exhibited good antimicrobial and antioxidant activities.
In TLC-DPPH assay four clear zones of antioxidant components were observed in ethyl acetate and butanol
fractions. The fractions were subjected to open column chromatography under gravitational force (2.5cm×73cm
using 150 g silica, particle size 0.063–0.200 nm, Merck 70–230 mesh ASTM) using the acidified solvent mixture
of dichloromethane: methanol starting with 100% dichloromethane, 98:2, 96:4, 94:6, 92:8, 90:10, 85:15, 80:20,
70:30, 60:40, 50:50 as mobile phase. The eluents were monitored with DPPH-TLC antioxidant assay and
vanillin/H2SO4 spray reagent. The fractions with the same compounds were combined and subjected to further
cleaning by re-chromatography using silica gel or Sephadex L20 until single spots were obtained on TLC
chromatogram using three different mobile phases. Schematic representation of the isolation procedure is
presented in Fig 9.2.
Bauhinia galpinii leaf powder (365.75 g)
Extraction with mixture of acidified
70% acetone and n-hexane (70.09 g)
Decanting
127
Chlorophyll and terpenoid-enriched hexane
fraction (4.56 g)
Phenolic-enriched crude extracts (66.53 g)
Liquid-liquid fractionation
Open column chromatography (Silica gel:
Stationary phase [100 g].
Gradient solvent starting with 100% hexane
and 1% increasing amount of ethyl acetate
Butanol fraction
(18.94 g)
Water fraction
(30.89 g)
Open column chromatography (Silica gel:
Stationary phase [150 g].
Gradient solvent starting with 100%
dichloromethane and 2% increasing
amount of methanol
Fraction 2 (eluted
with 96:4 (20.23 mg)
Sephadex-L20
Compound 2 (5.11 mg)
Ethyl acetate
fraction (7.59 g)
Open column chromatography (Silica gel:
Stationary phase [150 g].
Compound 1 (eluted with hexane:ethyl
acetate [96:4]. Repeated column
chromatography with 100:0, 98:2, 96:4,
92:8 yielded pure compound 1 [115 mg]
Fraction 1 (eluted with
98:2) (71.03 mg)
Dichloromethane
fraction (4.3 g)
Fraction 3
(eluted with 94:6)
(107.01 mg)
Fraction 1
(eluted with
90:10) (30 mg
Sephadex-L20
Compound 3 (31.3
mg) and 4 (15.2 mg)
Gradient solvent starting with 100%
dichloromethane and 5% increasing amount
of methanol
Fraction 4
(eluted with
92:8) (88 mg)
Fraction 2
(eluted with
85:15 (109
mg)
Sephadex-L20
Compound 5 (35 mg)
Fraction 3
(eluted with
80:20) (89
mg)
Fraction 4
(eluted with
70:30) (205
mg)
Fraction 5
(eluted with
60:40) (120
mg)
Sephadex-L20
Compound 6 (40 mg)
Fig. 9.2. Extraction, fractionation and isolation of bioactive compounds from the leaf extract of Bauhinia galpinii
9.4 Characterization of the compounds
9.4.1 NMR spectroscopy
One dimensional (1D) (1H and 13C) and two dimensional (2D) NMR spectra (1H-1H COSY, HMQC and HMBC)
NMR spectra were recorded on a Varian-NMR-vnmrs 600 spectrometer with tetramethylsilane (TSM) as internal
standard. Standard pulse sequences were used for homo- and heteronuclear correlation experiments. 1H NMR
spectra were measured at 599 MHz whereas 13C NMR spectra were run at 150 MHz. Multiplicities of 13C NMR
resonances were determined by DEPT experiments. All NMR experiments were performed at constant
temperature (27 °C) using software supplied by the manufacturer, employing deuteriochloroform,
deuteriomethanol, or deuteriodimethylsulphoxide as solvent on the basis of solubility of the sample and literature
data.
9.4.2 Mass Spectroscopy:
128
Electrospray ionization mass spectrometric analyses (negative and positive mode) were carried out to obtain the
molecular weight and fragmentations patterns of the isolated compound(s) using TOF mass spectrometer
(WATERS HPLC).
9.4.3 UV spectroscopy:
The UV-spectrum of the isolated bioactive compound(s) was recorded using Agilent 1200 UV-Visible
spectrophotometer.
9.5 Results
9.5.1 Identification of the chemical structures of isolated compounds from Combretum vendae
The n-Hexane and ethyl acetate fractions from the acidified 70% acetone leaf extract of Combretum vendae A.
E. van Wyk through bioassay guided fractionation were repeatedly subjected to gravity column chromatography
to yield one pure, mixture of four position isomer antimicrobial triterpenoids and two stilbene glucopyranoside.
The structures of the compounds were determined by extensive NMR techniques and chemical methods mainly
by 1D NMR (1H, 13C and DEPT) and 2D NMR (HSQC, HMBC and COSY), ESIMS, UV-visible spectra and by
comparison with the literature data.
Compound 1 was obtained as an amorphous white powder. Detailed analyses of the 1D and 2D NMR spectra
indicated the presence of 30 carbons which revealed 7 methyl, 9 methylene, 6 methane, 6 quaternary, 1
carboxylic acid at δC 178.9, an olefinic broad triplet proton at δH 5.25 (H-12) coupled to a carbon at δC 125.4, a
quaternary carbon at δ 138.8.0 (C-13) and a β-18 proton at δH 2.05 characteristic of signal of an ursol-12-en
skeleton (Appendix 9.1). The Comparison of these NMR data with the literature confirms the compound as ursol12-en-28-oic acid (ursolic acid) (Mahato and Kundu, 1994).
Compound 2 and 3 were obtained as position isomeric mixture; the TLC fingerprint indicated unresolved single
spot with three different mobile phases. However, the 1D and 2D NMR spectra exhibited chemical shift
characteristic of both olean-12-ene and ursol-12-ene. 13C NMR (DMSO): δ 179.7(C-28, olean), 178.0 (C-28, urs),
145.08 (C-13, olean), 122.72 (C-12, olean), 139.42 (C-13, urs), 125.55 (C-12, urs), 41 (C-18, olean), 53 (C-18,
urs), 27.97 (C-19, olean), 39.07 (C-19, urs) and 46.13 (C-20, olean), 39.02 (C-20, urs) (Appendix 9.2).
Comparing the data with the literature values, the two compounds in the mixture were identified to be corosolic
acid and maslinic acid (Mahato and Kundu, 1994)
Compound 4 and 5 were also obtained as position isomeric mixture. The spectra 1D and 2D were similar to the
spectra of compound 2 and 3 except the presence of additional hydroxyl at C-24) (appendix 9.3). From the
correlation data and literature values, compound 4 and 5 were identified to be asiaitic acid and arjunolic acid
respectively (Mahato and Kundu, 1994).
129
Compound 6 was obtained as an amorphous powder from the ethyl acetate fraction. The proton NMR spectrum
shows signal for apigenin: δH 6.19 (d, J=2.0 Hz, H-6), 6.38 (d, J=2.0 Hz, H-8), 7.2 (d, J=2.1 Hz, H-3), 6.50 (d,
J=8.9 Hz, H-3’ and H-5’), and 7.30 (d, J=2.1 Hz, 8.9, 2H’ and H-6’).
Compound 7 was obtained as creamy glass-like solid mass and deduced to have molecular formula C23H29O11
with molecular weight 481 by EIMS. The 13C-NMR spectrum indicated 17 carbon signals due to aglycone and six
carbon signals of a glycoside group. The signal consist twelve aromatic signals, three methoxy and two aliphatic
(Appendix 5). The 1H-NMR spectra revealed the presence of two aromatic benzylic rings. The 1H detected
heteronuclear multiple-bond connectivity (HMBC) spectrum indicated long-range correlations from 1H-1a’(δH 2.9,
3.0) to C-1’ (δC 128.55), C-2’ (δC 144.35), C-6’ (δC 118.95), C-1a (δC 36.96) and C-1 (δC 132.72). Long range
correlation observed between 1H-1a (δH 2.7) and C-1 (δC 132.72, C-2 (δC 106.16), C-6 (δC 106.16), C-1a’ (δC
31.73) and C-1’ (δC 128.55). Additional long-range correlation were observed between 1H-2, 6 (δH 6.5) and C-1
(δC 132.72), C-3, C-5 (δC 148.16), C-4 (δC 133.79), C-1a (δC 36.96); 1H-5’ (δH 6.7) to C-1’ (δC 128.55), C-3’ (δC
139.71), C-6’ (δC 118.95), C-4’ (δC 147.26) ; and 1H-6’ (δH 6.6) to C-1a’ (δC 31.73), C-3’ (δC 139.71), C-4’ (δC
147.26). Long-range correlation also observed between methoxyl signal (δH 3.7) and the C-3, C-5 (δC 148.16), C4’ (δC 147.26) (appendix 9.4). Based on these data, the aglycone of compound 6 was determined to be stilbenes
derivative. The signals for anomeric proton and carbon (δH 4.5 and δC 106.26) indicated the presence of a sugar
moiet. The long-range correlation in the HMBC experiment between the anomeric proton signal (δH 4.5) of the βD-glucopyranosyl group and the C-2’ signal (δC 144.35) confirmed the position of the attachment of the
glycopyranosyl moiety on the phenolic ring. The structure of compound 6 was determined to be combretastatin
B5-O-2’-β-D-glucopyranoside. The EIMS, 1D (1H and 13C, DEPT), and 2D (HSQC, HMBC, COSY) data correlate
with literature information on the compound (Pettit et al., 1985).
Compound 8 had molecular weight of 496 and the molecular formula was deduced to be C24H32O11 by EIMS. The
compound differs from compound 6 by the presence of extra methyl group. This was confirmed by 1D and 2D
NMR spectra. From HSQC one additional methoxyl signal (δH 3.6 and δC 59.69) having long range correlation C4 (δC 135) (appendix 9.5). The long range correlation from HMBC experiment between 1H-4 proton of the
glycopyranosyl moiety (δH 3.7) and the C-2’ signal (δC 143.78) confirmed the position of the structure of
compound 7 to be combretastatin B1-O-2’-β-D-glucopyranoside. The EIMS, 1D (1H and 13C, DEPT), and 2D
(HSQC, HMBC, COSY) data correlate with literature information on the compound (Schwikkard et al., 2000).
130
C H3
C H3
H 3C
H 3C
O
C H3
H 3C
OH
C H3
HO
H 3C
C H3
C H3
C oroso lic a cid (2)
U rso lic a cid (1)
H 3C
OH
HO
O
C H3
OH
C H3
HO
H 3C
C H3
HO
C H3
O
C H3
HO
OH
O
C H3
OH
C H3
O
HO
H 3C
A pigenin (6)
C H3
H 3C
Ma slinic acid (3)
H 3C
HO
O
C H3
H 3C
HO
O
C H3
H 3C
C H3
C H3
OH
C H3
HO
H 3C
OH
C H3
HO
H 3C
OH
A rjuno lic acid (5)
OH
A siatic acid
(4)
OH
OH
HO
HO
HO
HO
OH
O
O
Me
Me
O
O
HO
Me
O
Me
O
Me
C ombretastatin B 5-O-2'-beta glucopyran oside (7)
OH
O
Me
O
O
HO
O
HO
O
Me
C o mb retastatin B 1-O-2'-beta glucopyra noside (8)
Fig 9.3 Chemical structures of isolated bioactive compounds from 70% acetone leaf extract of Combretum
vendae
9.5.2 Antimicrobial assay of isolated compound from C. vendae
Antimicrobial activities of the isolated compounds against standard and clinical isolate pathogens expressed as
MIC are presented in Table 9.1. Some of the compounds exhibited good microbial growth inhibitory potential
worthy of pharmacological considerations with MIC ranging from 3.9-31 µg/ml.
Table 9.2: Minimum inhibitory concentration (µg/ml) of the isolated compounds from the leaf extract of
Combretum vendae
131
Microorganisms
Compound 1
Compounds 2
Compounds
and 3
4 and 5
Compound 7
Compound
8
E.coli
62
250
250
250
250
E. faecalis
31
31
31
31
31
S. aureus
62
125
125
125
62
P. aeruginosa
125
125
62
250
250
C. albicans (M0825)
15
7.8
7.8
31
31
C. albicans (M0824)
62
62
31
125
125
C. albicans (1051604)
3.9
3.9
3.9
3.9
3.9
C. albicans (1051608)
7.8
7.8
7.8
15
15
C. albicans (ATCC 10231)
7.8
15
7.8
31
31
C. neoformans
15
15
15
125
125
A. fumigatus
31
31
31
250
250
1: Ursolic acid, 2 and 3: mixture of maslinic and corosolic acid, 4 and 5: mixture of asiatic and arjulonic acid, 6: combretastatin B5-O-2’-βD-glucopyranoside.7: combretastatin B1-O-2’-β-D-glucopyranoside. C. albicans (M0824), C. albicans (M0825), C. albicans (1051604), C.
albicans (1051608) are clinical isolate obtained from National Health Laboratory Service, Pretotia, South Africa.
9.5.3 Identification of the isolated bioactive compounds from Bauhinia galpinii
The combine TLC fingerprint, TLC-DPPH assay and bioactivity guided fractionation of acidified 70% acetone leaf
extract of B. galpinii detected four major flavonoids from ethyl acetate fraction with antioxidant activity.
Bioautography against fungal and bacterial pathogens revealed two microbial inhibitory growth spot in Hexane
fraction and one in DCM fraction respectively. The bioactive compounds were isolated from each fraction using
open column chromatography with silica gel as stationary phase. The phenolics compounds were further purified
using Sephadex L-20 as stationary phase and acetone/methanol (50:50) as mobile phase at a rate of 2ml/5min.
The chemical structures of the compounds were determined by detailed nuclear magnetic resonance (NMR)
techniques including the one dimensional (1D) NMR (proton (1H), carbon-13 (13C) and distortion enhancement
DEPT) and two dimensional (2D) NMR (HSQC, HMBC and COSY). Mass spectrometry and the fragmentation
patterns of the compounds were extensively used for the structural elucidation.
Compound 9 obtained as white amorphous powder from hexane fraction was characterized by 1H NMR spectra
(in CDCl3), 13C-NMR spectra (in CDCl3), HSQC, HMBC, DEPT and COSY. The 1H NMR spectrum of 9 showed a
one-proton doublet at δH 5.33 (J¼5.5 Hz) assigned to a vinylic H-6 proton. A one-proton broad multiplet at δH 3.46
with half-width of 18.5 Hz was attributed to carbinol H-3 proton. Two three-proton broad signals at δH 0.67 and
1.01 were attributed to a tertiary C-18 and C-19 methyl protons. A six-proton broad signal at δH 0.83 was
associated with C-29 and C-20 methyl protons. Two three proton doublets at δH 0.91 (J¼6.2 Hz) and 0.85 (J¼6.3
Hz) were due to secondary C-26 and C-27 methyl protons. The remaining methylene and methine protons
appeared between δH 2.50 and 1.27. The presence of all the methyl signals in the range δH 1.01–0.67 suggested
that all these functionalities were located on the saturated carbons. The 13C NMR spectrum of 9 exhibited signals
for vinylic carbons at δC 140.97 (C-5) and 121.96 (C-6). The two carbinol signals appeared at δC 72.04 (C-3) and
132
76.86 (CH3 CH2O-) respectively. The carbon signals in the upfield region at δC 12.20, 21.30, 19.25, 20.04, 19.62,
12.08 and 19.00 were associated with the methyl functionalities. The remaining methylene and methine carbon
resonated between δC 56.99 and 23.29. The 1H–1H COSY spectrum of 9 showed correlations of H-6 with H2-7
and H-8; H-3 with H2-2, H2-4 (appendix 9.6). The 1H and 13C NMR spectral data of steroidal nucleus of 9 were
compared with related steroidal constituents (Alam et al., 1994). On the basis of the foregoing discussion, the
structure of 9 was elucidated as 3β-ethoxy stigmast-5-en-ol (3β-ethoxy sitosterol).
Compound 10 and 11 was isolated from the ethyl acetate fraction as a yellow powder by repeated gravitational
column chromatography on silica gel and Sephadex L-20 stationary phases. The 1H NMR spectrum in
deuterated methanol of 10 showed signals for quercetin: δH 6.19 (d, J=2.0 Hz, H-6), 6.38 (d, J=2.0 Hz, H-8), 7.57
(d, J=2.1 Hz, H-2’, δC ), 6.86 (d, J=8.9 Hz, H-5’, δC), and 7.56 ( d, J=2.1 Hz, 8.9, H-6’, δC). Carbon 13 NMR (100
MHz, in ppm, methanol-d4) shows 15 signals and the data (appendix 6) correlated well with literature for 3, 5, 7,
3’, 4’-pentahydroxyflavone (Quercetin) (Said et al., 2009)
The 1H NMR of compound 11 in deuterated methanol at 599.74MHz revealed the presence of myricetin
aglycone: δH 7.383 (s, H-2’,6’, δC 107.09) for the B phenyl ring, and 6.369 (d, H-8, δC 92.95), 6.338 (d, H-8, δC
92.95), 6.172 (d, H-6, δC 97.80) and 6.170 (d, H-6, δC 97.80) for the meta substituted A phenyl ring. The structure
was confirmed by
13C-NMR
which showed 15 signals without methoxy or glycoside substituent and 2D
correlation (HSQC and HMBC) (appendix 9.7). The data correlated well with literature data for 3, 5, 7, 3’, 4’, 5’hexahydroxyflavone (myricetin) (Said et al., 2009)
The NMR spectrum of compound 12 was typical for a flavone with one meta-substituted and para-substituted
phenolic moiety. The UV spectrum showed λmax 261.27 and 369.27 nm which compared favourably with 263 and
367 nm reported by Abdurrahman and Moon, 2007 for isoetin moiety. The compound was assigned molecular
formula C16H10O7 with the aid of a peak observed in the ESI-MS experiment at 315.054. The carbon chemical
shifts were assigned by the combination1H-13C HSQC and long range couplings in the 1H-13C HMBC
experiments. The 1H-NMR spectrum was indicative of five aromatic protons, one methoxyl group. The 1H and 13C
NMR spectra data correlated with those of isoetin flavone moiety (Abdurrahman and Moon, 2007; Pauli and
Junior, 1995; Voirin et al., 1975) (Table 1). The 1H-13C HSQC correlations were used to assign signal at δH 7.19,
6.27 and 6.40 to the protons at C-3 (s, δC 108.85), C-6 (d, J = δC 99.97 ) and C-8 (d, J = δC 94.86) positions of A
ring, and the signal at δH 6.65 and 7.38 to the proton at C-3’ (s, δC 101.36) and C-6’ (s, δC 114.45) positions of B
ring of the isoetin moiety respectively. The long range coupling in the HMBC presented in appendix 9.10 also
supported the isoetin flavone moiety. The methoxy protons signal at δH 3.8 was correlated with the quaternary
carbons (C-2’) at δC 153.28 indicating the attachment of methoxy group at the carbon (appendix 9.8). From all
the correlations, compound 13 was determined to be a new flavone named as 5, 7, 4’ 5’ tetrahydroxy-2’methoxyflavone (isoetin 2’-methyl ether) or 5, 7, 2’ 5’ tetrahydroxy-4’-methoxyflavone (isoetin 4’-methyl ether)
133
Compound 13 and 14 obtained as yellow powder respectively. UV spectra in MeOH showed λmax of 203.27,
255.27, 355.27 nm for compound 14, and 207.27, 257, 354.27 nm for compound 13 respectively. The ESI–MS
peaks of compound 13 and compound 14 in negative mode, were observed at m/z 463 [M-H] - and m/z 479 [MH] - respectively. The molecular ion of compound13 was 16 mass units smaller than that of compound14, which
corresponds to the difference in the number of hydroxyl groups on the B-ring of the flavonol aglycone. The 1H
NMR spectrum in deuterated methanol of 13 showed signals for quercetin: δH 6.19 (d, J=2.0 Hz, H-6, δC 99.17),
6.38 (d, J=2.0 Hz, H-8, δC 94.09), 7.57 (d, J=2.1 Hz, H-2’, δC ), 6.86 (d, J=8.9 Hz, H-5’, δC), and 7.56 ( d, J=2.1
Hz, 8.9, H-6’, δC) and anomeric protons at δH 5.192 (s, δC 104.14) and 5.22 (s, δC 104.14) characteristic of
galactopyranose. The 1H NMR of 14 in deuterated methanol at 599.74MHz revealed the presence of myricetin
aglycone with five aromatic proton: δH 6.19 (s, H-6, δC 98.48), 6.38 (s, H-8, δC 93.25), 7.37 (s, H-2’, H-6’, δC
108.52) and similar anomeric protons to those in 13 (appendix 9.9). The 13C NMR and HMBC, HSQC spectra in
deuterated methanol at 150MHz of 13 and 14 were very similar, except for the signals corresponding to the
flavonol aglycone (appendix 9.8). The spectral data and correlation with literature information revealed the two
compounds to be quercetin-3-O-β-galactopyranoside (Rayyan et al., 2004; Yan et al., 2002) and myricetin-3-O-βgalactopyranoside (Yan et al., 2002) respectively.
134
CH3
OH
O
CH3
HO
O
OH
CH3
H3C
O
OH
3 beta-O -ethyl sitosterol
O
5, 7, 4', 5' tetrahydroxy-2-methoxyflavone
(Isoetin 2'-methyl ether) (12)
(9)
OH
OH
OH
OH
HO
O
OH
HO
O
OH
OH
OH
OH
O
O
3, 5, 7, 3' 4', 5' Hexahydroxyflavone
(Myricetin) (11)
3, 5, 7, 3' 4' pentahydroxyflavone
(Quercetin) (10)
OH
OH
OH
HO
HO
O
OH
O
OH
OH
OH
O
OH
O
O
HO
OH
HO
OH
O
O
O
OH
OH
Quercetin-3-O-galactopyranose (13)
OH
Myricetin-3-O-galactopyranose (14)
Fig. 9.4. Chemical structure of bioactive compound isolated from the leaf extract of bauhinia galpinii
9.5.4. Antimicrobial assay of isolated compounds from B. galpinii
Antimicrobial potential of the isolated compounds against some diarrhoeal pathogens and organisms of other
important infectious diseases are presented in Table 9.2 as minimum inhibitory concentration (MIC) (µg/ml).
Table 9.3. Minimum inhibitory concentration (µg/ml) of the isolated compounds from the leaf extract of Bauhinia
galpinii and positive control
Microorganisms
9
10
11
13
14
Gentamicin
Amphoteric B
135
E.coli
125
31
31
31
62
15.5
-
E. faecalis
31
31
7.8
62
62
7.75
-
S. aureus
62
62
15
31
31
3.87
-
P. aeruginosa
125
62
15
15
15
3.87
-
C. albicans (M0825)
15
3.9
3.9
31
31
-
3.9
C. albicans (M0824)
31
31
31
31
31
-
3.9
C. albicans (1051604)
3.9
3.9
3.9
3.9
3.9
-
0.8
C. albicans (1051608)
62
3.9
3.9
15
15
-
1.93
C. albicans (ATCC 10231)
62
3.9
3.9
31
31
-
1.93
C. neoformans
125
31
31
125
125
-
7.75
A. fumigatus
125
31
31
125
125
-
7.75
1: 3β-ethoxyl sitosterol; 2 Quercetin; 3 Myricetin, 4 Quercetin-3-O-β-galactopyranose; 5 Myricetin- 3-O-β-galactopyranose. C. albicans (M0824), C. albicans
(M0825), C. albicans (1051604), C. albicans (1051608) are clinical isolate obtained from National Health Laboratory Service, Pretotia, South Africa.
9.6. Discussion
9.6.1. Bioactive compounds from Combretum vendae
Bioactivity guided investigation of Combretum vendae afforded ursolic acid ((3β)-3-hydroxyurs-12-en-28-oic
acid), mixture of asiatic and arjunolic acid, mixture of maslinic and corosolic acid, apigenin, Combretastatin B5-2’O-β-glucopyranoside and Combretastatin B1-2’-O-β-glucopyranoside all exhibiting broad based microbial growth
inhibitory potentials.
Ursolic acid and its derivatives biosynthetically derived from the cyclization of squalene (and other triterpene
acids) have been extensively studied as pharmacological active molecules in many in vitro and in vivo studies.
Some of the biological activities include antioxidant, hepatoprotective, anti-inflammatory, anticancer, anti-HIV,
vasorelaxant (Aguirre-Crespo et al., 2006) and antidiabetic activities. Several mechanisms have been proposed
to explain its anti-inflammatory activity, including inhibition of secretory PLA2 enzymes, IL-1β secretion, iNOS and
COX-2. Ursolic acid have no antispasmolytic effect (Estrada-Soto et al., 2007), however the broad base
antimicrobial activities (3.9-125 µg/ml) obtained in this work and other reports of antioxidant, anti-inflammatory
and antidiabetic provide pharmacological bases for further investigation of the compound as antidiarrhoeal agent.
Biological activities such as α-glucosidase inhibition of arjunolic acid (18.63±0.32 µg/ml), asiatic acid
(30.03±0.41 µg/ml), maslinic acid (5.52±0.19 µg/ml) and corosolic acid (3.53±0.27 µg/ml) isolated from
Lagerstroemia speciosa were reported. The α-glucosidase inhibition of these compounds shows their antidiabetic
and antiadhesion potential against microbial pathogens both of which are also important in antidiarrhoea therapy.
However the compounds have no α-amylase inhibitory activities (Hou et al., 2009). Mixture of arjunolic acid and
asiatic acid isolated from Combretum nicholsonii have antifungal activities with MIC of 0.2-1.5 µg/ml. However,
antimicrobial activities obtained in the work is slightly higher for the mixture (3.9-250 µg/ml), the difference in
result may be due to experimental variable such as concentrations of the culture media, composition of the
136
mixture, incubation time and strains. These results indicate that individual compound or the mixtures have
pharmacological potentiality against infectious pathogens.
Apigenin have been isolated previously from the acetone leaf extract of C. vendae. The antibacterial activity of
the compound was evaluated (Eloff et al., 2008).
Combretastatin B5-2’-O-β-glucopyranoside have been isolated previously from the seed of Combretum kraussii.
The aglycone moiety of this compound was isolated from the acetone leaf extract of Combretum woodii reported
to have antibacterial activity. This is the first report on the isolation of the compound from the leaf extract and its
antimicrobial activities.
Combretastatin B1-2’-O-β-glucopyranoside have been previously isolated from the seeds of Combretum kraussii
(Pettit et al, 1987) and wood bark of Combretum erythrophylum (Schwikkard et al., 2000). The stilbenes have
been reported to have cytotoxic activity with effect on tubulin polymerization, the primary protein component of
microtubules in cancer hence the potential of the compound as anticancer drug is being explored. The
compound has been evaluated for selective inhibitory activity against the DNA-damaging repair-deficient strain of
Saccharomyces cerevisiae deficient in the RAD52 recombination repair gene and exhibited no activity while the
derivative with unsaturated bond at 1aC- 1a’C (combretastatin A1-2’-O-β-glucopyranoside) was active
(Schwikkard et al., 2000). This indicated the importance of the unsaturated bond in the structure-activity
relationship for cytotoxicity effects. However, there is no literature report on other biological activity potential such
as antimicrobial, antioxidant, anti-inflammatory and antidiarrhoea. This is the first report on the isolation of the
compound from the leaf extract and its antimicrobial activities. The non cytotoxic effect of the compound against
the cancer cells lines (Schwikkard et al., 2000) indicates that the compound can be exploited for other biological
activities.
9.6.2. Bioactive compounds from Bauhinia galpinii
Isoetin (5, 7, 2’ 4’ 5’-pentahydroxyl flavone) and its various derivatives are rare compounds formed by insertion of
a 2’ hydroxyl group into luteolin to give characteristic yellow pigments in some plant part Lattanzio et al., 2006).
5, 7, 4’ 5’ tetrahydroxy-2’-methoxyflavone (isoetin 2’-methyl ether) is a new compound (unfortunately the quantity
obtained was not enough for bioassays). A related compound 5, 7, 2’ 4’ tetrahydroxy-5’-methoxyflavone (isoetin
5’-methyl ether) isolate from Trihosanthes kirilowii (Cucurbitaceae) was reported to be cytotoxic against human
lung cell line A549 (IC50 0.92 µg/ml), human melanoma Sk-Mel-2 (IC50 8.0 µg/ml), and mouse melanoma B16F1
cell lines (IC50 7.23 µg/ml). High cytotoxicity (IC50 2.5 µg/ml) of the acetone root extract of B. galpinii against Vero
cell lines has been reported (Samie et al., 2009). Isolation of more 5, 7, 4’ 5’ tetrahydroxy-2’-methoxyflavone
(isoetin 2’-methyl ether) from B. galpinii for further studies on cytotoxicity effect important.
Quercetin and myricetin are the common flavonol present in dietary and constitute the active component of
medicinal plant with characteristic hydroxyl substitutions at the 3, 5, 7, 3’, 4’ (Quercetin), and 3, 5, 7, 3’, 4’ and 5’
137
(myricetin) positions of flavone ring. The compounds occur in nature mostly as glycoside with D-glucose, Lrhamnose, D-galactose or arabinose rather than free aglycone. Biological effects of phenolic compounds depend
on their bioavailability which is determined by the lipophilicity of each molecule. The mechanisms involved in
digestion and absorption of phenolic compound is complex but passive transports through the membrane have
been proposed. The glycosides and methylated phenolics are not readily absorbed in native form but need to be
hydrolysed by intestinal enzymes or colonic microflora to aglycone before absorption.
Quercetin and myricetin alongside their galactopyranoside derivatives were isolated from the acidified 70%
acetone leaf extract of Bauhinia galpinii. Quercetin galactopyranose and myricetin galactopyranose were
previously isolated from this plant and their antioxidant activity and cytotoxicity was evaluated (Aderogba et al.,
2007).
A wide range of biological activities related to diarrhoeal pathogenesis including antimicrobial (Naz et al., 2007),
anti-inflammatory and spasmolytic due to their antioxidant and/or free radical scavenging (Aderogba et al., 2007)
as well as ability to interfere with several enzymatic pathways have been reported for the compounds. Quercetin
and myricetin are active against microorganisms of the genera Bacillus, Corynebacterium, Salmonella, Shigella,
Staphylococcus, Streptococcus, and against Escherichia coli and Vibrio cholerae (Naz et al., 2007).
Both compounds also have protective and promotive effects on intestinal TJ barrier function through interaction
with intracellular signaling molecules, tyrosine kinases and protein kinase C δ (PKCδ) (Suzuki and Hara, 2010).
The intercellular TJs are the major determinant of the intestinal physical barrier regulating the paracellular
movement of ions, solute, and water through the intestinal epithelium. Impaired intestinal TJs functions are
involved in several intestinal and metabolic diseases, such as diarrhoea, inflammatory bowel disease and food
allergy (Suzuki and Hara, 2010). Myricetin inhibits the generation of MDA a cytotoxic by-product of lipid
peroxidation of arachidonic acid liberated from membrane phospholipids (Robak et al., 1986).
Myricetin has potential as an antiviral agent by its ability to inhibit the reverse transcriptase from Moloney murine
leukaemia virus, Rauscher murine leukaemia virus and human immunodeficiency virus (Ono et al., 1990).
Myricetin also has antidiabetic activity with ability to stimulate lipogenesis and enhanced glucose uptake into
adipocytes. The mechanisms postulated include changes in lipid-protein interaction or increase membrane
fluidity. Myricetin inhibits the intraluminal accumulation of fluid and prevent diarrhoea induced by castor oil.
9.7. Conclusion
All the isolated compounds from the two plant species have biological activities with relevance against one or
more diarrhoeal pathophysiology. The antimicrobial of some of the compounds are worthy of pharmacological
consideration. In order to exploit the full potentials of these compounds some in vitro and in vivo studies are
required to determine the mechanism of action.
138
CHAPTER TEN
General conclusion and future prospects
10. Introduction
Diarrhoea is one of the major health challenges facing the world and especially developing countries. The
problem is aggravated due to the increasing number of immunocompromised people infected by HIV, with
associated opportunistic infections and other health complications manifesting as diarrhoeal symptoms. The
emergence of more virulent strains resulting from drug resistant pathogens and the apparent side effects of
some conventional drugs currently in use is also serious concerns in diarrhoeal control and management. In
animal production, diarrhoeal outbreaks usually cause serious economic losses due to reduced productivity, cost
of treatment, lower level of reproduction and increased mortality.
However, the success of oral rehydration therapy in reducing mortality and lack of commercial interest in drugs
for developing countries has slowed the progress in the development of novel agents for treating diarrhoeal
diseases. Therefore, there is an urgent need for new therapeutic drugs or herbal products with lower cost, high
efficacy and little or no side effects. Plants and plant preparations have been used ethnopharmacologically in
treating diarrhoea successfully, although their efficacies, mechanisms of action and safety have generally not
been proven scientifically. Thus, the overall aim of this project was to systematically determine the efficacy, mode
of action and safety of some plants used traditionally in South African traditional medicine as diarrhoea therapy.
The following objectives were identified to attain this aim:
To conduct comprehensive literature works on diarrhoeal aetiologies and mechanisms and, medicinal
plants use for treating diarrhoea symptoms in Southern Africa.
To determine the phenolic compositions of the crude extract.
To evaluate the effects of some selected medicinal plant species against pathogenic microbes known to
induce diarrhoea.
To determine the antioxidative properties of the selected plants using various standard protocols.
To determine the anti-inflammatory potentials of the selected plants using various standard protocols.
To evaluate the toxicity risk of the crude extracts.
To determine the intestinal motility modulatory effects of the most promising extracts on the contractility
process of the isolated rat ileum induced by spasmogens and ion channels activators.
To isolate and characterize the component(s) that exhibit antimicrobial and antioxidant properties from
the most promising extracts.
The achieved objectives of the study are outlined as follows:
10.1. Identification of diarrhoeal pathogenesis and medicinal plants used as therapeutic agents
139
The data generated from the literature work indicate that diarrhoea has a number of pathogenesis such as
microbial infection, chronic inflammation, oxidative injury to intestinal mucosal lining, and deranged intestinal
motility. The mechanisms involved include one or combination of ionic and water secretion into the lumen and
reduced absorption of fluid from the intestine. The compendium of the medicinal plants used as antidiarrhoeal
agents (254 species) in Southern Africa also revealed the diarrhoeal challenges and the wide acceptability of
medicinal for cure.
10.2. Antimicrobial evaluation of the extracts against infectious pathogens
The results obtained by the antimicrobial screening indicate the presence of many compounds with potent
antibacterial and antifungal activity against the standard strains of microbes responsible for infectious diarrhoea
and other important infectious diseases in humans and animals. The significant inhibitory activity exhibited by the
water fraction of the C. vendae against S. aureus can be considered as important for the traditional use of this
plant, where water is the main extractant available. Generally, the results revealed that the antimicrobial potential
of the extracts are potentiated in the hexane and cdichloromethane fractions. Future investigation of the potent
extracts and fractions against resistant and virulent pathogens might indicate new mechanisms for the growth
inhibition of the microorganisms.
10.3. Antioxidant evaluation of the extracts
The crude extracts and the polar fractions of ethyl acetate and butanol had significant radical scavenging activity,
through hydrogen and proton donation mechanisms. These activities are ascribed to the presence of large
quantity of phenolics. In view of the oxidative stress in the pathogenesis of diarrhoea through tissue injury by
ROS/RNS involvement in lipid peroxidation, exacerbation of inflammatory processes and some of the reactive
species serving as secretagogues, the strong antioxidant activity could indicate the presence of compounds with
potentially important mechanisms of pharmacological relevance in reducing the deleterious effects of the
oxidative species in diarrhoea. In vitro results however, cannot be literally translated into in vivo situation due the
problem of bioavailability, absorption and possible metabolic transformations of the bioactive compounds in the
intestine. Further research are needed to verify using other models with different mechanisms against substrates
which are generated in human or animal cells as well as in vivo studies to evaluate their efficacy and safety.
10.4. Anti-inflammatory potential of the extracts
Inflammation is regarded as the hallmark of many diseases aetiology and the siginificant mediators are
eicosanoids (prostaglandins, prostacyclin) from cyclooxygenase (COX) and leukotrienes from lipoxygenase
(LOX) pathways. These two enzymes are the target for modulating the inflammatory process. The result obtained
indicated that the polar extracts of Bauhinia, Carissa, and Syzygium species used for COX inhibitory assay were
140
active against COX-1 with no activity against COX-2 while the Combretum species were inactive against both
enzymes. COX-1 selective inhibitors are considered to cause GIT injury while selective COX-2 inhibitors are
more beneficial against inflammatory processes, therefore these plant polar extracts should be used with caution
because possible intestinal injury. Most of the plant extracts however, had good LOX inhibitory activity. Current
research on anti-inflammatory agent focus on dual activity as COX and LOX inhibitors since both pathways uses
the same substrate. If one pathway is closed down, more substrate will be available to the other unperturbed
pathway, thus increasing its products and consequently promoting some other inflammatory mechanisms.
Additional work is required to determine the fraction(s) in which the active component is present and the
probable mechanisms of action. Since the polar extract are not active against COX-2, the non-polar extracts or
fractions still have to be tested. The plant extracts also have to be tested against other inflammatory biomarkers
and mediators including an in vivo studies using laboratory animal model.
10.5. Toxicity risk of the extracts
The toxicity risk assessment using MTT assay (Mosmann, 1983) using Vero African green monkey kidney cell
lines indicated that the extracts of Combretum species except C. bracteosum were highly toxic. The other
extracts have varying degree of toxicity with Ozoroa mucronata being the least toxic. These results are important
because they show that there are risks of toxicity with an inappropriate use of some of these extracts as
therapeutics for any ailments except perhaps cancer. The toxicity of medicinal plants depends on many factors
such as the plant part used, and the solvents used as extractant which are determinant of the compositional
characteristics and biological activity of extracts. Most of the highly toxic extracts also contain a high quantity of
hydrolysable tannin. Poor handling of raw or processed materials may produce exogenous toxic contaminants
not inherent as plant phytochemicals. It should be kept in mind that the results of cytotoxicity testing may vary
considerably depending on the cell type used, the initial cell density to which the extracts are exposed, and the
duration of exposure. Vero cells were selected as these are readily available and are commonly used in
cytotoxicity tests. In this study, a low cell density was used and the cells were exposed for a long time, 5 days
(McGaw et al., 2007). Hence, relatively low LC50 values were obtained and differences in cytotoxicity between
the extracts were maximised. Further work is needed to test the extracts, fractions and subfractions against other
cell lines and, also to conduct acute and chronic toxicity assays with a view of determining the toxic constituents
present in the plants.
It is a pity that one of the two plant species selected for in depth work C. vendae had a high cellular toxicity. In
future studies toxicity of extracts should be investigated at an early stage.
10.6 Motility modulatory effects of Bauhinia galpinii and Combretum vendae
Considering the wide ethnopharmacological use of B. galpinii and C. vendae against GIT disorders and their
excellent activity in some of the preliminary screening, the two plants were chosen for motility modulatory assays
despite the toxicity potential of C. vendae. This was with the view that the toxic component(s) will be determined
141
and separated from the other active components. The data generated by the study indicate that B. galpinii has a
dual-mechanism of action (prokinetic and relaxant) on gastro-intestinal motility while C. vendae extracts exhibited
spasmolytic (relaxant) effects on isolated rat ileum through multiple mechanisms. These results were important
as they indicate that B. galpinii extract can clinically be relevant as therapeutic agent in diarrhoea and
constipation which are both diseases with aetiology based on motility disturbances to a large extent while the
presence of multiple acting spasmolytic activities in the C. vendae extract might be contributing towards its
effectiveness in diarrhoea and abdominal spasm therapy. Further work are needed for the identification of the
specific ENS receptors through which these extract acts as well as the phytochemical compounds responsible for
their activities.
10.7. Isolation and characterisation of antimicrobial compounds
Bioassay-guided protocols for antibacterial and antioxidant activity were adopted for the identification and
isolation of 14 compounds (8 from C. vendae and 6 from B. galpinii) using open column chromatography with
silica gel and Sephadex LH 20 as stationary phases. However, some of the compounds are mixtures of position
isomers which are extremely difficult to separate. The compounds were characterised as ursol-12-en-28-oic acid
(ursolic acid), a mixture of corosolic acid and maslinic acid, and a mixture of asiatic acid and arjunolic acid, two
stilbenoid glycosides (combretastatin B5-O-2’-β-D-glucopyranoside and combretastatin B1-O-2’-β-Dglucopyranoside) and one flavone (apigenin) from the Combretum vendae.
One phytosterol (β-3 ethoxy sitosterol), one new flavone (5, 7, 4’ 5’ tetrahydroxy-2’-methoxyflavone (isoetin 2’methyl ether) or 5, 7, 2’ 5’ tetrahydroxy-4’-methoxyflavone (isoetin 4’-methyl ether)), two known flavonols (3, 5, 7,
3’, 4’-pentahydroxyflavone (Quercetin) and 3, 5, 7, 3’, 4’, 5’-hexahydroxyflavone (myricetin)) and their galactoside
derivatives (quercetin-3-O-β-galactopyranoside and myricetin-3-O-β-galactopyranoside) were isolated from
Bauhinia galpinii.
The results from this study indicate that medicinal plants used in ethnopharmacology are reservoirs of bioactive
compounds. Some of the medicinal plants may serve as potential sources of novel active compounds or lead
molecule for synthesis of more potent drugs. There is also a distinct possibility of developing plants extracts that
could be used by poor rural people or sophisticated herbal medicines from some of the species investigated in
this study. The information gained from this work provides a baseline study for other scientist to explore other
medicinal plant species in depth with possible commercial application.
142
References
Abbas, A. K., Lichtman, A. H., 2003. Cellular and Molecular Immunology. Elsevier, Amsterdam.
Abdel-Hameed, E. S. S., 2009. Total phenolic contents and free radical scavenging activity of certain Egyptian Ficus species
leaf samples. Food Chemistry 114 (4): 1271-1277.
Abdur Rahman, M, A., Moon, S-S., 2007. Isoetin 5’-Methyl Ether, A Cytotoxic Flavone from Trichosanthes kirilowii. Bulletin
of Korean Chemical Society 28 (8), 1261-1264.
Abo K. A. Ashidi J. S., 1999. Antimicrobial screening of Bridelia micrantha, Alchormea cordifolia and Boerhavia diffusa.
African Journal of Medicine & Medical Sciences 28(3-4), 167-169.
Abrosca, B. D., Fiorentino, A., Monaco, P., Oriano, P. Pacifico, S., 2006. Annurcoic acid: A new antioxidant ursane
triterpene from fruits of cv. Annurca apple. Food Chemistry 98, 285–290.
Acharya, S., Datra, A., Bag, P. K., 2009. Evaluation of the antimicrobial activity of some medicinal plants against enteric
bacteria with particular reference to Multi-Drug Resistant Vibrio cholerae. Tropical Journal of Pharmaceutical
Research 8, 231-237.
Adedapo, A. A., Sofidiya, M. O., Masika, P. J., Afolayan, A. J., 2008. Anti-Inflammatory and Analgesic Activities of the
Aqueous Extract ofAcacia karroo Stem Bark in Experimental Animals. Basic & Clinical Pharmacology & Toxicology
103, 397–400.
Adedapo, A. A., Sofidiya, M. O., Masika, P. J., Afolayan, A. J., 2008. Safety evaluations of the aqueous extract of Acacia
karroo stem bark in rats and mice. Scientific Commons
Aderibigbe, A.O., Emudianughe, T.S., Lowal, B.A., 2001. Evaluation of antidiabetic action of Mangifera indica in mice.
Phytotherapy Research 15, 456–458.
Aderogba, M. A., McGaw, L. J., Ogundaini, A. O., Eloff, J. N., 2007. Antioxidant activity and cytotoxicity study of the flavonol
glycosides from Bauhinia galpinii. Natural Product Research 21, 591-599.
Adewusi, E. A., Afolayan, A. J., 2009a. Antibacterial, antifugal and antioxidant activity of the root and leaves of Pelargonium
reniforme Curtis (Geraniaceae). African Journal of Biotechnology, 8(22), 6425-6433.
Adewusi, E. A., Afolayan, A. J., 2009b. Safety evaluation of the extract from the roots of Pelargonium reniforme Curtis in
male Wistar rats. African Journal of Pharmacy and Pharmacology 3(8), 368-373.
Adzu, B., Abubakar, M. S., Izebe, K. S., Akumka, D. D., Gamaniel, K. S., 2005. Effect of Annona senegalensis rootbark
extracts on Naja migricotlis nigricotlis venom in rats. Journal of Ethnopharmacology 96, 507–513.
Afolayan, A. J., Yakubu, M. T., 2009. Effect of Bulbine natalensis Baker stem extract on the functional indices and histology
of the liver and kidney of male Wistar rats. Journal of Medicinal Food. 12(4): (in press), DOI:10.1089/jmf.2008.0221.
Agnihotri, V. K., Elsohly, H. N., Smillie, T. J., Khan, I. A., Walker, L. A., 2009. Constituents of Leonitis leonurus flowering
tops. Phytochemistry letters 2:103-105.
Aguirre-Crespo, F., Vergara-Galicia, J., Villalobos-Molina, R., L´opez-Guerrero, J. J., Navarrete-V´azquez, G., Estrada, S.,
2006. Ursolic acid mediates the vasorelaxant activity of Lepechinia caulescens via NO release in isolated rat
thoracic aorta. Life Science 79, 1062–1068.
Ahmed, A. S., Igwe, C. C., Eloff, J. N., 2009. Preliminary studies of the antibacterial activities of Combretum vendae leave
extract. African Journal of Traditional Medicine 366-367.
Akah, P. A., 1996. Antidiarrhoeal activity of Kigalia africana in Experimental animal.Journal of Herbs and Spices Medicinal
plants 4, 31-38.
143
Akah, P. A., Nwafor, S. V., 1999. Studies on anti-ulcer properties of Cissampelos mucronata leaf extract. Indian Journal of
Experimental Biology 37 (9), 936 –938.
Akula, U. S., Odhav, B., 2008. In Vitro 5-lpiooxygenase inhibition of polyphenolic antioxidant from undomesticated plants of
South Africa. Journal of Medical plant Research 2(9), 207-212.
Alam, M. S., Chopra, N., Ali, M., Niwa, M., Sakae, T., 1994. Ursane and sterol derivatives from Pluchea lanceolata.
Phytochemistry, 37, 521–524.
Albuquerque, U. P., Hanazaki, N., 2006. As pesquisas etnodirigidas na descoberta de novos farmacos de interesse medico
e farmaceutico: fragilidades e pespectivas. Brazilian Journal of Pharmacognosy 16, 678–689.
Al-Khatib, M. S., Khyami-Horani, H., Badran, E., Shehabi, A. A., 2007. Incidence and characterization of diarrheal
enterotoxins of fecal Bacillus cereus isolates associated with diarrhea. Diagnostic Microbiology and Infectious
Disease 59, 383–387.
Almeida, C. E., Karnikovski, M. G., Foleto, R., Baldisserotto, B., 1995. Analysis of antidiarrheic effect of plants used in
popular medicine. Rev. Saude Publica 29,428-433.
Amabeoku, G. J., 2009. Antidiarrhoeal activity of Geranium incanum Burm. f. (Geraniaceae) leaf aqueous extract in mice.
Journal of Ethnopharmacology 123,190–193.
Amabeoku, G. J., Bamuamba, K., 2010. Evaluation of the effects of Olea europaea L.subsp. africana (Mill.) P. S. Green
(Oleaceae) leaf methanol extract aginst Castor oil-induced diarrhoea in mice. Journal of Pharmcy and Pharmacology
62(3), 368-373.
Amara, A. A., El-Masry, M. H., Bogdady, H. H., 2008. Plant crude extracts could be the solution: Extracts showing in vivo
antitumorigenic activity. PakistanJouranl of Pharmaceutical Sciences, 21(2), 159-171.
Amusan, O. O. G., Sukati, N. A., Dlamini, P. S., and Sibandz, F. G., 2007. Some Swazi phytomedicines and their
constituents. African Journal of Biotechnology 6(3), 267-272.
Angeh, J. E., Huang, X., Sattler, I., Swan, G. E., Dahse, H., Hartl, A., Eloff, J. N., 2007. Antimicrobial and anti-inflammatory
activity of four known and one new triterpenoid from Combretum imberbe (Combretaceae). Journal of
Ethnopharmacology 110, 56–60.
Angeh, J. E., Huang, X., Swan, G. E., Mollman, U., Sattler, I., Eloff, J. N., 2007. Novel antibacterial triterpenoin from
Combretum padoides (Combretaceae). ARKICOC (ix) 113-120.
Annese, V., Janssens, J., Vantrappen, G. et al., 1992. Erythromycin accelerates gastric emptying by inducing antral
contractions and improved gastroduodenal coordination. Gastroenterology 102, 823–828.
Appidi, J. R., Grierson, D. S., Afolayan, A. J., 2008. Ethnobotanical study of plants used for the treatment of diarrhoea in the
Eastern Cape, South Africa. Pakistan Journal of Biological Sciences 11, 1961–1963.
Arnold, H-J., Gulumian, M., 1984. Pharmacopoeia of traditional medicinein Venda. Journal of Ethnopharmacology 12, 35–
74.
Arts, I. C. W., Hollman, P. C. H., 2005. Polyphenols and disease risk in epidemiologic studies. American Journal of Clinical
Nutrition, 81(1), 317S–325S.
Aruoma, O. I., Colognato, R., Fontana, I., Gartlon, J., Migliore, L., Koike, K., Coecke, S., Lamy, E., Mersch-Sundermann, V.,
Laurenza, I., Benzi, L., Yoshino, F., Kobayashi, K., Lee, M. C., 2006. Molecular effects of fermented papaya
preparation on oxidative damage, MAP kinase activation and modulation of the benzo[a]pyrene mediated
genotoxicity. Biofactors 26, 147–159.
144
Asase, A. A., Oteng-Yeboah, G. T., Odamtten Simmonds, M. S., 2005. Ethnobotanical study of some Ghanaian anti-malarial
plants. Journal of Ethnopharmacology 99, 1221-1229
Asres, K., Bucar, F., Knauder, E., Yardley, V., Hendrick, H., Croft, S. L., 2001. In vitro antiprotozoal activity of extract and
compound from the stem bark of Combretum molle. Phytotherapy Research 15(7), 613-617.
Athanasiadou, S., Houdijk, J., Kyriazakis, I., 2008. Exploiting synergisms and interactions in the nutritional approaches to
parasite control in sheep production systems. Small Ruminant.Research 76, 2–11.
Atta, A. H., Mouneir, S. M., 2005. Evaluation of some medicinal plant extracts for antidiarrhoeal activity. Phytotherapy
Research 19(6), 481–485.
Auroma, O. I., Halliwell, B., Laughton, M. J., Quinlan, G. J., Gutteridge, J. M. C., 1989. The mechanism of initiation of lipid
peroxidation. Evidence against a requirement for an iron(II)-iron(III) complex. Biochemistry Journal 258:617– 620.
Aworet-Samseny, R. R., Souza, A., Kpahé, F., Konaté, K., Datté, J. Y., 2011. Dichrostachys cinerea (L) Wight et Arn.
(Mimosaceae) hydro-alcoholic extract action on the contractility of tracheal smooth muscle isolated from guinea-pig.
BMC Complementary and Alternative Medicine 11, 23.
Ayinde, B. A., Owolabi, O. J., 2009. Effect of the aqueous extract of Ficus capensis Thunb (Moraceae) leaf on
gastrointestinal motility. Journal of Pharmacognosy and Phytotherapy 1(3), 031-035.
Babajide, O. J., Mabusela, W. T., Green, I. R., Ameer, F., Weitz, F., Iwouha, E. I., 2010. Phytochemical screening and
biological activity studies of five South African indigenous medicinal plants. Journal of Medicinal Plant Research
2(18), 1924-1932.
Baker, J. T., Borris, R. P., Carte, B., Cardell, G. A., Soejarto, D. D., Gragg, G. M., Gupta, M. P., Iwu, M. M., Madilid, D. R.,
and Tyler, V. E., 1995: . Natural product drug discovery and development: New perspectives on international
collaboration. Journal of Natural products 58(9), 1325-1357.
Baldi, F., Bianco, M. A., Nardone, G., Pilotto, A., Zamparo, E., 2009. Focus on acute diarrhoeal disease. World Journal of
Gastroenterology 15(27), 3341-3348.
Bamuamba, K., Gammon, D. W., Meyers, P., Dijoux-Franca, M.-G., Scott, G., 2008. Anti-mycobacterial activity of five plant
species used as traditional medicines in the Western Cape Province (South Africa). Journal of Ethnopharmacology
117, 385–390.
Bandaranayake, W. M., 2006. Quality control, Screening Toxicity and Regulation of Herbal Drugs. In: Modern
phytomedicine. Turning medicinal plants into drugs (Edited by Ahmed F Aqil and Owais M) WILEY-VCH Verlag
GmbH and Co KGaA, Weinheim Pp 25-53.
Barbosa, E., Calzada, F., Campos, R., 2007. In vivo antigiardial activity of three flavonoids isolated of some medicinal plants
used in Mexican traditional medicine for the treatment of diarrhea. Journal of Ethnopharmacology 109, 552–554.
Barbour, E. K., Sharif, M. A., Sagherian, V. K., Habre, A. N., Talhouk, R. S., Talhouk, S. N., 2004. Screening of selected
indigenous plants of Lebaonon for antimicrobial activity. Journal of Ethnopharmacology 93, 1-7.
Barton, M. D., 2000. Antibiotic use in animal feed and its impact on human health. Nutritional Research Review 13, 279-299.
Batawila, K., Kokou, K., Koumaglo, K., Gbeassor, M., de Foucault, B., Bouchet, Ph. Akpagana, K., 2005. Antifungal
activities of five Combretaceae used in Togolese traditional medicine. Fitoterapia 76(2), 264-268.
Beaugerie L, Flahault A, Barbut F et al. Antibiotic-associated diarrhoea and Clostridium difficile in the community. Alimentary
Pharmacology and Therapeutics 2003; 17: 905–912
Beaugerie, L., Pettit, J-C., 2004. Antibiotic-associated diarrhoea. Best Practice & Research Clinical Gastroenterology 18,
337–352.
145
Beavis, K. G., Weymouth, L. A., 1996. Cellular and Molecular pathology of Infectious diseases. In: Cellula and Molecular
Pathogenesis. Editor, Alphonse E. Sirica: Lippincott-Raven Publishers, 277 East Washington Square,
Philadelphia, Pennsylvania 19106-3780 Pp 219-244.
Berker, K. I., Guclu, K., Tor, I., Apak, R., 2007. Comparative evaluation of Fe(III) reducing power-based antioxidant capacity
assays in the presence of phenathroline, batho-phenanthtroline, tripyridyltriazine (FRAP) and ferricyanide reagent.
Talanta 72, 1157-1165.
Berlin, E.A., Berlin, B., 2005. Some field methods in medical ethnobiology. Field Methods 17, 235–268.
Bessong, P. O., Obi, C. L., Andreola, M. L., Rojas, L. B., Pouysegu, L., Igumbor, E., Marion Meyer, J. J., Quideau, S.,
Litvak, S., 2005. Evaluation of selected South African medicinal plants for inhibitory properties against human
immunodeficiency virus type 1 reverse transcriptase and integrase. Journal of Ethnopharmacology 99, 83–91.
Bhan, M. K., 2000. Current and Future: Management of childhood diarrhoea. International Journal of Antimicrobial Agents
14, 71-73.
Bhargav, B., Rupal, A. V., Reddy, A. S., Narasimhacharya, A. V. R. L., 2009. Antihyperglycaemic and hypolipidemic effect of
Adansonia digitata L. On Alloxan induced diabetic rtas. Journal of Cells and Tissue Research 9(2), 1879-1882.
Bhat, R. B., Jacobs, T. V., 1995. Traditional herbal medicine in Transkei. Journal of Ethnopharmacology, 48, 7-12.
Bienvenu, E., Amabeoku, G. J., Eagles, P. K., Scott, G., Springfield, E. P., 2002. Anticonvulsant activity of aqueous extract
of Leonotis leonurus. Phytomedicine 9, 217–223.
Bisi-Johnson, M. A., Obi, C. L., Kambizi, L., Nkomo, M., 2010. A survey of indigenous herbal diarrhoeal remedies of O.R.
Tambo district, Eastern Cape Province, South Africa. African Journal of Biotechnology 9(8), 1245-1254.
Bizimenyera, E. S., Githiori, J. B., Eloff, J. N.,Swan, G. E., 2006. In vitro activity of Peltophorum africanum (Fabaceae)
extracts on the egg hatching and larval development of the parasitic nematode Trichostrongylus colubriformis.
Veterinary Parasitology 142, 336-343.
Blikslager, A. T., 2010. Mucosal epithelial barrier repair to maintain pig health. Livestock Science 133, 194–199.
Bohn, L. M., Raehal, K. M., 2006. Opioid receptors signalling: relevance for gastrointestinal therapy. Current Opinion in
Pharmacology 6, 559-563.
Borghetti, P., Saleri, R., Mocchegiani, E., Corradi, A., Martelli, P., 2009. Infection, Immunity and the Neuroendocrine
Response. Veterinary Immunology and Immunopathology 130, 141–162.
Borrelli, F., Capasso, F., Capasso, R., Ascione, V., Aviello, G., Longo, R., Izzo, A. A., 2006. Effects of Boswellia serrata on
intestinal motility in rodents: inhibition of diarrhoea without constipation. British Journal of Pharmacology 148, 553560
Botes, L., Van der Westhuizen, F. H., Loot du, T., 2008. Phytochemical and antioxidant capacity of two Aloe greatheadii var.
davyana extract. Molecules 13(9), 2169-2180.
Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity.
Lebensmittel Wissenschaftund Technologie 28, 25-30.
Brendler, T., Van Wyk, B. E., 2008. A historical, scientific and commercial perspective on the medicinal use of Pelargonium
sidioides (Geraniaceae). Review. Journal of Ethnopharmacology 119, 420–433.
Breytenbach, J. C., Malan, S. F., 1989. Pharmacological properties of Combretum zeyheri. South African Journal of
Sciences 85(6), 372-374.
146
Brijesh, S., Daswani, P. G., Tetali, P., Rojatkar, S. R., Antia, N. H., Birdi, T. J., 2006. Studies on Pongania pinnata (L) Pierre
leaves: Understanding the mechanism(s) of action in infectious diarrhea. Journal of Zhegiang University SCIENCE B
7(8), 665-674.
Brown, J.H., Taylor, P., 1996. Muscarinic receptor agonists and antagonists. In: Gilman, A.G., Hardman, J.G., Limbird, L.E.,
Molinoff, P.B., Ruddon, R.W. (Eds.), Goodman & Gillman’s: The Pharmacological Basis of Therapeutics. McGrawHill, New York, pp. 141–159.
Buettner, G.R., 1993. The pecking order of free radicals and antioxidants lipid peroxidation alpha-tocopherol and ascorbate,
Archive Biochemisstry Biophysics 300, 535–543.
Buret, A. G. 2007. Mechanisms of epithelial dysfunction in giardiasis. GUT 56(3), 328–335.
Butler, K. M., 2008. Enterococcal Infection in Children. Seminars in Pediatric Infectious diseases 17, 128-139
Buwa, L. V., Van Staden, J., 2006. Antibacterial and Antifungal activity of traditional medicinal plants used against venereal
diseases in South Africa. Journal of Ethnopharmacology 103(1), 139-142.
Cai, Y. Z., Sun, M., and Corke, H., 2003. Antioxidant activity of betalains from plants of the Amaranthaceae. Journal of
Agricultural and Food chemistry 51(8), 2288-2294.
Calis, I., Yürüker, A., Tas¸ demir, D., Wright, A.D., Sticher, O., Luo, Y.D., Pezzuto, J. M., 1997. Cycloartane triterpene
glycosides from the roots of Astragalus melanophrurius. Planta Medica 63, 183–186.
Calzada, F., Yépez Mulia, L., Aguilar, A., 2006. In vitro susceptibility of Entamoeba histolytica and Giardia lamblia to plants
used in Mexican traditional medicine for the treatment of gastrointestinal disorders. Journal of Ethnopharmacology
108, 367–370.
Caparroz-Assef, S. M., Grespan, R., Batista, R. C. F., Bersani-Amado, F. A., Baroni, S., Dantas, J. A., Cuman, R. K. N.,
Bersani-Amado, C.A., 2005. Toxicity studies of Cordia salicifolia extract. Acta Scientiarum Health Science 27, 41–
44.
Casburn-Jones, A. C., and Farthing, M. J. D., 2004. Management of infectious diarrhoea. GUT 53, 296-305.
Castillo, N. A., de LeBlanc, A. M., Galdeano, C. M., Perdigón, G., 2011. Probiotics: An alternative strategy for combating
salmonellosis Immune mechanisms involved. Food Research International doi:10.1016/j.foodres.2011.04.031
Catalá, A., 2006. An overviewof lipid peroxidation with emphasis in outer segments of photoreceptors and the
chemiluminescence assay. Int. J. Biochem. Cell Biol. 38, 1482–1495, Review.
Catala, A., 2009. Lipid peroxidation of membrane phospholipids generates hydroxy-alkenals and oxidized phospholipids
active in physiological and/or pathological conditions. Chemistry and Physics of Lipid 157: 1-11.
Catnach, S. M., Fairclough, P. D., 1992. Erythromycin and the gut. Gut 33, 397–401.
Cellek, S., John, A. K., Thangiah, R., Dass, N. B., Bassil, A. K., Jarvie, E.M., Lalude, O., Vivekanandan, S., Sanger, G. J.,
2006. 5-HT4 receptor agonists enhance both cholinergic and nitrergic activities in human isolated colon circular
muscle. Neurogastroenterology and Motility 18, 853–861.
Ceylan, E., Fung, D. Y. C., 2004. Antimicrobial activity of spices. Journal of RapidMethods and Automation in Microbiology,
12(1), 1–55.
Chacin-Bonilla, L., 2010. Epidemiology of Cyclospora cayetanensis: A review focusing in endemic areas. Acta Tropica 115,
181–193.
Chang, S-L., Chiang, Y-M., Chang, C. L-T., Yeh, H-H., Shyur, L-F., Kou, Y-H., Wu, T-K., Yang, W-C., 2007. Flavonoid,
Centaurein and Centaurenidin, from Bidens pilosa stimulate IFN-γ expression. Journal of Ethnopharmacology, 112,
232-236.
147
Charlier, C., Michaux, C., 2003. Dual inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) as a new strategy
to provide safer non-steroidal anti-inflammatory drugs. European Journal of Medicinal Chemistry 38, 645-659.
Chaturongakul, S., Raengpradub, S., Wiedman, M., Boor, K. J., 2008. Modulation of stress and virulence in Listeria
monocytogenes. Trend in Microbiology 16, 388-396.
Chen, J-C., Chang, Y-S., Wu, S-L., Chao, B-C., Chang, C-S., Li, C-C., Ho, T-Y., Hsiang, C-Y., 2007. Inhibition of Escherichi
coli heat labile enterotoxins induced diarrhoea by Chaenomeles speciosa. Journal of Ethnopharmacology 113, 233239.
Chen, J-C., Ho, T-Y., Chang, Y-S., Wu, S-L., Li, C-C., Hsiang, C-Y., 2009. Identification of Escherichia coli enterotoxin
inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses. Journal of Ethnopharmacology
121, 372–378.
Chen, K., Suh, J., Carr, A. C., Morrow, J. D., Zeind, J., Frei, B., 2000. Vitamin C suppresses oxidative lipid damage in vivo,
even in the presence of iron overload. American Journal of Physiology, Endocrinology and Metabolism 279,
E1406–E1412.
Chen, X-W., Serag, E. S., Sneed, K. B., Zhou, S-F., 2011. Herbal bioactivation, molecular targets and the toxicity relevance.
Chemico-Biological Interactions 192, 161–176.
Chetty, N., Irving, H. R., Coupar, I. M., 2006. Activation of 5-HT3 receptors in the rat and mouse intestinal tract: a
comparative study. British Journal of Pharmacology 148, 1012–1021.
Chi, J., VanLeeuwen, J. A., Weersink, A., Keefe, G. P., 2002. Direct production losses and treatment costs from bovine viral
diarrhoea virus, bovine leukosis virus, Mycobacterium avium subspecies paratuberculosis, and neospora caninum.
Preventive veterinary medicine 55, 137-153.
Chiang, Y-M., Chang, C, L-T., Yang, S-L., Shyur, L-F., 2007. Cytopiloyne, a novel polyacetylenic glycoside from Biden
pilosa, function as a T-helper cell modulator. Journal of Ethnopharmacology 110(3), 532-538.
Chinsamy, M., Finnie, J. F., Van Staden, J., 2010. The ethnobotany of South African medicinal orchids. South African
Journal of Botany
Choma, I. M., Grzelak, E. M., 2011. Bioautography detection in thin-layer chromatography. Journal of Chromatography A
1218, 2684–2691.
Christophersen, O. A., Haug, A., 2005. Possible roles of oxidative stress, local circulatory failure and nutrition factors in the
pathogenesis of hypervirulent influenza: Implications for therapy and global emergency preparedness. Microbial
Ecology in Health and Disease 17, 189-199.
Cimanga, R. K., Kambu, K., Tona, L., Hermans, N., Apers, S., Totte, J., Pieters, L., Vlietinck, A. J., 2006. Cytotoxicity and in
vitro susceptibility of Entamoeba histolytica to Morinda Morindoides leaf extract and its isolated constituents. Journal
of Ethnopharmacology 107, 83-90.
Cimanga, R. K., Mukenyi, P. N. K., Kambu, O. K., Tona, G. L., Apers, S., Totté, J., Pieters, L., Vlietinck, A. J., 2010. The
spasmolytic activity of extracts and some isolated compounds from the leaves of Morinda morindoides (Baker)
Milne-Redh. (Rubiaceae). Journal of Ethnopharmacology 127, 215–220
Cimpoiu, C., 2006. Analysis of some natural antioxidants by Thin-Layer Chromatography and High performance Thin-layer
Chromatography. Journal of liquid chromatography and related Technologies, 29: , 1125-1142.
Clarke, S. C., 2001. Diarrhoeagenic Escherichia coli-an emerging problem? Diagnostic Microbiology and infectious disease
41, 93-98.
Coates-Palgrave, M., 2002. Keith Coates-Palgrave Trees of Southern Africa, 3rd edition, imp. Struik Publishers. Cape Town.
148
Conforti, F., Sosa, S., Marrelli, M., Menichini, F., Statti, G. A., Uzunov, B., Tubaro, A., Menichini, F., Loggia, R. D., 2008. In
vivo anti-inflammatory and in vitro antioxidant activities of Mediterranean dietary plants. Journal of
Ethnopharmacology 116, 144-151.
Coopoosamy, R. M., 2011. Traditional information and antibacterial activity of Four Bulbine species (Wolf). African Journal of
Biotechnology 10(2), 220-224.
Cos, P., Vanden Berghe, D.; De Bruyne, T.; Vlietinck, A.J. Curr. Org. Chem., 2003, 7, 1163.
Cotton, J. A., Beatty, J. F., Buret, A. G., 2011. Host parasite interactions and pathophysiology in Giardia infections.
International J.ournal for Parasitology xxx , xxx–xxx
Coutino, R. R., Hernandez, C. P., Giles, R. H., 2001. Lectins in fruits having gastrointestinal activity: their participation in the
hemagglutinating property of Escherichia coli O157:H7. Archives of Medical Research 32, 251–257.
Cowan, M. M., 1999. Plant products as antimicrobial agents. Clinical Microbiology Review 12, 564–582.
Cuello, S., Alberto, M. R., Zampini, I. C., Ordonez, R. M., Isla, M. I., 2011. Comparative study of antioxidant and antiinflammatory activities and genotoxicity of alcoholic and aqueous extracts of four Fabiana species that grow in
mountainous area of Argentina. Journal of Ethnopharmacology xxx , xxx– xxx
Cui, J., Wang, Z. Q., Xu, B. L., 2011. Review: The epidemiology of human tirchinellosis in China during 2004-2009. Acta
tropica 118, 1-5.
Cuzzocrea, S., Mazzon, E., Serraino, I., Dugo, L., Centorrino, T., Ciccolo, A., Sautebin, L., Caputi, A.P., 2001. Celecoxib, a
selective cyclo-oxygenase-2 inhibitor reduces the severity of experimental colitis induced by dinitrobenzene sulfonic
acid in rats. European Journal of Pharmacology. 431, 91–102.
da Silva, G., Tamia, M., Rocha, J., Serrona, R., Gomes, E. T., Sepodea, B., Silva, O., 2010. In vivo Anti-inflammatory effect
and. toxicological screening of Maytenus heterophylla and Maytenus senegalensis extracts. Human and
Experimental Toxicology 000(00) 1-8 DOI: 10: 1177/0960327110379242
Dambisya, Y. M., Tindimwebwa, G., 2003. Traditional remedies in children around Eastern Cape, South Africa. East African
Medical Journal 80, 401–405.
Danna, P. L., Urban, C., Bellin, E et al., 1991. Role of candida in pathogenesis of antibiotic-associated diarrhoea in elderly
patients. Lancet 337, 511–514.
de Boer, H. J., Kool, A., Broberg, A., Mziray, W. R., Hedberg, I., Levenfors, J. J., 2005. Antifungal and antibacterial activity
of some herbal remedies from Tanzania. Journal of Ethnopharmacology 96(3), 461-469.
de Oliveira, C. E. V., Stamford, T. L. M., Neto, N. J. G., de Souza, E. L., 2010. Inhibition of Staphylococcus aureus in broth
and meat broth using synergies of phenolics and organic acids. International Journal of Food Microbiology 137, 312316.
De Villiers, B. J., Van Vuuren, S. F., Van Zyl, Z. L., Van Wyk, B–E., 2010. Antimicrobial and antimalarial activity of Cussonia
species (Araliaceae). Journal of Ethnopharmacology 129: 189-196.
de Wet, H., Nkwanyana, M. N., van Vuuren, S. F., 2010. Medicinal plants used for the treatment of diarrhoea in northern
Maputaland, KwaZulu-Natal Province, South Africa. Journal of Ethnopharmacology 130. 284–289.
Deeni, Y. Y., Hussain, H. S. N., 1994. Screening of Vernonia kotschyana for antimicrobial activity and alkaloids. International
Journal of Pharmacognosy 32, 388–395.
Dekker, T. G., Fourie, T. G., Matthee, E., Snycker, F. O., Ammann, W., 1987. Studies of South African medicinal plants. Part
4: Jaherin, a new daphnane diterpene with antimicrobial properties from Jatropha zeyheri. South African Journal of
Chemistry, 40, 74–76
149
Delaporte, R. H., Sanchez, G. M., Cuellar, A. C., Giuliani, A., Palazzo de Mello, J. C., 2002. Anti-inflammatory activity and
lipid peroxidation inhibition of iridoid lamiide isolated from Bouchea fluminensis (vell.) Mold. (Verbenaceae). Journal
of Ethnopharmacology 82: 127-130.
Delves, P., Martin, S., Burton, D., Roitt, I., 2006. Roitt’s Essential Immunology, 11th ed. Wiley-Blackwell, Hoboken, NJ, USA.
Desire, O., Riviere, C., Razafindrazaka, R., Goosens, L., Moreau, S., Guillon, J., Uverg-Ratsimamanga, S., Andriamadio, P.,
Moore, N., Randriantsoa, A., Raharisololalao, A., 2010. Antispasmodic and antioxidant activities of fraction sand
bioactive constituent davidigenin isolated from Mascarenhasia arborescens. Journal of Ethnopharmacology 130,
320-328.
Deutschlander, M. S., Van de Venter, M., Louw, J., Lall, N., 2009. Hypoglycaemic activity of four plant extracts traditionally
used in South Africa for diabetes. Journal of Ethnopharmacology, 124(3), 619-624.
Dickman, K. G., Hempson, S. J., Anderson, J., Lippe, S., Zhao, L., Burakoff, R., et al. 2000. Rotavirus alters paracellular
permeability and energy metabolism in Caco-2 cells. American Journal of Physiology, Gastrointestinal and Liver
Physiology, 279(4), G757–G766.
Dobbins, W. O., Herrero, B. A., Mansbach, C. M., 1968. Morphologic alterations associated with neomycin induced
malabsorption. The American Journal of Medical Sciences 255, 63–77.
Dold, A. P., Cocks, M. L., 2001. Traditional veterinary medicine in the Alice district of the Eastern Cape Province, South
Africa. South African Journal of Science 97, 375-379.
Du Toit, K., Elgorashi, E. E., Sarel, F. M., Drewes, S. E., Van Staden, J., Crouch, N. R., Mulholland, D. A., 2005. Antiinflammatory activity and QSAR studies of compounds isolated from Hyacinthaceae and Tachiadenus longiflorus
Griseh (Gentianaceae). Bioorganic and medicinal chemistry 13, 2561-2568.
Dudeja, P. K., and Ramaswamy, K., 2006. Physiology of Gastrointestinal Tract, Fourth edition, Edited by Leonard R.
Johnson. Academic Press, Chicago USA.
Dupont, H. L., Marshall, G. D., 1995. HIV-associated diarrhoea and wasting. Lancet 346, 352-356.
Dweyer, D. J., Kohanski, M. A., Collins, J. J. 2009. Role of reactive oxygen species in antibiotic action and resistance.
Current Opinion in Microbiology 12, 482–489.
Ebert, E. C., 2005. Gastrointestinal Complications of Diabetes Mellitus. Dis Mon 51, 620-663.
Ehling-Schulz, M., Fricker, M., Scherer, S., 2004. Bacillus cereus, the causative agent of an emetic type of food-borne
illness. Molecular Nutrition and Food Research 48, 479–487.
Eldeen, I. M. S., Eldeen, I. M., Van Heerden, F. R., Van Staden, J., 2008. Isolation and Biological activities of Termilignan B
and arjunic acid from Terminalia sericea. Planta Medica 74(4), 411-413.
Eldeen, I. M. S., Elgorashi, E. E., Mulholland, B. A., Van Staden, J., 2006. Anolignan B: A bioactive compound from the
roots of Terminalia sericea. Journal of Ethnopharmacology 103(1), 135-138.
Eldeen, I. M. S., Elgorashi, E. E., Van Staden, J., 2005. Antibacterial, antiinflammatory, anti-cholinesterase and mutagenic
effects of extrats obtained from some trees used in South African traditional medicine. Journal of
Ethnopharmacology 102, 457-464
Eldeen, I. M. S., Van Heerden, F. R., Van Staden, J., 2007. Biological activities of cycloart-2, 3-ene-3, 25-diol isolated from
the leaves of Trichilia drageana South African Journal of botany 73(3), 366-371.
Eldeen, I. M. S., Van Heerden, F. R., Van Staden, J., 2010. In vitro biological activities of niloticane, a new bioactive
cassane diterpene from the bark of Acacia nilotica subsp. Kraussiana. Journal of Ethnopharmacology 128, 555–560.
150
Elgayyar, M., Droughon, F. A., Golden, D. A., Mount, J. R., 2001. Antimicrobial activity of essential oils from plants against
selected pathogenic and saprophytic microorganisms. Journal of Food Protection 64, 1019-1024.
Elgorashi, E. E., Taylor, J. L. S., Maes, A., van Van Staden, J., De Kimpe, N., Verschaeve, L., 2003. Screening of medicinal
plants used in South African traditional medicine for genotoxic effects. Toxicology Letters 143, 195–207.
Ellman, G. L., Coutney, D., Andies, V., Featherstone, R. M., 1961. A new and rapid colourimetric determination of
acetylcholinesterase activity. Biochemical Pharmacology 7, 88–95.
Eloff, J. N. 2001. Antibacterial activity of Marula (Sclerocarya birrea (A. Rich.) Hochst. subsp. caffra (Sond.) Kokwaro)
(Anacardiaceae) bark and leaves. Journal of Ethnopharmacology 76, 305-308.
Eloff, J. N., 1998. A sensitive and quick micro-plate method to determine the minimal inhibitory concentration of plant
extracts for bacteria. Planta Medica, 64: 711-713.
Eloff, J. N., Famakin, J. O., Katerere, D. R., 2005. Isolation of antibacterial stilbenes from Combretum woodii
(Combretaceae) leaves. African Journal of Biotechnology 4, 1166–1171.
Eloff, J. N., Katerere, D. R., McGaw, L. J., 2008. The Biological Activity and Chemistry of the Southern African
Combretaceae. Journal of Ethnopharmacology 119, 686–699.
Elsinghorst, E. A., 2002. Enterotoxigenic Escherichia coli, In Escherichia coli: Virulence Mechanisms of a Versatile
Pathogen. Elsevier Science (USA) Pp 155-187.
Epple, H. J., Schneider, T., Troeger, H., Kunkel, D., Allers, K., Moos, V., et al., 2009. Impairment of the intestinal barrier is
evident in untreated but absent in suppressively treated HIV-infected patients. Gut 58, 220–227.
Erde‘lyi, K., Kiss, A., Bakondi, E., Bai, P., Szabó, C., Gergely, P., Erdó di, F., Virá g, L., 2005.Gallotannin inhibits the
expression of chemokines and inflammatory cytokines in A549 Cells. Molecular Pharmacology 68, 895–904.
Ernst, E., 2004. Risks of herbal medicinal products. Pharmacoepidemiology and Drug Safety 13, 767–771.
Escandell, J. M., Recio, M. C., Giner, R. M., Mánez, S., Cerdá-Nicolás, M., Merfort, I., Ríos, J. L., 2010. Inhibition of
delayed-type hypersensitivity by cucurbitacin R through the curving of lymphocyte proliferation and cytokine
expression by means of NF-AT translocation to the nucleus. Journal of Pharmacology and Experimental
Therapeutics 332, 352–363.
Escandell, J. M., Recio, M. C., Mánez, S., Giner, R. M., Cerdá-Nicolás, M., Gil-Benso, R., Ríos, J. L., 2007.
Dihydrocucurbitacin B inhibits delayed-type hypersensitivity reaction by suppressing lymphocyte proliferation.
Journal of Pharmacology and Experimental Therapeutics 322, 1261–1268.
Essop, A. B., van Zyl, R. L., van Vureen, S. F., Mulholland, D and Vilijoen, A. M. 2008. The in vitro phamacological activities
of South African Hermania species. Journal of Ethnopharmacology 119, 615-619.
Esterbauer, H., Schaur, R.J., Zollner, H., 1991. Chemistry and biochemistry of 4- hydroxynonenal, malonaldehyde and
related aldehydes. Free Radical Biolology and Medicine 11, 81–128.
Estrada-soto, S., Rodriguez-Avilez, A., Castaneda-Avila, C., Castillo-Espana, P., Navarrete-Vazquez, G., Hernandez, L.,
and Aguirre-Crespo, F., 2007. Spasmolytic action of Lepechinia caulescens is through calcium blockage and NO
release. Journal of Ethnopharmacology 114:364-370.
Fabry, W., Okemo, P., Ansorg, R., 1996. Activity of East African medicinal plants against Helicobacter pylori. Chemotherapy
42, 315–317.
Fallman, M., Gustavsson, A., 2005. Cellular Mechanisms of Bacterial Internalization Counteracted by Yersinia. International
Review of Cytology 246, 135-188.
Farthing, M. J. G., 2002. Novel target for the control of secretory diarrhoea. Gut, 50(Suppl III):iii15–iii18
151
Farthing, M. J. G., Casburn-Jones, A., Banks, M. R., 2003. Getting control of intestinal secretion: thoughts for 2003.
Digestive and Liver Disease 35, 378–385.
Fawole, O. A., Finnie, J. F., Van Staden, J., 2009. Antimicrobial activity and mutagenic effects of twelve traditional medicinal
plants used to treat ailments related to the gastro-intestinal tract in South Africa. South African Journal of Botany 75,
356–362.
Fennell, A. W., Lindsey, K. L., McGaw, L. J., Sparg, S. G., Stafford, G. I., Elgorashi, E. E., Grace, O. M., van Staden, J.
2004. Assessing African medicinal plants for efficacy and safety: Pharmacological screening and toxicology. Journal
of Ethnopharmacology 95, 205-217.
Field, M., 2003. Intestinal ion transport and the pathophysiology of diarrhea. Journal of Clinical Investigation 111, 931–943.
Fiorucci, S., Meli, R., Bucci, M., Cirino, G., 2001. Dual inhibitors of cyclooxygenase and 5-lipoxygenase. A new avenue in
anti-inflammatory therapy? Biochemical Pharmacology 62, 1433–1438.
Fitzgerald, G. A., 2004. Coxibs and cardiovascular disease. New England Journal of Medicine 351, 1709−1711.
Forbes, V.S. (Ed.), 1986. Carl Peter Thunberg Travels at the Cape of Good Hope 1772–1775. Van Riebeeck Society, Cape
Town, ISBN: 0620109815.
Forgacs, I., Patel, V., 2011. Diabetes and gastrointestinal tract. Medicine 39 (5), 288-292.
Fouche, G., Cragg, G.M., Pillay, P., Kolesnikova, N., Maharaja, V.J., Senabe, J., 2008. In vitro anticancer screening of
South African plants. Journal of Ethnopharmacology 119, 455–461.
From, C., Hormazabal, V., Granum, P. E., 2007. Food poisoning associated with pumilacidin-producing Bacillus pumilus in
rice. International Journal of Food Microbiology 115, 319–324.
Gadewar, S., Fasano, A., 2005. Current concepts in the evaluation, diagnosis and management of acute infectious diarrhea.
Current Opinion in Pharmacology 5, 559–565.
Gaginella, S. T., Kachur. J. F., Tamai, H., Kershavarzian, A., 1995. Reactive oxygen and Nitrogen metabolites as mediators
of Secretory Diarrhea. Gastroenterology 109, 2019-2028.
Gale, G. A., Kirtikara, K., Pittayakhajounwut, P., Sivichai, S., Thebtaranonth, Y., Thongpanchang, C., Vichai, V., 2007. In
search of cyclooxygenase inhibitors, anti-Mycobacterium tuberculosis and anti-malarial drugs from Thai flora and
microbes. Pharmacology and Therapeutics 115, 307-351.
Galvez, J., Zarzuelo, A., Crespo, M. E., Lorente, M. D., Ocete, M. A., Jimenez, J., 1993. Antidiarrhoeic activity of Euphorbia
hirta extract and isolation of an active flavonoid constituent. Planta Medica 59(4), 333-336.
Gambhir, I. S., Nath, G., Jaiswal, J., Gopal, A., 2006. Candida Associated Acute Diarrhoea in Elderly. Journal of the Indian
Academy of Geriatrics 2, 57-60.
Gangoue-Pieboji, J., Baurin, S., Frere, J. M., Ngassam, P., Ngameni, B., Azebaze, A., Pegnyemb, D. E., Watchueng, J.,
Goffin, C., Galleni, M., 2007. Screening of some medicinal plants from cameroonCameroon for beta-lactamase
inhibitory activity Phytotherapy Research 21(3), 284-287.
Gautam, R., Saklani, A., Jachak, S., 2007. Indian medicinal plants as a source of antimycobacterial agents. Journal of
Ethnopharmacology 110, 200–234.
Gelberg, B. H., 2007. Pathology of organ systems: Alimentary system. In: Pathologic Basis of Veterinary Diseases. (Edited
by McGavin MD and Zachary JF) Mosby Elsevier 11830 West line Industrial Drive, St Louis, Mussolini 63146 Pp
301-391.
Geronikaki, A. A., Gavalas, A. M., 2006. Antioxidants and anti-inflammatory Diseases: synthetic and natural antioxidants
with anti-inflammatory activity. Combinatorial chemistry and High Throughput Screening 9, 425-442.
152
Gertsch, J., Viverose-Paredes, J. M., Taylor, P., 2010. Plant immunostimulants—Scientific paradigm or myth?. Journal of
Ethnopharmacology xxx, xxx–xxx.
Giess, W., Snyman, J. W., 1986. The naming and utilization of plant life by the Zu'hoasi Bushmen of the Kau-Kauveld. In:
Vossen, R., Keuthmann, K. (Eds.), Contemporary Studies on Khoisan I and II. In honour of Oswin Köhler on the
Occasion of His 75th Birthday. Buske, Hamburg, pp. 237–346.
Gilani, A. H., Bashir, S., Janbaz, K. H. and Khan, A., 2005a. Pharmacological basis for the use of Fumaria indica in
constipation and diarrhoea. Journal of Ethnopharmacology 96, 585–589.
Gilani, A. H., Bashir, S., Jambaz, K. H., Shah, A. J., 2005b. Presence of cholinergic and calcium channel blocking activities
explains the traditional use of Hibiscus rosasinensis in constipation and diarrhoea. Journal of Ethnopharmacology
102, 289–294.
Gilani, A. H., Bashir, S., Jambaz, K. H., Shah, A. J., 2005. Presence of cholinergic and calcium channel blocking activities
explains the traditional use of Hibiscus rosasinensis in constipation and diarrhoea. Journal of Ethnopharmacology
102, 289–294.
Gilani, A. H., Rahman, A., 2005. Trends in Ethnopharmacology. Journal of Ethnopharmacology, 100, 43–49.
Godfraind, T., Miller, R., Wibo, M., 1986. Calcium antagonism and calcium entry blockade. Pharmacological Reviews 38,
321–416.
Goncalves, J. L., Lopes, R. C., Oliviera, D. B., Costa, S. S., Miranda, M. M., Romanos, M. T., Santos, N. S., Wigg, M. D.,
2005. In vitro anti-rotavirus activity of some medicinal plants used in Brazil against diarrhea. Journal of
Ethnopharmacology 99, 403–407.
Gonzalez, A. G., Bazzocchi, I. L., Moujir, L., Jimenez, I. A., 2000. Ethnobotanical uses of Celastraceae: Bioactive
metabolites studies. Natural Products Chemistry 23, 649-738.
Gorowara, S., Sapru, S., Ganguly, N. K., 1998. Role of intracellular second messengers and ROS in the pathophysiology of
V. Cholerae 0139 treated rabbit ileum. Biochimica Biophysic Acta 1407, 21–30.
Gould, M., Sellin, J. H., 2009. Diabetic diarrhea. Current gastroenterology reports 11950, 354-359.
Granot, E., Kohen, R., 2004. Oxidative stress in childhood-in health and disease states. Clinical Nutrition 23, 3–11.
Granum, E. P., 2006. Bacterial toxins as food poisons: Clinical, immunological aspects and applications of Bacterial protein
toxins: The comprehensive sourcebook of Bacterial protein toxins (Ed. Alouf and Popoff) Pp. 949-958.
Greca, M. D., Manaco, P., Previtera, L., 1990. Stagmasterol from Typa latifolia. Journal of Natural Products, 53, 1430–1435.
Green, D. R., Reed, J. C., 1998. Mitochondria and apoptosis. Science 281, 1309– 1312.
Green, E., Samie, A., Obi, C. L., Bessong, P. O., Ndip, R. N., 2010. Inhibitory properties of selected South Africa medicinal
plants against Mycobacterium tuberculosis. Journal of Ethnopharmacology 130, 151-157.
Greenwood, D. (ed.) 1989. Antibiotic sensitivity testing. In: Antimicrobial Chemotherapy. Oxford University, Oxford, p.91100.
Griendling, K. K., 2005. ATVB in focus: redox mechanisms in blood vessels. Arterios Thrombotic and Vascular Biol. 25,
272–273.
Grierson, D. S., Afolayan, A. J., 1999. An ethnobotanical study of plants used for the treatment of wounds in the Eastern
Cape, South Africa. Journal of Ethnopharmacology 67, 327–332.
Groschwitz, K. R., Hogan, S. P., 2009. Intestinal barrier function: Molecular regulation and disease pathogenesis. Journal of
Allergy and Clinical Immunology 124, 3–20 quiz 21–22.
153
Grosser, T., Fries, S., FitzGerald, G. A., 2006. Biological basis for the cardiovascular consequences of COX-2 inhibition:
Therapeutic challenges and opportunities. Journal of Clinical Investigation 116, 4-15.
Guerrant, R. L., Oria, R., Bushen, O. Y., Patrick, P. D., Houpt, E., Lima, A. A. M., 2005. Global impact of diarrhoeal diseases
that are sampled by Traveller: The rest of the Hippopotamus. Clinical Infectious Diseases 4, 5524-5530.
Guerrant, R. L., Steiner, T. S., Lima, A. A. M., Bobak, D. A., 1999, How Intestinal Bacteria Cause Disease. The Journal of
Infectious Diseases 179(2), S331-S337.
Gupta, S., Ali, M., Alam, M. S., Niwa, M., Sakai, T., 1992. 2, 4 β-Ethylcholest-4-en-3β-ol from the roots of Lawsonia inermis.
Phytochemistry 31, 2558–2560.
Gupta, T. P., Ehrinpreis, M. N., 1990. Candida-associated diarrhea in hospitalized patients. Gastroenterology 98, 780–785.
Gutierrez, R. M. P., Mitchell, S., Solis, R. V., 2008. Psidium guajava: A review of its traditional uses, phytochemistry and
pharmacology. Journal of Ethnopharmacology 117, 1–27
Gutierrez, S. P., Sanchez, M. A. Z., Gonzalez, C. P., Garcia, L. A., 2007. Antidiarrhoeal activities of different plants used in
traditional medicine. African Journal of Biotechnology 6(25), 2988-2994.
Gutteridge, J.M.C., 1988. Lipid peroxidation: some problems and concepts, in: B. Halliwell, (Ed.), Oxygen Radicals and
Tissue Injury. FASEB, Bethesda, MD, pp. 9–19.
Guttman, J. A., Finlay, B. B., 2008. Subcellular alterations that lead to diarrhea during bacterial pathogenesis. Trends in
Microbiology 16(11), 535-542.
Guttman, J. A., Samji, F.N., Li, Y., Vogl, A. W., Finlay, B. B., 2006. Evidence that tight junctions are disrupted due to intimate
bacterial contact and not inflammation during attaching and effacing pathogen infection in vivo. Infections and
Immunology 74, 6075–6084.
Ha, D. T., Kim, H., Thuong, P. T., Ngoc, T. M., Lee, I., Hung, N. D., Bae, K., 2009. Antioxidant and lipoxygenase inhibitory
activity of oligostilbenes from the leaf and stem of Vitis amurensis. Journal of Ethnopharmacology 125, 304–309.
Ha, T. J., Kubo, I., 2005. Lipoxygenase Inhibitory Activity of Anacardic Acids. Journal of Agricultural and Food Chemistry
53(11), 4350-4354.
Haeggström, J. Z., Rinaldo-Matthis, A., Wheelock, C. E., Wetterholm, A., 2010. Advances in eicosanoid research, novel
therapeutic implications. Biochemical and Biophysical Research Communications 396, 135–139.
Halliwell, B., 1997. Antioxidants and human disease: a general introduction. Nutrition Reviews 55, 544-552.
Halliwell, B., 2008. Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies?
Archives of Biochemistry and Biophysics 476, 107–112.
Halliwell, B., Gutteridge, J. M., 1990. Role of free radicals and catalytic metal ions in human disease: an overview. Methods
in Enzymology. 186, 1–85.
Hamza, O. J. M., van den Bout-van den Beukel, C. J. P., Mates, M. I. N., Moshi, M. J., Mikx, F. H. M., Selemani, H. O.,
Mbwambo, Z. H., Van der Van, A. J. A. M., Verweij, P. E., 2006. Antifungal activity of some Tanzania plants used
traditionally for the treatment of fungal infection. Journal of Ethnopharmacology 108, 124-132.
Han, T., Li, H-L., Zhang, Q-Y., Han, P., Zheng, H-C., Rahman, K., Qin, L-P., 2007. Bioactivity guided fraction for
antinflammatory and analgesic properties and constituent of Xynthium strumarium .L. Phytomedicine, 14(12), 825829.
Havagiray, R., Ramesh, C., Sadhna, K., 2004. Study of antidiarrhoeal activity of Calotropis gigantea r.b.r. in experimental
animals. Journal of Pharmacology and Pharmaceutical Science 7, 70–75.
154
Havsteen B. H., 2002. The biochemistry and medical significance of the flavonoids. Pharmacology and Therapeutics 96, 67–
202 Sci. 57, 6–15.
Heimler, D., Vignolini, P., Dini, M. D., Vincieri, F. F., Romani, A., 2006. Antiradical activity and polyphenol composition of
local Brassicaceae edible varieties. Food Chemistry, 99, 464-469.
Henry-Stanley, M. J., Hess, D. J., Erickson, E. A., Garni, R. M., Wells, R. M., 2003. Effect of Lipopolysaccharide on
Virulence of Intestinal Candida albicans. Journal of Surgical Research 113, 42–49.
Hermon-Taylor, J., 2006. Gut pathogens: Invaders and turncoats in a complex cosmos. Gut pathogens 1:3
hppt:/www.gutpathogen.com/content/1/1/3
Hodges, K., Gill, R., 2010. Infectious diarrhea Cellular and molecular mechanisms. Gut Microbes 1, 4-21.
Hofmann, A. F., 1977. Bile acids, diarrhea, and antibiotics: data, speculation, and a unifying hypothesis. Journal Infectious
Diseases 135(supplement), S126–S132.
Holxzapfel, C., Van Wyk, B., Castro, A., Marias, W., Herbst, M., 1995. A Chemotaxonomic Survev of Kaurene Derivatives in
the Genus Alepidea (Apiaceae). Biochemikal .Qstematics and Ecology, Vol. 23, No. 718, pp. 799-803.
Holzer, P. 2004. Gastrointestinal afferents as targets of novel drugs for the treatment of functional bowel disorders and
visceral pain. European Journal of Pharmacology 429, 177–193.
Homans, A. L., Fuchs A., 1970. Direct biautography on thin layer chromatograms as a method for detecting fungitoxic
substances. Journal of Chromatography 51, 327–329.
Hoogerwerf, W. A., 2006. Prokineticin 1 inhibits spontaneous giant contractions in the murine proximal colon through nitric
oxide release. Neurogastroenterology and Motility 18, 455–463.
Hopkins, M. J., Macfarlane, G. T., 2003. Nondigestible oligosaccharides enhance bacterial colonization resistance against
Clostridium difficile in vitro. Applied and Environmental Microbiology 69, 1920–1927.
Hostettmann, K., Marston, A., 2002. Twenty years of research into medicinal plants: Results and perspectives.
Phytochemistry Review1, 275-285.
Hostettmann, K., Terreaux, C., Marston, A., Potterat, O., 1997. The role of planar chromatography in the rapid screening
and isolation of bioactive compounds from medicinal plants. Journal of Planar Chromatography-Mod. TLC, 10, 251–
257.
Hostettmann-Kaldas, M., Nakanishi, K., 1979. Moronic acid, a simple triterpenoids keto acid with antimicrobial activity
isolated from Ozoroa mucronata. Planta Medica 37(4), 358-360.
Hou, W., Li, Y., Zhan, Q., Wei, X., Peng, A., Chen, L., Wei, Y., 2009. Triterpene Acids Isolated from Lagerstroemia speciosa
Leaves as α-Glucosidase Inhibitors. Phytother. Res. 23, 614–618
Hu, L., Tall, B. D., Curtis, S. K., Kopecko, D. J., 2008. Enhanced microscopic definition of Campylobacter jejuni 81-176
adherence to, invasion of, translocation across, and exocytosis from polarized human intestinal Caco-2 cells.
Infection and Immunity 76(11), 5294–5304.
Hutchings, A., Scott, A. H., Lewis, G., 1996. Zulu Medicinal Plants: An Inventory. University of Natal Press, Durban, p. 266–
267.
Huycke, M. M., Moore, D. R., 2002. In vivo production of hydroxyl radical by enterococcus faecalis colonizing the intestinal
tract using aromatic hydroxylation. Free Radical Biology & Medicine, 33(6), 818–826.
Imanishi, K., 1993. Aloctin A, an active substance of Aloe arborescens Miller as an immunomodulator. Phytotherapy
Research, 7(7), S20-S22.
155
Iwalewa, E. O., McGaw, L. J., Naidoo, V., Eloff, J. N., 2007. Inflammation: the foundation of diseases and disorders. A
review of phytomedicines of South African origin used to treat pain and inflammatory conditions. African Journal of
Biotechnology 6 (25), 2868-2885.
Iwalokun, B. A., Gbenle, G. O., Adewole, T. A., Akinsinde, K. A., 2001. Shigellocidal properties of three Nigerian medicinal
plants: Ocimum gratissimum, Terminalia avicennoides, and Momordica balsamina. Journal of Health, Population
and Nutrition 19(4), 331-335.
Jager, A. K., Van Staden, J., 2005. Cyclooxygenase inhibitory activity of South African plants used aganist inflammation.
Phytochemistry Review 4, 39-46.
Jay, J. M., 1996. Modern Food Microbiology 5th edn New York, NY: Chapman and Hall.
Jeller, A. H., Silva, D. H. S., Liao, L. M., Bolzani, V. S., Furlan, M., 2004. Phytochemistry 65, 1977.
Johns, T, Faubert, G. M., Kokwaro, J. O., Muhunnah, R. L. A., Kimanam, 1995. Anti-giardial activity of gastrointestinal
remedies of the Luo of East African. Journal of Ethnopharmacology 46, 17-23.
Johnson, A. M., Kaushik, R. S., Hardwidge, P. R., 2010. Disruption of transepithelial resistance by enterotoxigenic
Escherichia coli. Veterinary Microbiology 141, 115-119.
Jordan, S. A., Cunningham, D. G., Marles, R. J., 2010. Assessment of herbal medicinal products: challenges, and
opportunities to increase the knowledge base for safety assessment, Toxicol. Ogy and Appl. ied Pharmacol. ogy 243
(2010) 198–216.
Joseph, C. C., Moshi, M. J., Innocent, E., Nkunya, M. H. H., 2007. Isolation of a Stilbenes Glucoside and Other constituent
of Terminalia sericea. African Journal of Traditional and Complementary, Alternative Medicine, 4(4), 383-386.
Kaikabo, A. A., Suleiman, M. M., Samuel, B. B., Eloff, J. N., 2008. Antibacterial activity of eleven South African plants use in
treatment of diarrhoea in folkloric medicine. African Journal of Traditional, Complementary and Alternative Medicine
5, 315–316.
Kamatou, G. P. P., Viljoen, A. M., Steenkamp, P., 2010. Antioxidant, anti-inflammatory activities and HPLC analysis of
South African Salvia species. Food Chemistry 119, 684–688.
Kane, A. B., 1996. Mechanisms of Cell and Tissue Injury. In: Cellula and Molecular Pathogenesis. Editor, Alphonse E.
Sirica: Lippincott-Raven Publishers, 277 East Washington Square, Philadelphia, Pennsylvania 19106-3780 Pp 122.
Katerere, D. R., Gray, A. I., Nash, R. J., Waigh, R. D., 2003. Antimicrobial activity of pentacyclic triterpenes isolated from
African Combretaceae. Phytochemistry 63, 81-88.
Katsoulis, L. C., Veale, D. J. H., Havlik, I., 2000. The pharmacological action of Rhoicissus tridentata on isolated rat uterus
and ileum. Phytotherapy 14(6), 460-462.
Kaviarasan, S., Naik, G. H., Gangabhagirathi, R., Anuradha, C. V., Priyadarsini, K. I., 2007. In vitro studies on antiradical
and antioxidant activities of fenugreek (Trigonella foenum graecum) seeds. Food Chemistry 103, 31–37.
Kayser, O., Kolodziej. H., 1997. Antibacterial activity of extracts and constituent of Pelargonium sidoides and Pelargonium
reniforme Curtis
Kayser, O., Kolodziej. H., Kiderlen, A. F., 2001. Immunomodulatory principle of Pelargonium sidoides. Phytotherapy
Research 15(2), 122-126.
Kehrer, J. P., Biswal, S. S., 2000. The molecular effects of acrolein. Toxicological Sciences 57, 6–15.
Kenny, J. M., Kelly, P., 2009. Protozoal gastrointestinal infections. Medicine 39, 599-602.
156
Kermanshai, R., McCamy, B. E., Rosenfeld, J., Summers, P. S., Weretilnyk, E. A., Sorger, G. J., 2001. Benzyl
isothiocyanate isthe chief or sole anthelmmintic Papaya seed extracts. Phytochemistry 57(3), 427-435.
Khajuria, A., Gupta, A., Garai, S., Wakhloo, B. P., 2007. Immunomodulatory effects of two sapogenins 1 and 2 isolated from
Luffa cylindrica in Balb/C mice. Bioorganic and Medicinal Chemistry Letters 17, 1608–1612.
Khaled, M. A., 1994. Oxidative stress in childhood malnutrition and diarrheal disease. Journal of Diarrheal Disease
Research 12, 165–72.
Kim, H. S., 2009. 5-Hydroxytryptamine 4 receptor agonists and colonic motility. Journal of Smooth Muscle Research 45, 25–
29.
Kinghorn, A. B., Chai, H-B., Sung, C-K., Keller, W-J., 2011. The classical drug discovery approach to defining bioactive
constituents of botanicals. Fitoterapia 82, 71-79.
Kishida, E., Tokumaru, S., and Ishitani, Y., 1993. Comparison of the formation of malondialdehyde and thiobarbituric acid
reactive substances from autoxidized fatty acids based on oxygen consumption. Journal of Agricultural and Food
Chemistry, 41, 1598-1600.
Koduru, S., Grierson, D. S., Afolayan, A. J., 2006. Antimicrobial activity of Solanum aculeastrum. Pharmaceutical Biology
44(4), 283-286.
Koduru, S., Grierson, D. S., Van der Venter, M., Afolayan, A. J., 2007: Anticancer activity of steroid alkaloids isolated from
Solanum aculeastrum. Pharmaceutical Biolog 45(8), 613-618.
Koff, R. S., 1992. Clinical manifestation and diagnosis of hepatitis A virus infection. Vaccine 10, S15-S17.
Koff, R. S., 1998. Hepatitis A. Lancet 341, 1643–1649.
Komane, B. M., Olivier, E., Alvaro, I. Viljoen, M., 2011. Trichilia emetica (Meliaceae) – A review of traditional uses, biological
activities and Phytochemistry. Phytochemistry Letters 4, 1–9.
Koopmans, M., 2008. Progress in understanding norovirus epidemiology. Current Opinion on Infectious Diseases 21, 544–
552.
Kosar, M., Bozan, B., Temelli, F., Baser, K. H. C., 2007. Antioxidant activity and phenolic composition of sumac (Rhus
coriaria L.) extracts. Food Chemistry 103, 952-959.
Kougan, G. B., Miyamoto, T., Mirjolet, J-F., Duchamp, O., Sondengam, B. L., Lacaille-Dubios, M-A., 2009. Arboreasides AE, Triterpene Saponins from the Bark of Cussonia arborea. Journal of Natural Products 72, 1081–1086.
Krakauer, T., Li, B. Q., Young, H. A., 2001. The flavonoids baicalin inhibits superantigens-induced inflammatory cytokines
and chemokines. Federation of European Biochemical Societies Letter 500, 52-55.
Krishnaiah, D., Sarbatly, R., Nithyanandam, R., 2011. A review of the antioxidant potential of medicinal plant species. food
and bioproducts processing 89, 217–233.
Kubo, I., Kim, M., Naya, K., Komatsu, S., Yamagiwa, Y., Ohashi, K., Sakamoto, Y., Hirakawa, S., Kamikawa, T., 1987.
Prostaglandin synthetase inhibitors from the African medicinal plant” Ozoroa mucronata. Chemistry letters, 11011104.
Kuete, V. Ngameni, B., Fotso Simo, C. C., Kengap Tankeu, R., Tchaleu Ngadjui¸ B., Meyer d, N., Lall, J. J. M., Kuiate, J. R.,
2008. Antimicrobial activity of the crude extracts and compounds from Ficus chlamydocarpa and Ficus cordata
(Moraceae). Journal of Ethnopharmacology 120, 17–24.
Kuete, V., Krusche, B., Youns, M., Voukenga, I., Fankam, A. G., Tankeo, S., Lacmata, S., Efferth, T., 2011. Cytotoxicity of
some Cameroonian spices and selected medicinal plant extracts. Journal of Ethnopharmacology 134, 803–812.
157
Kuete, V., Nana, F., Ngameni, B., Mbaveng, A. T., Keumedjio, F., Chaleu Ngadjui, B. T., 2009. Antimicrobial activity of the
crude extract, fractions and compounds from stem bark of Ficus ovata (Moraceae). Journal of Ethnopharmacology
124, 556–561.
Kunkel, S. L., Lukacs, N., Strieter, R. M., 1996. Cytokines and Inflammatory disease. In: Cellula and Molecular
Pathogenesis. Editor, Alphonse E. Sirica: Lippincott-Raven Publishers, 277 East Washington Square,
Philadelphia, Pennsylvania 19106-3780 Pp 23-36.
Kunle, O. O., Shittu, A., Nasipuri, R. N., Kunle, O. F., Wambembe, C., Akah, P. A., 1999. Gastrointestinal activity of Ficus
sur. Fitoterapia 70(6), 542-547.
Lajubutu, B. A., Pinney, R. L., Roberts, M. F., Odelola, H. A., Oso, B. A., 1995. Antibacterial activities of diosquinone and
plumbagin from the root of Diospyros mespiliformis (Hostch) (Ebenaceae). Phytotherapy Research, 9(5), 346-350.
Lall, N., Meyer, J. J. M., 2001. Inhibition of drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis by
Diospyrin, isolated from Euclea natalensis. Journal of Ethnopharmacology 78(2-3), 213-216.
Laohachai, K. N., Bahadi, R., Hardo, M. B., Hardo, P. G., Kourie, J. I., 2003. The role of bacterial and non-bacterial toxins in
the induction of changes in membrane transport: implications for diarrhea. Review: Toxicon 42, 687–707.
Lansky, E. P., Newman, R. A., 2007. Review: Punica granatum (pomegranate) and its potential for prevention and treatment
of inflammation and cancer. Journal of Ethnopharmacology 109, 177–206.
Lattanzio, V., Lattanzio, V. M. T., Cardinali, A., 2006. Role of phenolics in the resistance mechanisms of plants against
fungal pathogens and insects. Phytochemistry: Advances in Research 23-67.
Lauwaet, T., Oliveira, M. J., Callewaert, B., De Bruyne, G., Mareel, M., Leroy, A., 2004. Proteinase inhibitors TPCK and
TLCK prevent Entamoeba histolytica induced disturbance of tight junctions and microvilli in enteric cell layers in
vitro. International Journal for Parasitology 34(7), 785–794.
Le Bouguenec, C., 2005. Adhesins and Invasins of pathogenic Escherichia coli. International journal of medical microbiology
295, 471-478.
Lee, C. W., Sarma, S. K., Singaram, C. Casper, M. A., 1997. Ca2+ channel blockage by verapamil inhibits GMCs and
diarrhea during small intestinal inflammation. American Journal of Physiology 273, 785-794.
Lee, G. Y., Byeon, S. E., Kim, J. Y., Lee, J. Y., Rhee, M. H., Hong, S., Wu, C. J., Lee, H. S., Kim, M. J., Cho, D. H., Cho, J.
Y., 2007a. Immunomodulating effect of Hibiscus cannabinus extract on macrophage functions. Journal of
Ethnopharmacology 113, 62-71.
Lee, H. M., Lee, J. M., Jun, S. H., Lee, H. S., Kim, W. N., Lee, J. H., Ko, N. Y., Mun, S. H., Kim, B. K., Lim, B. O., Choi, D.
K., Choi, W. S., 2007b. The anti-inflammatory effects of Pyrolae herba extract through the inhibition of the
expression inducible nitric oxide synthase (iNOS) and NO production. Journal of Ethnopharmacology 112, 49-54.
Lee, J., Durst, R. W., Wrolstad, R. E., 2005. Determination of Total Monomeric Anthocyanin pigment content of fruit juices,
Beverages, Natural colourants, and Wines by the pH differential method: Collaborative study. Journal of AOAC
International 88, 1269-1278.
Lee, J., Rennaker, C., Wrolstad, R. E., 2008. Correlation of two anthocyanin quantification methods: HPLC and
spectrophotometric method. Food Chemistry 110, 782-786.
Lee, S.H., Oe, T., Blair, I.A., 2001. Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins.
Science 292, 2083–2086.
Lengsfeld, C., Faller, G., Hansel, A., 2007. Okra polysaccharides inhibit adhesion of Campylobacter jejuni to mucosa
isolated from poultry in vitro but not in vivo. Animal Feed Science and Technology 135, 113–125.
158
Levine, J., Dykoski, R. K., Janoff, E. N., 1995. Candida-Associated Diarrhea: A Syndrome in Search of Credibility. Clinical
Infectious Diseases 21 (4), 881-886.
Lewu, F. B., Afolayan, A. J., 2009. Ethnomedicine in South Africa: The role of weedy species. African Journal of
Biotechnology 8 (6), 929-934.
Li, Y., Trush, M. A., 1994. Reactive oxygen dependent DNA damage resulting from the oxidation of phenolic compounds by
a copper redox cycle. Cancer Research 54,1895s-1898s.
Lin, C-N., Haung, A-M., Lin, K-W., Hour, T-C., Ko, H-H., Yang, S-C., Pu, Y-S., 2010. Xanthine oxidase inhibitory terpenoids
of Amentotaxus formosana protect cisplatin-induced cell death by reducing reactive oxygen species (ROS) in normal
human urothelial and bladder cancer cells. Phytochemistry 71, 2140–2146.
Lin, J., Opoku, A. R., Geheeb-Keller, M., Hutchings, A. D., Terblanche, S. E., Jager, A. K., van Staden, J., 1999. Preliminary
screening of some traditional Zulu medicinal plants for anti-inflammatory and anti-microbial activities. Journal of
Ethnopharmacology 68, 267–274.
Lin, J., Puckree, T., Mvelase, T. P., 2002. Anti-diarrhoeal evaluation of some medicinal plants used by Zulu traditional
healers. Journal of Ethnopharmacology 79(1), 53-6.
Lindsey, K. L., Motsei, M. L., Jager, A. K., 2002. Screening of South African food plants for anti oxidant activity. Journal of
Food Science 64, 2129–2931.
Linscott, A. J., 2011. Food-borne illnesses. Clinical Microbiology Newsletter 33, 41-45.
Lis-Balchin, M., Hart, S., Simpson, E., 2001. Buchu (Agathosma betulina, Agathosma crenulata (Rutaceae)) essential oils:
their pharmacological activity on guinea pig ileum and antimicrobial activity on microorganism. Journal of Pharmacy
and Pharmacology 53(4), 579-582.
Liu, Y., Abreu, P., 2006. Tirucallane triterpenes from the roots of Ozoroa insignis. Phytochemistry67,1309–1315.
Lode, H, 2010. Safety and Tolerability of Commonly Prescribed Oral Antibiotics for the Treatment of Respiratory Tract
Infections. The American Journal of Medicine 123, S26-S38.
Lorrot, M., Vasseur, M., 2007. How do the rotavirus NSP4 and bacterial enterotoxins lead differently to diarrhea? Virology
Journal 4, 31
Lourens, A. C. U., Van Vuuren, S. F., Viljoen, A. M., Van Herdeen, F. R., 2011. Antimicrobial activity and in vitro cytotoxicity
of selected South African Helichrysum species. South African Journal of Botany, 77(1), 229-235.
Lourens, A. C. U., Viljoen, A. M., van Heerden, F. R., 2008. South African Helichrysum species: A review of the traditional
uses, biological activity and phytochemistry. Journal of Ethnopharmacology 119, 630–652.
Lund, T., DeBuyser, M. L., Granum, P. E., 2000. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis.
Molecular Microbiology 38, 254–261.
Luseba, D., Elgorashi, E. E., Ntloedibe, B. T., Van Staden, J., 2007. Antibacterial, anti-inflammatory and mutagenic effects
of some medicinal plants used in South Africa for the treatment of wounds and retained placenta in Livestock. South
African Journal of Botany 73, 378-383.
Luseba, D., Van der Merwe, D., 2006. Ethnoveterinary medicine practices among Tsonga speaking people of South Africa.
Onderstepoort Journal of Veterinary Research 73, 115–122.
Lutterodt, G. D., Ismail, A., Basheer, R. H., Baharuddin, M. H., 1999. Antimicrobial effects of Psidium guajava extracts as
one mechanism of its antidiarrhea action. Malaysian Jouranl of medicinal Sciences. Makkar HPS, Francis G, Becker
K. 2007: Bioactivity of phytochemicals in some lesser-known plants and their effects and potential applications in
livestock and aquaculture production system. Animal 1(9), 1371- 1391, 17-20.
159
Lutterodt, G.D., 1992. Inhibition of Microlax-induced experimental diarrhoea with narcotic-like extracts of Psidium guajava
leaf in rats. Journal of Ethnopharmacology 37, 51–157
Lyckander, I. M., Malterud, K. E., 1992. Lipophilic flavonoids from Orthosiphon spicatus as inhibitors of 15-lipoxygenase.
Acta Pharmaceutica Nordica 4, 159–166.
Mabogo, D. E. N., 1990. The ethnobotany of the VhaVenda. M. Sc. dissertation, University of Pretoria.
MacNaughton, W. K., 2006. Mechanisms and Consequences of Intestinal Inflammation. Physiology of Gastrointestinal
Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press.
Mahato, S. B., Kundu, A. P., 1994. 13C-NMR spectra of pentacyclic triterpenoids. A compilation and some salient features.
Phytochemistry 37, 1517–1575.
Majhenic, L., Skerget, M., and Knez, Z., 2007. Antioxidant and antimicrobial activity of guarana seed extracts. Food
Chemistry 104, 1258-1268.
Makhlouf, M. G., 1994. Neuromuscular function of the small intestine, In: Johnson, Leonard R. (Ed.), Physiology of the
Gastrointestinal Tract, Third ed. Raven Press, New York, pp. 977–990.
Makkar, H. P. S., 2003. Quantification of tannins in tree and foliage- a laboratory manual. Klumer Academic Press
Dordrecht, The Netherland.
Makkar, H. P. S., Francis G, Becker K. 2007. Bioactivity of phytochemicals in some lesser-known plants and their effects
and potential applications in livestock and aquaculture production system. Animal 1(9), 1371- 1391.
Males, Z., Medic-Saric, M., 2001. Journal of Pharmacology and Biomedical Analysis 24 (2001) 353.
Mamphiswara, N. D., Mashela, P. W., Mdee, L. K., 2010. Distribution of total phenolics and antioxidant in fruit, leaf, stem
and root of Monsonia burkeana. African Journal of Agricultural Research, 5(18), 2570-2575.
Mandal, S., Nayak, A., Kar, M., Banerjee, S. K., Das, A., Upadhyay, S. N., Singh, R. K., Banerji, A., Banerji, J., 2010.
Antidiarrhoeal activity of carbazole alkaloids from Murraya koenigii Spreng (Rutaceae) seeds. Fitoterapia 81, 72-74.
Mansfield, L. S., Gajadhar, A. A., 2004. Cyclospora cayetanensis a food-and water-borne mcoccidian parasite. Veterinary
Parasitology 126, 73–90.
Maregesi, S. M., Pieters, L., Ngassapa, O. D., Apers, S., Vingerhoets, R., Cos Paul, Berghe, Van den Dirk, A., Vlietinck, A.
J., 2008. Screening of some Tanzanian medicinal plants from Bunda district for antibacterial, antifungal and
antiviral activities. Journal of Ethnopharmacology 119, 58-66.
Marino, S. D., Gala, F., Borbone, N., Zollo, F., Vitali, S., Visioli, F., Lorizzi, M., 2007. Phenolic glycoside from Foeniculum
vulgare fruit and evaluation of antioxidative activity. Phytochemistry 68(13), 1805-1812.
Marquez-Martin, A., Puerta, R. D. L., Fernandez-Arche, A., Ruiz-Gutierrez, V., Yaqoob, P., 2006. Modulation of cytokine
secretion by pentacyclic triterpenes from olive pomace oils in human mononuclear cells. Cytokine, 36(5-6), 211-217.
Marston, A., 2011. Review: Thin-layer chromatography with biological detection in Phytochemistry. Journal of
Chromatography A, 1218, 2676–2683.
Martinez-Augustin, O., Romero-Calvo, I., Suarez, M. D., Zarzuelo, A., and Sanohez de Medina, F., 2009. Molecular bases of
impaired water and ion movement in inflammatory bowel diseases. Inflammatory Bowel Diseases 15, 114-127.
Masika, P. J., Afolayan, A. J., 2002. Antimicrobial activity of some plants used for the treatment of livestock disease in the
Eastern Cape, South Africa. Journal of Ethnopharmacology 83, 129-134.
Masika, P. J., Sultana, N., Afolayan, A. J., 2004. Antibacterial activity of two flavonoids isolated from Schotia latifolia.
Pharmaceutical Biology 42(2), 105-108.
160
Masoko, P., Picard, J., Eloff, J. N., 2005. Antifungal activities of six South African Terminalia species (Combretaceae).
Journal of Ethnopharmacology 99, 301-308.
Masoko, P., Picard, J., Eloff, J. N., 2007. The antifungal activities of twenty-four Southern southern Africa Combretum
species (Combretaceae).South African Journal of Botany 73, 173-183.
Mastroeni, P., Maskell, D., 2006. Salmonella infections: Clinical, immunological, and molecular aspects. Cambridge
University Press.
Mathabe, M. C., Nikolova, R. V., Lall, N., Nyazema, N. Z., 2006. Antibacterial activities of medicinal plants used in the
treatment of diarrhea disease in Limpopo Province, South Africa. Journal of Ethnopharmacology 105, 286-293.
Mativandlela, S. P. N., Lall, N., Meyer, J, J, M., 2006. Antibacteria, antifungal, antitubercular activityactivities of the root of
Pelargonium reniforme Curtis and Pelargonium sidoides (DC) (Geraniaceae) root extract. South African Journal of
Botany 72(2), 233-237.
Mativandlela, S. P. N., Meyer, J, J, M., Hussein, A. A., Houghton, P. J., Hamilton, C. J., Lall, N., 2008. Activity against
Mycobacterium smegmatis and Mycobacterium tuberculosis by South African Medicinal plants. Phytotherapy
Research 22(6), 841-845.
Matsui, M., Motomura, D., Fujikawa, T., Jiang, J., Takahashi, S., Manabe, T., Taketo, M.M., 2002. Mice lacking M2 and M3
muscarinic acetylcholine receptors are devoid of cholinergic smooth muscle contractions but still viable. The Journal
of Neuroscience 22, 10627–10632.
Mattison, K., 2010. Norovirus as a food-borne disease hazard. Advances in Food and Nutrition Research 62, 1-39.
Matu, E. N., van Staden, J., 2003. Antibacterial and anti-inflammatory activity of some plants used for medical purposes in
Kenya. Journal of Ethnopharmacology 87, 35-41.
Mbagwu, H. O. C., Adeyemi O. O., 2008. Anti-diarrhoeal activity of the aqueous extracts of Mezoneuron benthamianum Baill
(Caesalpiniaceae) Journal of Ethnopharmacology 116, 16-20.
Mbatchi, S. F., Mbatchi, B., Banzouzi, J. T., Basmba, T., Nsonde Ntandou, G. F., Ouamba, J–M., Berry, A., Benoit-Vical, F.,
2000. In vitro antiplasmodial activity of 18 plants used in Congo Brazzaville traditional medicine. Journal of
Ethnopharmacology 104, 168-174.
McGaw, L. J., Eloff, J. N., 2008. Ethnoveterinary use of Southern African plants and scientific evaluation of their medicinal
properties. Journal of Ethnopharmacology 119: 559–574.
McGaw, L. J., Jäger, A. K., van Staden, J., 2000. Antibacterial, anthelmintic and anti-amoebic activity in South African
medicinal plants. Journal of Ethnopharmacology 72, 247–263.
McGaw, L. J., Jager, A. K., van Staden, J., 2002. Isolation of antibacterial fatty acids from Schotia brachypetala. Fitoterapia
73(5), 431-433.
McGaw, L. J., Rabe, T., Sparg, S. G., Jager, A. K., Eloff, J. N., van Staden, J., 2001. An investigation on the biological
activity of Combretum species. Journal of Ethnopharmacology 75: 45–50.
McGaw, L. J., Van der Merwe, D., Eloff, J. N., 2007. In vitro anthelmintic, antibacterial and cytotoxic effects of extracts from
plant used in South African ethnoveterinary medicine. The veterinary Journal 173, 366-372.
Meghashri, S., Vijar Kumar, S., Gopal, S., 2010. Antioxidant properties of aof novel flavonoids from leaves of Leucas
aspera. Food Chemistry 122(1), 105-110.
Mello, D. L., ALves, A. A., Macedo, D. V., Kbota, L. T., 2005. Peroxidase-based biosensor as a tool for a fast evaluation of
antioxidant capacity of tea. Food Chemistry 92, 515-519.
161
Michelangeli, F., Ruiz, M. C., 2003. Physiology and pathology of the gut in relation to viral diarrhoea. Viral gastroenteritis: U.
Desselberger and J. Gray (Editors). Elservier Science BV. Pp 23-50.
Middleton, E., Jr., Kandaswami, C., and Theoharides, T. C., 2000. The effects of plant flavonoids on mammalian cells:
Implications for inflammation heart disease and cancer. Pharmacology Reviews, 52(4), 673–751.
Miliauskas, G., van Beek, T. A., de Waard, P., Venskutonis, R. P., and Sudholter, E. J. R. 2005. Identification of radical
scavenging compounds in Rhaponticum carthamoides by means of LC-DADSPE- NMR. Journal of Natural
Products, 68(2), 168–172.
Mishra, A., Kumar, A., 2000. Medicinally important trees of Rajasthan. International Journal of Mendel 1–8, 37–38.
Mlambo, N. P., 2008. The screening of medicinal plants traditionally used to treat diarrhoea, in Ongoye area, Kwazulu Natal.
A dissertation submitted to the Faculty of Science at the University of Zululand in fulfillment of the requirement for
the degree of Master of Science.
Mølgaard, P., Nielsen, S. B., Rasmussen, D. E., Drummond, R. B., Makaza, N., Andreassen, J., 2001. Anthelmintic
screening of Zimbabwean plants traditionally used against schistosomiasis. Journal of Ethnopharmacology 74,
257–264.
Molla, A., Viljoen, A. M., 2008. Buchu- Agathosma betulina and Agathosma crenulata (Rutaceae): A Review. Journal of
Ethnopharmacology 119: 413–419.
More, G., Tshikalange, T. E., Lall, N., Botha, F., Meyer, J. J. M., 2008: . Antimicrobial activity of medicinal plants against oral
microorganisms. Journal of Ethnopharmacology, 119, 473–477.
Moshi, M. J., Mbwambo, Z. H., 2005. Some pharmacological properties of extract of Terminalia sericea roots. Journal of
Ethnopharmacology 97(1), 43-47.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity
assays. Journal of Immunological Methods 65, 55–63.
Motlhanka, D. M. T., 2008. Free radical scavenging activity of selected medicinal plants of Eastern Botwana. Pakistan
Journal of Biological Sciences 11(5), 805-808.
Moundipa, P. F., Flore, K. G. M., Bilong Bilong, C. F., Bruchhau, I., 2005. In vitro amoebicidal activity of some medicina
plants of the Bamu region (Cameroon). African Journal of Traditional and Complimentary Medicine 2(2), 113-121.
Moure, A., Cruz, J. M., Franco, D., Dominquez, J. M., Sineiro, J., Dominquez, H., Nunez, M. J., and Parajo, J. C., 2001.
Natural antioxidant from residual sources. Food Chemistry 72, 145-171.
Mueller, C.F., Laude, K., McNally, J.S., Harrison, D.G., 2005. ATVB in focus: redox mechanisms in blood vessels.
Arterioscler. Thromb. Vasc. Biol. 25, 274–278.
Muhammed, I., El-Sayed, K. A., Mossa, J. S., Al-Said, M. S., El-Feraly, F. S., Clark, A. M., Hufford, C. D., Oh, S., Mayer, A.
M. S., 2000. Bioactive 12-Olaenene triterpene and secotriterpene acids from Maytenus undata. Journal of Natural
Products 63, 605-610.
Mulaudzi, R. B., Ndhalala, A. R., Kulkarni, M. G., Finnie, J. F., Van Staden, J., 2011. Antimicrobial properties and phenolic
contents of medicinal plants used by Venda people for conditions related to venereal diseases. Journal of
Ethnopharmacology, DIO: 10: 1016/J.jep2011.03.022.
Mulaudzi, R. B., Ndhlala, A. R., Finnie, J. F., Van Staden, J., 2009. Antimicrobial, anti-inflammatory and genotoxicity activity
of Alepidea amatymbica and Alepidea natalensis (Apiaceae). South African Journal of Botany 75, 584–587.
Murata, T., Miyase, T., Muregi, F. W., Naoshima-Ishibashi, Y., Umshara, K., Warashina, T., Kanou, S., Mkoji, G. M., Terada,
M., Ishih, A., 2008. Antiplasmodial triterpenoids from Ekebergia capensis, Journal of Natural Product 71, 167-174.
162
Murayama, T., Eizuru, Y., Yamada, R., Sadanari, H., Matsubara, K., Rukung, G., Tolo, F. M., Mungai, G. M., Kofi-Tsekpo,
M., 2007. Anticytomegalovirus activity of pristimerin, a triterpenoids quinine methide isolated from Maytenus
heterophylla (Eckl. & Zeyh.). Antiviral Chemistry and Chemotherapy 18(3), 133-139.
Mutanyatta, J., Bezabih, M., Abegaz, B. M., Dreyer, M., Brun, R., Kocher, N., Bringmann, G., 2005. The first 6’-O-sulfated
phenylanthraquinones: Isolation from Bulbine frutescens, structural elucidation enantiomeric purity and partial
synthesis. Tetrahedron 61, 8475-8484.
Muthaura, C. N., Rukunga, G. M., Chhabra, S. C., Omar, S. A., Guantai, A. N., Gathirwa, J. W., Tolo, F. M., Mwitari, P. G.,
Keter, L. K., Kirira, P. G., Kimani, C. W., Mungai, G. M., Njagi, E. N. M., 2007. Antimalarial activity of some plants
traditionally used in treatment of malaria in Kwale district of Kenya. Journal of Ethnopharmacology 112, 545-551.
Naczk, M., Shahidi, F., 2004. Extraction and analysis of phenolics in food. Journal of Chromatography A, 1054, 95-111.
Naidoo, L. A. C., Drewes, S. E., Van Staden, J., Hutching, J., 1992. Exocarpic acid and other compounds from tubers and
inflorescences of Sarcaphyte sanguine. Phytochemistry 31(11), 3929-3931.
Naidoo, V., McGaw, L. J., Bisschop, S. P. R., Duncan, N., Eloff, J. N., 2008. The value of plant extracts with antioxidant
activity in attenuating coccidiosis in broiler chickens. Veterinary Parasitology 153, 215–219.
Naidoo, V., Zweygarth, E., Eloff, J. N., Swan, G. E., 2005. Identification of anti-babesial activity for four ethnoveterinary
plants in vitro Veterinary Parasitology 130(1-2), 9-13, 2005.
Naidoo, V., Zweygarth, E., Swan, G. E., 2006. Determination and quantification of the in vitro activity of Aloe marlothii (A.
Berger) subsp. marlothii and Elephantorrhiza elephantina (Burch.) skeels acetone extracts against Ehrlichia
ruminantium. Onderstepoort Journal of Veterinary Research 73, 175–178.
Nanayakkara, N. P., Burandt, C. L. Jr., Jacob, M. R., 2002. Flavonoid with activity against methicilin-resistnt Staphylococcus
aureus from Dalea scandens var. paucifolia. Planta medica 68, 519-522.
Nardi, G. M., Siqueira Junior, J. M., Monache Belle, F., Pizzolatti, M. G., Ckless, K., Ribeiro-do-Valle, R. M., 2007.
Antioxidant and anti-inflammatory effects of products from Croton celtidifolius Bailon on carrageenan-induced
pleurisy in rats. Phytomedicine 14, 115-122.
Nathan, C., 1992. Nitric Oxide as a secretory product of mammalian cells. FASEB J. 6: 3051-3064.
National Committee for Clinical Laboratory Standards (NCCLS), 2002. Performance Standards for Antimicrobial
Susceptibility testing. Twelfth Information Supplement M100 - S12 NCCLS, Wayne.
Naz, S., Siddiqi, R., Ahmad, S., Rasool, S. A., Sayeed, S. A., 2007. Antibacterial activity directed isolation of compounds
from Punica granatum. Journal of Food Science 72, M341–M345.
Ndamba, J., Nyazema, N., Makaza, N., Anderson, C., Kaondera, K. C., 1994. Traditional herbal remedies used for the
treatment of urinary schistosomiasis in Zimbabwe. [Comparative Study] Journal of Ethnopharmacology 42(2), 12532
Ndawonde, B. G., Zobolo, A. M., Dlamini, E. T., Siebert, S. J., 2007. A survey of plants sold by traders at Zululand muthi
markets, with a view to selecting popular plant species for propagation in communal gardens. African Journal of
range and Forage Siecence 42(2), 103-107(5).
Ndhlala, A. R., Finnie, F. R., Van staden, J., 2010. Plant composition, Pharmacological properties and mutagenic evaluation
of commercial Zulu herbal mixture: Imbiza ephuzwato. Journal of Ethnopharmacology 133(2), 663-674.
Neish, A. S., 2009. Microbes in Gastrointestinal Health and Disease. Gastroenterology 136, 65–80.
163
Nergard, C. S., Diallo, D., Michealsen, T. E., Malteru, K. E., Kiyohara, H., Matsumoto, T., Yamada, H., Paulsen, B. S., 2004.
Isolation, partial characterization and immunomodulating activity of polysaccharides from Vernonia kotschyana Sch.
Bip. Ex. Walp., Journal of Ethnopharmacology 91(1), 141-152.
Neuwinger, H. D., 1996. African ethnobotany: poisons and drugs: chemistry, pharmacology, toxicology. Chapman and Hall,
Germany.
Nguemeving, J. R., Azebaze, A. G. B., Kuete, V., Carly, N. N. E., Beng, V. P., Meyer, M., Blond, A., Bodo, B., Nkengfack, A.
E., 2006. Laurentixanthones A and B, antimicrobial xanthones from Vismia laurentii. Phytochemistry 67, 1341–1346.
Ngueyem, T. A., Brusotti, G., Caccialanza, G., Vita Finzi, P., 2009. The genus Bridelia: A phytochemical and
ethnopharmacological review. Journal of Ethnopharmacology 124, 339–349.
Nhiem, N. X., Tai, B. H., Quang, T. H., Kiem, P. V., Minh, C. V., Nam, N. H., Kim, J-H., Im, L-R., Lee, Y-M., Kim, Y. H., 2011.
A new ursane-type triterpenoid glycoside from Centella asiatica leaves modulates the production of nitric oxide
and secretion of TNF-a in activated RAW 264.7 cells. Bioorganic & Medicinal Chemistry Letters 21, 1777–1781.
Nigam, S., Schewe, T., 2000. Phospholipase A2 and lipid peroxidation. Biochimie and Biophysics Acta 1488, 167–181.
Nissanka, A. P. K., Karunaratne, V., Bandara, B. M. R., Kumar, V., Nakanishi, T., Nishi, M., Inada, A., Tillekeratne, L. M. V.,
Wijesundara, D. S. A., Gunatilaka, A. A. L., 2000. Antimicrobial alkaloids from Zanthophylum tetraspermum and
caudatum. Phytochemistry 56, 857-861.
Noreen, Y., Ringbom, T., Perera, P., Danielson, H., Bohlin, L., 1998. Development of a radiochemical Cyclooxygenase-1
and -2 in vitro assay for identification of natural products as inhibitors of prostaglandin biosynthesis. Journal of
Natural Products 61, 2-7
Nose, K., 2000. Role of reactive oxygen species in regulation of physiological functions. Biological and Pharmaceutical
Bulletin 22, 897-903.
Nyiredy, S. Z.; Glowniak, K., 2001. Planar chromatography in medicinal plant research. In Planar Chromatography; Nyiredy,
Sz. Ed.; Springer Scientific Publisher: Budapest, Hungary 550.
O’Hara, S. P., Chen, X-M., 2011. The cell biology of cryptosporidium Cryptosporidium infection. Microbes and Infection xx,
1-10.
Oben, J. E., Assi, S. E., Agbor, G. A., Musoro, D. F., .2006. Effect of Eremomastax speciosa on experimental diarrhea.
African Journal of Traditional Complimentary Alternative Med. 3(1), 95-100.
Obi, C. L., Potgieter, N., Bessong, P. O., Masebe, T., Mathebula, H., Molobela, P., 2003. In vitro antibacterial activity of
Venda medicinal plants. South African Journal of Botany 69(2), 1-5.
Odhav, B., Kanasamy, T., Khumalo, N., Baijnath, H., 2010. Screening of African traditional vegetables for their alphaamylase inhibitory effect. Journal of Medicinal Plant Research, 4(14), 1502-1507.
Ofek, I., Sharon, N., 1990. Adhesins as lectins: Specificity and role in infection. In: K, Jann B (eds) Bacterial capsules and
adhesins: Fact and Principles. Current topic in Microbiology and Immunology. Vol 151, Berlin: Springer, 91-113.
Ogoina, D., Onyemelukwe, G. C., 2009. The role of infections in the emergence of non-communicable diseases (NCDs):
Compelling needs for novel strategies in the developing world. Journal of Infection and Public Health 2, 14-29.
Ojewole, J. A. O., 2004. Evaluation of the Antidiabetic, Anti inflammatory and Antidiabetic properties of Sclerocarya birrea
(A. Rich) Hochst. Stem bark aqueous Extract in Mice and Rats. Phytotherapy Research 18, 601-608.
Ojewole, J. A. O., 2005. Antinociceptive, antiinflammatory and antidiabetic effects of Leonotis leonurus (L) R. BR.
[Lamiaceae] leaf aqueous extract in mice and rats. Methods and Findings in Experimental and Clinical
Pharmacology 27, 257–264
164
Ojewole, J. A. O., 2006. Antinococeptive, anti-inflammatory and antidiabetic properties of Hypoxis hemerocallidea Fisch.
and C. A. Mey.(Hypoxidaceae) Corm (‘African potato’) in mice and rats. Journal of Ethnopharmacology 103(1), 126134
Ojewole, J. A. O., Awe, E. O., Nyinawumuntu, A., 2009. Antidiarrhoeal Activity of Hypoxis hemerocallidea Fisch. and C. A.
Mey.(Hypoxidaceae) Corm (‘African potato’) Aqueous Extract in Rodents. Phytotherapy Research, 23, 965–971.
Ojewole, J. A. O., Mawoza, T., Chiwororo, W. D. H., Owira, P. M. O., 2010. Sclerocarya birrea (A. Rich.) Hochst. [‘Marula’]
(Anacardiaceae): a review of its phytochemistry, pharmacology and toxicology and its ethnomedicinal uses.
Phytotherapy Research 24, 633–639.
Okokon, J. E., Ita, B. N., Udokpoh, A. E., 2006. The in vivo antimalarial activities of Uvaria chamae and Hippocratea
africana. Annals of Tropical Medicine and Parasitology 100, 585–590.
Orabi, K. Y., Al-Qasoumi, S., El-Olemy, M. M., Mosaa, J. S., Muhammed, I., 2001. Dihydragarofuran alkaloid and triterpenes
from Maytenus hetrophylla and Maytenus arbutifolia. Phytochemistry 58(3), 475-480.
Oskay, M., Oskay, D., Kalyoncu, F., 2009. Activity of some plants extracts against multi drug resistant human.pathogens.
Iranian Journal of Pharmaceutical Research 8, 293-300.
Oskay, M., Sari, D., 2007. Antimicrobial screening of some Turkish medicinal plants. Pharmaceutical Biology 45(3), 176-181.
Ovenden, P. B., Yu, J., Bernays, J., Wanss Christophidis, L. J., Sberna, G., Tait, R. A., Wildman, H. G., Labeller, D.,
Lowther, J., Walsh, N. G., Meurer-Grimes, B. M., 2004. Physaloside A, an Acylated sucrose ester from Physalis
viscose.
Owolabi, O. J., Omogbai, E. K. I., 2007. Analgesic and anti-inflammatory activities of the ethanolic stem bark extract of
Kigelia africana ) Bignoniaceae). African Journal of Biotechnology, 6(5), 582-585.
Owolabi, O. J., Omogbai, E. K. I., Obasuyi, O., 2007. Antifunal and antibacterial activities of the ethanolic and aqueous
extracts of Kigelia africana ) Bignoniaceae). African Journal of Biotechnology 6(14), 1677-1680.
Owolabi, O.J., Nworgu, Z. A., Odushu, K., 2010. Antidiarrhoeal evaluation of the eythanol extract of Nauclea latifolia root
bark. Methods and Findings in Experimental and Clinical Pharmacology 32(8), 551-555.
Ozyurek, M., Bektasogu, B., Guclu, K., Apak, R., 2008. Hydroxyl radical scavenging assay of phenolics and flavonoids with
a modified cupric reducing antioxidant capacity (CUPRAC) method using catalase for hydrogen peroxide
degradation. Analytica Chimica Acta 616, 196–206.
Pallant, C. A., and Steenkamp, V., 2008. In-vitro bioactivity of Venda medicinal plants used in the treatment of respiratory
conditions. Human & Experimental Toxicology 27, 859–866.
Palombo, E. A., 2006. Phytochemicals from traditional medicinal plants used in the treatment of diarrhoea: modes of action
and effects on intestinal function. Phytotherapy Research 20, 717–724.
Palombo, E. A., 2006. Traditional plants and Herbal Remedies used in the treatment of Diarrhea Disease: Mode of action,
Quality, Efficacy and Safety Consideration. In: Modern phytomedicine, turning medicinal plants into drugs. (Edited by
Ahmed F Aqil and Owais M) WILEY-VCH Verlag GmbH and Co KGaA, Weinheim. Pp 248-269.
Panda, D., Patra, C. R., Nandi, S., Swarup, D., 2009. Oxidative stress indices in gastroenteritis in dogs with canine
parvoviral infection. Research in veterinary science 86, 36-42.
Parekh, J., Chandra, S., 2007. Antibacterial and phytochemical studies on twelve species of Indian medicinal plants. African
Journal of Biomedical Research 10, 175-181.
Parola, M., Bellomo, G., Robino, G., Barrera, G., Dianzani, M. U., 1999. 4- Hydroxynonenal as a biological signal: molecular
basis and pathophysiological implications. Antioxidant and Redox Signalling 1, 255–284.
165
Pash, Y., Banks, M., Farthing, M., 2009. Causes and recommended management of acute diarrhoea. Prescriber, January,
16-34.
Pauli, G. F., Junior, P., 1995. Phenolic glucosides from Adonis Aleppica. Phytochemistry 38, 1245-1250.
Pavlick, K. P., Laroux, F. S., Fuseler, J., et al., 2002. Role of reactive metabolites of oxygen and nitrogen in inflammatory
bowel disease. Free Radical Biol. ogy and Medicine. 33, 311–322.
Peluso, I., Campolongo, P., Valeri, L. R., Palmery, M., 2002. Intestinal motility disorder induced by free radicals: a new
model mimicking oxidative stress in gut. Pharmacological Research 46 (6), 533-538.
Pendota, S. C., Yakubu, M. T., Grierson, D. S., Afolayan, A. J., 2010. Effect of administration of aqueous extracts of
Hippobromus pauciflorus leaves in male Wistar rats. African journal of Traditional, Complimentary and Alternative
Medicine 7(1): 40-46.
Pendota, S. C., Yakubu, M. T., Grierson, D. S., Afolayan, A. J., 2009. Antiinflammatory, analgesic, antipyretic activities of
the aqueous extract of Pippobromus puaciflorus (L. F.) Radk leaves in Male Wistar rats. African Journal of
Biotechnology, 8(10), 2036-2041.
Perez-Bosque, A. and Moreto, M., 2010. A rat model of mild intestinal inflammation induced by Staphylococcus aureus
enterotoxins B. The 3rd International Immunonutrition Workshop held in Spain on 21-24 Oct., 2009. Proceedings of
the Nutrition Society, 69, 447-453.
Petri Jr., W. A., Miller, M., Binder, H. J., Levine, M. M., Dillingham, R., Guerrant, R. L., 2008. Enteric infections, diarrhea, and
their impact on function and development. Journal of Clinical. Investigation 118:1277–1290.
Pharoah, D. S., Varsan, H., Tathan, R. W., 2006. Expression of the inflammatory chemokines CCL5, CCL3 and CXCL10 in
juvenile idiopathic arthritis and demonstration of CCL5 production by a typical subset of CD8+T cells. Arthritis
Research and Therapy 8, R50.
Picerno, P., Autore, G., Marzocco, S., Meloni, M., Sanago, R., Aquino, R. P., 2005. Anti-inflammatory activity of cutaneous
irritation of cell cullins and reconstituted Human epidermis. Journal of Natural Products 68(11): 1610-1614.
Pietta, P. G., 2000. Flavonoids as antioxidants. Journal of Natural Products, 63, 1035–1042.
Pillai, N. R., 1992. Antidiarrhoeal activity of Punica granatum in experimental animals. Pharmaceutical Biology 30(3), 201204.
Pillay. P., Maharaj, V. J., Smith, P. J., 2008. Investigating South African plants as a source of new antimalarial drugs.
Journal of Ethnopharmacology 119, 438-454.
Podewils, L. J., Mintz, E. D., Nataro, J. P., Parashar, U. D., 2004. Acute, Infectious Diarrhoea Among Children in Developing
Countries. Journal of seminars in Pediatric Infectious Diseases 155-168.
Polya, G. M., 2003. Biochemical Targets of Plant Bioactive Compounds. A Pharmacological Reference Guide to Sites of
Action and Biological Effects. CRC Press, Florida.
Ponou, B. K., Barboni, L., Teponno, R. B., Mbiantcha, M., Nguelefack, T. B., Park, H-J., Lee, K-T., Taponjou, L. A., 2008.
Polyhydrooleanane-type triterpenoids from Combretum molle and their anti-inflammatory activity. Phytochemistry
letters 1(4), 183-187.
Pooley, E., 1998. A Field Guide to Wildflowers KwaZulu-Natal and the Eastern Region. Natal Flora Publications Trust,
Durban.
Prior, R. L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of antioxidant capacity and phenolics in
foods and dietary supplements. Journal of Agriculture and Food Chemistry 53 (10), 4290–4302.
166
Prozesky, E. A., Meyer, J. J. M., Louw, A. L., 2001. In vitro antiplasmodial activity and cytotoxicity of ethnobotanically
selected South African plants. Journal of Ethnopharmacology.76 (3), 81-308.
Pujol J., 1990. Natur Africa: The Herbalist Handbook.Natural Healers Foundation, Durban.
Puyvelde, V. L., De Kimpe, N., Costa, J., Munyjabo, V., Nyirankuliza, S., Hakizamungu, E., Schamp, N., 1989. Isolation of
flavonoids and chalcone from Helichrysum odoratissimum and synthesis of Helichrysetin. Journal of Natural Product
52(3), 629-633.
Quais, E. Y., Elokda, A. S., Ghalyun, Y. Y. A., Abdullla, F. A., 2007. Antidiarrhoeal activity of the aqueous extract of Punica
granatum (Pomegranate) peels. Pharmaceutical Biology 45(9), 715-720.
Rabe, T., Van Staden, J., 1997. Antibacterial activity of South African plants used for medicinal purposes. Journal of
Ethnopharmacology 56, 81–87.
Raccach, M., 1984. The antimicrobial activity of phenolic antioxidant in foods: a review. Journal of Food Safety 6, 141-170.
Radi, Z. A., Khan, N. K., 2006. Effects of cyclooxygenase inhibition on the gastrointestinal tract. Experimental and
Toxicological pathology 58, 163-173.
Ragone, M. I., Sella, M., Cornforti, P., Volonte, M. G., Consolini, A. E., 2007. The spasmolytic effect of Aloysia citriodora,
Palau (South American cedron) is partially due to its vitexin but not isovitexin on rat duodenums. Journal of
Ethnopharmacology 113, 258–266.
Rahman, I., Adcock, I. M., 2006. Oxidative stress and redox regulation of lung inflammation in COPD. European Respiratory
Journal 28, 219-242.
Ralstron, K. S., Petri Jr., W. A., 2011. Tissue destruction and invasion by Entamoeba histolytica. Trends in Parasitology, 27,
253-262.
Ramahivhana, J. N., Moyo, S. R., Obi, C. l., 2010. The possible role of medicinal plants in tackling resistant microbial
pathogens in Limpopo Province, South Africa. Journal of Medicinal plant Research, 4(11), 999-1002.
Ramaroa, N., Lereclus, D., 2006. Adhesion and cytotoxicity of Bacillus cereus and Bacillus thuringiensis to epithelial cells
are FlhA and PlcR dependent, respectively. Microbes and Infection 8, 1483-1491.
Ramful, D., Bahorun, T., Bourdon, E., Tarnus, E., Aruoma, O. I., 2010. Bioactive phenolics and antioxidant propensity of
flavedo extracts of Mauritian citrus fruits: Potential prophylactic ingredients for functional foods application.
Toxicology xxx (2010) xxx–xxx.
Rao, V. S. N., Santos, F. A., Sobreika, T. T., Souza, M. F., Melo, L. L., Silveira, E. R., 1997. Investigations on the
gastroprotective and antidiarrhea properties of ternatin, a tetramethoxyflavone from Egletes viscose. Planta Medica
63(2), 146-149.
Rasoanaivo, O., Ramanittrahasimbola, D., Rafatro, H., Rakotondramanana, D., Robijaona, B., Rakotozafy, A.,
Ratsimamanga-Urverg, S., Labaied, M., Grellier P., Allorge, L., Mambu, L., Frappier, F., 2004. Screening of extracts
of Madagascan plants in search of antiplasmodial compounds, Phytotherapy Research, 18, 743-747.
Rasoanaivo, P., Ratsimamanga-Urverg, S., 1993. Biological Evaluation of Plants with Reference to the Malagasy Flora.
Monograph for the IFS-NAPRECA Workshop on Bioassays, Antananarivo, Madagascar, pp 9–43, 72–83.
Rauha, J. P., 2001. The research for biological activity in Finnish plant extracts containing phenolic compounds, PhD
Thesis, Pharmacognosy Department, Pharmacy Faculty of Science, University of Helsinki, pp. 31–45
Ravikumara, M., 2008. Investigation of chronic diarrhoea. Paediatrics and Child health 18(10), 441-447.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying improved
ABTS radical cation decolourization assay. Free Radical Biology and Medicine 26, 1231-1237.
167
Rea AI. Schmidt JM. Setzer WN. Sibanda S. Taylor C. Gwebu ET., 2003. Cytotoxic activity of Ozoroa insignis from
Zimbabwe Fitoterapia 74(7-8), 732-735.
Reid, K. A., Maes, J., Maes, A., van Staden, J., De Kimpe, N., Mulholland, D. A. Verschaeve, L., 2006. Evaluation of the
mutagenic and antimutagenic effects of South African plants. Journal of Ethnopharmacology 106, 44–50.
Rekka, E., Kourounakis, P. N., 1991. Effect of hydroxyethyl rutenosides and related compounds on lipid peroxidation and
free radical scavenging activity-some structural aspects. Journal of Pharmacy and Pharmacology 43, 486–491.
Reyes, C. P. Nunez, M. J. Jimenez, I. A. Busserolles, J., Alcaraz, M. J., Bazzocchi, I. L., 2006. Activity of lupane
triterpenoids from Maytenus species as inhibitors of nitric oxide and prostaglandin E2. Bioorganic & Medicinal
Chemistry 14, 1573–1579.
Rhee, I. K., Van De Meent, M., Ingkaninan, K., Verpoorte, R., 2001: . A screening for acetylcholinesterase inhibitors from
Amaryllidaceae using silica gel thin-layer chromatography in combination with bioactivity staining. Journal of
Chromatography A 915, 217–223.
Rietjens, I. M. C. M., Boersma, M. G., Van der Woude, H., Jeurissen, S. M. F., Schutte, M. E., Alink, G. M., 2005. Flavonoids
and alkenylbenzenes: mechanisms of mutagenic action and carcinogenic risk. Mutation Research 574, 124–138.
Rijke, E., Out, P., Niessen, W. M. A., Ariese, F., Gooijer C., Brinkman, U. A., 2006. Analytical separation and detection
methods for flavonoids. Journal of Chromatography A 1112, 31–63.
Ríos, J. L., 2008. Ganoderma lucidum, unhongo con potenciales propiedades inmunoestimulantes. Revista de Fitoterapia 8,
135–146.
Ríos, J. L., Bas, E., Recio, M. C., 2005. Effects of natural products on contact dermatitis.Current Medicinal Chemistry. Antiinflammatory and Anti-allergic Agents 4, 65–80.
Rios, J. L., Recio, M. C., 2005. Medicinal plants and antimicrobial activity. Journal of Ethnopharmacology 100, 80-84.
Risa, J., Risa, A., Adersen, A., Gauguin, B., Stafford, G. I., Van Staden, J., Jager, A. K., Screening of plants used in South
Africa for epilepsy and convulsions in the GABAA-benzodiazepine receptor assay. Journal of Ethnopharmacology
93(2-3), 177-182.
Robak, J., Duniec, Z., Rzadkowska Bodalska, H., Olechnowicz Stepien, W., Cisowski, W., 1986. The effect of some
flavonoids on non-enzymatic lipid oxidation and enzymatic oxidation of arachidonic acid. Pol. ish Journal of.
Pharmacognosy and Pharmaceutical 38, 483-491.
Robert, K., Egon, S., Daneila, B., Florian, D., Christoph, W., Gunter, J. K., Emil, C. R., 2001. Role of Candida in antibioticassociated diarrhea. Journal of infectious diseases 184, 1065-1069.
Roberts, M., 1990. Indigenous Healing Herbs. Southern Book Publishers, South Africa, pp. 1–285.
Rocha Martin, L. R., Brenzan, M. A., Nakamura, C. V., Dias Filho, B. P., Nakamura, T. U., Ranei Cortez, L. E., Garcia
Cortez, D. A., 2011. In vitro antiviral activity from acanthaspermum australei on herpesvirus and poliovirus.
Pharmaceutical Biology 49 (1), 26-31.
Rocha-Martins, L. R. R., Benzan, M. A., Nakamura, C. V., Filho, B. P. D., Nakamura, T. U., Cortez, E. R., Corteez, D. A. G.,
2010. In vitro antiviral activity of Acanthospermum australe on herpevirus and poliovirus. Pharmaceutical biology
49(1), 26-31.
Rosa, A., Pollastro, F., Afzeri, A., Appendino, G., Melis, M. P., Dieana, M., Incani, A., Loru, D., Dessi, M. A., 2011. Protective
role of arzanol against lipid peroxidation in biological systems. Chemistry and Physics of lipids 164, 24-32.
168
Rosengren, Å., Fabricius, A., Guss, B., Sylvén S., Lindqvist, R., 2010. Occurrence of foodborne pathogens and
characterization of Staphylococcus aureus in cheese produced on farm-dairies. International journal of Food
microbiology
Russo, A., Cardile, V., Lombardo, L., Vanela, L., Vanela, A., Garbarino, J. A., 2005. Antioxidant activity and antiproliferative
action of methanolic extract of Geum quellyon sweet roots in human tumor cells lines. Journal of
Ethnopharmacology 100, 323-332.
Saini, N. K., Singhal, M., Srivastava, B., Sachdeva, K., Singh, C. C., 2011. Antimicrobial activity of Tecomaria capensis
leaves extracts. International Journal of Pharmaceutical Sciences Review and Research 7(1), 121-124.
Sairam, K., Hemalatha, S., Kumar, A., Srinivasan, T., Ganesh, J., Shankar, M., Venkataraman, S., 2003. Evaluation of antidiarrhoeal activity in seed extracts of Magnifera indica. J. Ethnopharmacol. 84: 11-15.
Salvemini, D., Masferrer, J. L., 1996. Interactions of nitric oxide with cyclooxygenase in vitro, ex vivo and in vivo studies.
Methods of Enzymology 269:12-25.
Samie, A., Obi, C. L., Bessong, P. O., Namrita, L., 2005. Activity profiles of fourteen selected medicinal plants from rural
Venda communities in South Africa against fifteen clinical bacterial species. African Journal of Biotechnology, 4,
1443–1451.
Samie, A., Obi, C. L., Lall, N., Meyer, J. J. M., 2009. In-vitro cytotoxicity and antimicrobial activities, against clinical isolates
of Campylobacter species and Entamoeba histolytica, of local medicinal plants from the Venda region, in South
Africa. Annals of Tropical Medicine & Parasitology 103 (2), 159–170.
Samie, A., Tambani, J., Harshfield, E., Green, E., Ramalivhana, J. N., Bessong, P. O., 2010. Antifugal activities of selected
Venda medicinal plants against Candida albicans, Candida krussei and Cryptococcus neoformans isolated from
South African AIDS patients. African Journal of Biotechnology 9(20), 2965-2976.
Samy, R. P., Gopalakrishnakone, P., 2008. Therapeutic Potential of Plants as Anti-microbials for Drug Discovery: Review
eCAM 1-12.
Sanders, K. M., 2001. Invited review: mechanisms of calcium handling in smooth muscles. Journal of Applied Physiology 91,
1438–1449.
Saunders, D. R., Wiggins, H. S., 1981. Conservation of mannitol, lactulose, and raffinose by the human colon. American
Journal of Physiology 1241, G397–E402.
Savi, L. A., Caon, T., de Oliveira, A. P., Sobottka, A. M., Werner, W., Reginatto, F. H., Schenkel, E. P., Barardi, C. R. M.,
Simões, C. M. O., 2010. Evaluation of antirotavirus activity of flavonoids. Fitoterapia xxx, xxx–xxx.
Saxena, G., Towers, G. H. N., Farmer, S., Hancock, R. E. W., 1995. Use of specific dyes in the detection of antimicrobial
compounds from crude plant extracts using thin layer chromatography agar overlay technique. Phytochemical
Analysis 6: 125-129.
Schenk, M., Mueller, C., 2008. The mucosal immune system at the gastrointestinal barrier. Best Practice & Research
Clinical Gastroenterology 22, 391–409.
Schiller, L. R., 1999. Secretory diarrhea. Current Gastroenterology Reports 1, 389-397.
Schmitz, H., Rokos, K., Florian, P., Gitter, A. H., Fromm, M., Scholz, P., et al., 2002. Supernatants of HIV-infected immune
cells affect the barrier function of human HT-29/B6 intestinal epithelial cells. AIDS 16(7), 983–991.
Schuier, M., Sies, H., Illek, B., Fischer, H., 2005. Cocoa-Related Flavonoids Inhibit CFTR-Mediated Chloride Transport
across T84 Human Colon Epithelia. Journal of Nutrition, 2320-2325.
169
Schulzke, J. D., Troger, H., Amasheh, M., 2009. Disorders of intestinal secretion and absorption. Best Practice & Research
Clinical Gastroenterology 23, 395–406.
Schwikkard, S., Zhou, B.-N., Glass, T. E., Sharp, J. L., Mattern, M. R., Johnson, R. K., Kingston, D. G. I., 2000. Bioactive
compounds from Combretum erythrophyllum. Journal of Natural Products 63, 457–460.
Sebothoma, C., 2010. Isolation and characterization of antibacterial compounds from Searsia (Rhus) leptodictya Diels
(Anacardiaceae). M. Sc. Thesis submitted to University of Limpopo
Selvarani, V., Hudson, J. B., 2009. Multiple inflammatory and antiviral activities in Adansonia digitata (Baobab) leaves, fruit,
and seeds. Journal of Medicinal plant Research 3(8), 576-582.
Shai, L. J., McGaw, L. J., Aderogba, L. J., Mdee, L. K., Eloff, J. N., 2008b. Four pentacyclic triterpenoids with antifungal and
antibacterial activity from Curtisia dentata (Burm.f) C.A. Sm. Leaves. Journal of Ethnopharmacology 118, 238–244.
Shai, L. J., McGaw, L. J., Eloff, J. N., 2009. Extracts of the leaves and twigs of the threatened tree Curtisia dentata
(Cornaceae) are more active against Candida albicans and other microorganisms than the stem bark extract. South
African Journal of Botany 75, 363–366.
Shai, L. J., McGaw, L. J., Masoko, P., Eloff J. N., 2008a. Antifungal and antibacterial activity of seven traditionally used
South African plant species against C.albicans. South African Journal of Botany 74, 677-684.
Shale, T. L., Stirk, W. A., van Staden, J., 2005. Variation in antibacterial and anti-inflammatory activity of different growth
forms of Malva parviflora and evidence for synergism of the anti-inflammatory compounds. Journal of
Ethnopharmacology 96, 325-330.
Sharkey, K. A., Mawe, G. M., 2002. Neuroimmune and epithelial interactions in intestinal inflammation. Current Opinion in
Pharmacology 2, 669-677.
Sibandze, G. F., Van Zyl, R. L., Van Vuuren, S. F., 2010. The anti-diarrhoeal properties of Breonadia salicina, Syzygium
cordatum and Ozoroa sphaerocarpa when used in combination in Swazi traditional medicine. Journal of
Ethnopharmacology 132(2), 506-511.
Sikander, A., Rana, S. V., Prasad, K. K., 2009. Role of serotonin in gastrointestinal motility and irritable bowel syndrome.
Clinica Chimica Acta 403, 47–55.
Simmons, D. L., Botting, R. M., Hla, T., 2004. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and
inhibition. Pharmacological Review 56, 387-437.
Simpson, A. J., McNally, D. J., Simpson, M. J., 2011. NMR spectroscopy in environmental research: From molecular
interactions to global processes. Progress in Nuclear Magnetic Resonance Spectroscopy 58, 97–175.
Slater, T. F., Cheeseman, K. H., Davies, M. J., Proudfoot, K., Xin, W., 1987. Free radical mechanisms in relation to tissue
injury. Proceedings of the Nutrition Society 46, 1-12.
Smid, S. D., Svensson, K. M., 2009. Inhibition of cyclooxygenase-2 and EP1 receptor antagonism reduces human colonic
longitudinal muscle contractility in vitro. Prostaglandins and other lipid mediators 88, 117-121.
Smirnoff, N., Cumbes, O. J., 1996. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28, 1057–
1060.
Soderholm, J. D., Perdue M. H., 2006. Effect of stress on intestinal mucosal function. Physiology of the gastrointestinal tract,
Fourth Edition, edited by Leonard R. Johnson. Academic Press.
Sousa, A., Ferreira, I. C. F. R., Calhelha R., 2006. Phenolic and antimicrobial activity of traditional stoned table olives
“alcaparra”. Bioorganic and Medicinal Chemistry 14, 8533-8538.
170
Spickett, A. M. Van Der Merwe, D., Matthe, O., 2007. The effect of orally administered Aloe marlothii leaveson Boophilus
decoloratus tick burdens on cattle. Experimental and Applied Acarol 41, 139–146.
Spiller, R. C., 2004. Inflammation as a basis for functional GI disorder. Best Practice and Research Clinical
Gastroenterology 18, 641–661.
Spiller, R., Garsed, K., 2009. Infection, inflammation and irritable bowel syndrome. Digestive and Liver disease, 41, 844849.
Sprague, A. H., Khalil, R. A., 2009. Inflammatory cytokines in vascular dysfunction and vascular disease. Biochemical
Pharmacology 78, 539–552.
Springfield, E. P., Amabeoku, G., Weitz, F., Mabusela, W., Johnson, Q., 2003. An assessment of two Carpobrotus species
extracts as potential antimicrobial agents. Phytomedicine 10, 434–439.
Stables, M. J., Gilroy, D. W., 2010. Old and new generation lipid mediators in acute inflammation and resolution. Progress in
Lipid Research, doi:10.1016/j.plipres.2010.07.005.
Stanzel, R. D. P., Lourenssen, S., Blennerhassett, M. G., 2008. Inflammation causes expression of NGF in epithelial cells of
the rat colon. Experimental Neurology 211, 203–213.
Steenkamp, V., Gouws, M. C., 2006. Cytotoxicity of Six South African medicinal plant extracts used in the traditional of
Cancer. South African Journal of Botany 72(4), 630-633.
Steenkamp, V., Gouws, M. C., Gulumian, M., Elgorashi, E. E., Van Staden, J., 2006. Studies on antibacterial,
antinflammatory and antioxidant activity of herbal remedies used in the treatment of benign prostatic and prostatitis.
Journal of Ethnopharmacology 103(1), 71-75.
Steenkamp, V., Mathivha, E., Gouws, M. C., Van Rensburg, C. E. J., 2004. Studies on antibacterial, antioxidant and
fibroblast growth stimulation of wound healing remedies from South Africa. Journal of Ethnopharmacology 95(2-3),
353-357.
Stehbens, W. E., 2004. Oxidative stress in viral hepatitis and AIDS. Experimental and Molecular Pathology 121-132.
Sticher, O., 2008: Natural product isolation. Natural product report 25, 517-554.
Stoddart, B., Wilcox, M. H., 2002. Clostridium difficile. Current Opinion in Infectious. Diseases 15, 513–518.
Stojiljkovic, V., Todorovic, A., Pejic, S., Kasapovic, J., Saicic, Z. S., Radlovic, N., Pajovic, S., 2009. Antioxidant status and
lipid peroxidation in small intestinal mucosa of children with celiac disease. Clinical Biochemistry 42, 1431–1437
Suleiman, M. M., McGaw, L. J., Naidoo, V., Eloff, J. N., 2010. Evaluation of several tree species for activity against the
animal fungal pathogen Aspergillus fumigatus. South African Journal of Botany 76, 64-71.
Sun, J., Hu, X.-L., Le, G.-W., Shi, Y.-L., 2010. Lactobacilli prevent hydroxy radical production and inhibit Escherichia coli and
Enterococcus growth in system mimicking colon fermentation. Letters of Applied Microbiology 50, 264-269.
Suzuki, T., Hara, H., 2011. Role of flavonoids in intestinal tight junction regulation. Journal of Nutritional Biochemistry 22,
401–408.
Svenningsen, A. B., Madsen, K. D., Liljefors, T., Stafford, G. I., Staden, J., Jäger, A. K., 2006. Biflavones from Rhus species
with affinity for the GABA(A)/benzodiazepine receptor. Journal of Ethnopharmacology 103, 276–280.
Taganna, J. C., Quanico, J. P., Perono, R. M. G., Amor, E. C., Rivera, W. L., 2011. Tannin-rich fraction from Terminalia
catappa inhibits quorum sensing (QS) in Chromobacterium violaceum and the QS-controlled biofilm maturation
and LasA staphylolytic activity in Pseudomonas aeruginosa. Journal of Ethnopharmacology 134, 865–871
Taguri, T., Tanaka, T., Kouno, I., 2004. Antimicrobial activity of 10 different plant polyphenols against bacteria causing foodborne disease. Biol. Pharm. Bull. 27(12) 1965-1969.
171
Taiwo, O. B., Olajide, O. A., Soyannwo, O. O., Makinde, J. M., 2000. Antiinflammatory, antipyretic and antispasmodic
properties of Chromomaela odorata. Pharmaceutical Biology 38(5), 367-370.
Takeuchi K, Miyazawa T, Tanaka A, Kato S, Kunikata T., 2002. Pathogenic importance of intestinal hypermotility in NSAIDinduced small intestinal damage in rats. Digestion 66, 30–41.
Takeuchi, K., Tanaka, A., Kato, S., Amagase, K., Satoh, H., 2010. Roles of COX inhibition in pathogenesis of NSAIDinduced small intestinal damage. Clinica Chimica Acta 411, 459–466.
Takeuchi, T., Fujinami, K., Goto, H., Fujita, A., Taketo, M.M., Manabe, T., Matsui, M., Hata, F., 2005. Roles of M2 and M4
muscarinic receptors in regulating acetylcholine release from myenteric neurons of mouse ileum. Journal of
Neurophysiology 93, 2841–2848.
Taraporewala, I. B., Kauffman, J. M., 1990. Synthesis and structure-activity relation-ship of anti-inflammatory 9,10-dihydro 9oxo-2-acridine alkanoic acids and 4-(2-carboxyphenyl) aminobenzenealkanoic acids. Journal of Pharmacology
Science 79, 173–178.
Taylor, J. L. S., van Staden, J., 2001. COX-1 inhibitory activity in extracts from Eucomis L’Herit. species. Journal of
Ethnopharmacology 75, 257–265.
Teke, G. N., Kuiate, J. R., Ngouateu, O. B., Gatsing, D., 2007. Antidiarrhoeal and antimicrobial activities of Emilia coccinea
(Sims) G. Don extracts. Journal of Ethnopharmacology 112, 278-283.
Thamburan, S., Klaasen, J., Mabusela, W. T., Cannon, J. F., Folk, W., Johnson, Q., 2006. Tulbaghia alliacea Phytotherapy:
A Potential Anti-infective Remedy for Candidiasis. Phytotherapy Research 20, 844–850
Thapar, N., Sanderson, I. R., 2004. Diarrhoea in children: an interface between developing and developed countries. The
Lancet 363, 641–53.
Thiagarajah, J. R., Verkman, A. S., 2005. New drug targets for cholera therapy. TRENDS in Pharmacological Sciences
26(4), 172-174.
Thring, T. S. A., Springfield, E. P., Weitz, F. M., 2007. Antimicrobial activities of four plant species from the Southern
Overberg region of South Africa. African Journal of Biotechnology 6(15), 1779-1784.
Thring, T. S. A., Weitz, F. M., 2006. Medicinal plant use in the Bredasdorp/Elim region of the Southern Overberg in the
Western Cape Province of South Africa. Journal of Ethnopharmacology 103, 261–275.
Tobin, G., Giglio, D., Lundgren, O., 2009. Muscarinic receptor subtypes in the alimentary tract. J. ournal of Physiol. ogy and
Pharmacol. ogy 60 (1), 3–21.
Todd, E. C. D., Notermans, S., 2011. Surveillance of listeriosis and its causative pathogen, Listeria monocytogenes. Food
Control 22, 1484-1490.
Tona, L., Cimanga, R. K., Mesia, K., Musuamba, C.T., De Bruyne, T., Apers, S., Hernans, N., Van Miert, S., Pieters, L.,
Totte, J., Vlietinck, A.J., 2000. Antiamoebic and spasmolytic activities of extracts from some antidiarrhoeal traditional
preparations used in Kinshasa, Congo. Phytomedicine 2000 7(1), 31-8.
Tona, L., Kambu, K., Mesia, K., Cimanga, K., De Bruyne, T., Pieters, L., Totté, J., Vlietinck, A.J., 1999. Biological screening
of traditional preparations from some medicinal plants used as antidiarrhoeal in Kinshasa, Congo. Phytomedicine 6,
59–66.
Tona, L., Kambu, K., Ngimbi, N., Cimanga, R. K., Vlietinck, A. J., 1998. Antiameobic and phytochemical screening of some
Congolese medicinal plants. Journal of Ethnopharmacology 61(1), 57-65.
Troeger, H., Epple, H. J., Schneider, T., Wahnschaffe, U., Ullrich, R., Burchard, G. D., et al., 2007. Effect of chronic Giardia
lamblia infection on epithelial transport and barrier function in human duodenum. Gut 56(3), 328-335.
172
Troeger, H., Loddenkemper, C., Schneider, T., Schreier, E., Epple, H. J., Zeitz, M., et al., 2009. Structural and functional
changes of the duodenum in human norovirus infection. Gut 58, 1070-1077.
Tshibangu, J. N., Wright, A. D., Konig, G. M., 2003. HPLC isolation of the anti-plasmodially active bisbenzylisoquinone
alkaloids present in roots of Cissampelos mucronata. Phytochemical Analysis 14(1),13-22.
Tshikalange, T. E., Hussein, A. A., 2010. Cytotoxicity activity of compounds from Elaeodendron transvaalense ethanol
extract. Journal of Medicinal Plant Research 4(16), 1695-1697.
Tshikalange, T. E., Meyer, J. J. M., Hussein, A. A., 2005. Antimicrobial activity, toxicity and the isolation of a bioactive
compound from plants used to treat sexually transmitted diseases. Journal of Ethnopharmacology 96(3): ), 515-519.
Turel, I., Ozbek, H., Erten, R., Oner, A. C., Cengiz, N., Yilmaz, O., 2009. Hepatoprotective and anti-inflammatory activities of
Plantago major L. Indian Journal of Pharmacology 41(3), 120-124.
Uchida, K., 1999. Current status of acrolein as a lipid peroxidation product. Trends Cardiovascular Medicine 9, 109–113.
UNAIDS, 2006. Report on the Global AIDS Epidemic, May 2006.
Unno, T., Matsuyama, H., Sakamoto, T., Uchiyama, M., Izumi, Y., Okamoto, H., Yamada, M., Wess, J., Komori, S., 2005.
M2 and M3 muscarinic receptor-mediated contractions in longitudinal smooth muscle of the ileum studied with
receptor knockout mice. British Journal of Pharmacology 146, 98–108.
Valeur, J., Lappalainen J., Rita, H., Lin, A. H., Kovanen, P. T., Berstad, A., Eklund, K., Vaali K., 2009. Food allergy alters
jejuna circular muscle contractility and induces local inflammatory cytokines expression in a mouse model. BMC
gastroenterology 9:33.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., Telser, J., 2007. Free radical and antioxidants in normal
physiological functions and human diseases. The International Journal of Biochemistry and Cell Biology 39, 44-84.
Valko, M., Morris, H., & Cronin, M. T. D., (2005). Metals, toxicity and oxidative stress. Current MedicinalChemistry 12, 1161–
1208.
Van Damme, I. Habib, I., De Zutter, L., 2010. “Yersinia enterocolitica in slaughter pig tonsils: enumeration and detection by
enrichment versus direct plating culture,” Food Microbiology, 27(1), 158–161.
Van der Merwe, D., Swan, G. E., Botha, C. J., 2001. Use of ethnoveterinary medicinal plants in cattle by Setswana-speaking
people in the Madikwe area of the North West Province of South Africa. Journal of the South African Veterinary
Association 72, 189-196.
Van der Watt, E., Pretorious, J. C., 2001. Purification and identification of active antibacterial components in Carpobrotus
edulis. L. Journal of Ethnopharmacology 76, 87-91.
Van Dyk, S., Griffitto, S., Van Zyl, R. L., Malan, S. F., 2009. The importance of including toxicity assays when screening
plant extracts for antimalarial activity. African Journal of Biotechnology 8(20), 5595-5601.
Van Wyk, B., Van Wyk, P., 1997. Field Guide to Trees of Southern Africa. Struik Publishers (A Division of New Holland
Publishing, South Africa), Cape Town, South Africa, p. 496.
Van Wyk, B., Wink, M., 2004. Health disorders and Medicinal plants. In: Medicinal plants of the world. Published by Briza
Publications CK90/11690/23, P O Box 56569, Arcadia 0007, Pretoria, South Africa Pp 351-370.
Van Wyk, B.-E., 2008a. A review of Khoi-San and Cape Dutch medical ethnobotany. Journal of Ethnopharmacology 119,
331–341.
Van Wyk, B.-E., 2008b. A broad review of commercially important Southern African medicinal plants. Journal of
Ethnopharmacology 119, 342–355.
173
Van Wyk, B.-E., de Wet, H., Van Heerden, F. R., 2008. An ethnobotanical survey of medicinal plants in the Southeastern
Karoo, South Africa. South African Journal of Botany 74, 696–704.
Van Wyk, B.-E., Gericke, N., 2000. People’s Plants: A Guide to Useful Plants of Southern Africa. Briza Publications,
Pretoria, ISBN: 978 1 875093 19 9
Velazquez, C., Calzada, F., Esquivel, B., Barbosa, E., Calzada, S., 2009. Antisecretory activity from the flower of
Chiranthodendron pentadactylon and its flavonoids in intestinal fluid accumulation induced by Vibrio cholerae toxin
in rats. Journal of Ethnopharmacology, 126, 455-458.
Velázquez, C., Calzada, F., Torres, J., González, F., Ceballos, G., 2006. Antisecretory activity of plants used to treat
gastrointestinal disorders in Mexico. Journal of Ethnopharmacology 103, 66–70.
Venter, F., Venter, J.-A., 2002. Making the Most of Indigenous Trees, rev. edition. Briza Publications, Pretoria, South Africa,
p. 28
Vermerris, W., and Nicholson, R., 2006. Phenolic Compounds Biochemistry, Publisher Springer, New York. Pp 151-196.
Verschaeve, L., Van Staden, J. 2008. Mutagenic and antimutagenic properties of extracts from African South traditional
medicinal plants. Journal of Ethnopharmacology 119(3), 575-587.
Vital, P. G., Rivera, W. L., 2009. Antimicrobial activity and cytotoxicity of Chromolaena odorata (L. F.) King and Uncaria
perrottetii (A. Rich) Merr. Extracts. Journal of Medicinal plant Research 3(7), 511-518.
Vitali, F., Fonte, G., Saija, A., Tita, B., 2006. Inhibition of intestinal motility and secretion by extracts of Epilobium spp. in
mice. Journal of Ethnopharmacology 107, 342-348.
Viviers, P., Kolodziej, H., Young, D., Ferreira, D., Roux, D., 1983. Journal of Chemical Society, Perkin Trans 1, 2555.
Voirin, B.; Jay, M.; Hauteville, M., 1975. Isoetine, nouvelle flavone isolee de Isoetes delilei et de Isoetes durieui.
Phytochemistry 14, 257-259.
Von Koenen, E., 2001. Medicinal Poisonous and Edible Plants in Namibia. Klaus Hess Publishers, Windhoek and Göttingen,
ISBN: Namibia 99916-747-4-8; Germany 3-9804518-7-9.
Voravuthikunchai, S. P., and Limsuwan, S., 2006. Medicinal plant Extracts as anti-Escherichia coli 0157:H7 agent and their
effects on Bacterial Cell aggregation. Journal of Food Protection 69(10), 2336-2341.
Vundac, V. B., Brantner, A. H., Plazibat, M., 2007. Content of Polyphenolic constituent and antioxidant activity of some
stachys taxa. Food Chemistry 104, 1277-1281.
Wafo, P., Kamdeen, R. S. T., Ali, Z., Anjuin, S., Begum, A., Oluyemisi, O. O., Khan, S. N., Ngadjui, B. T., Etoa, X. F.,
Choudhany, M. I., Kaurene-typye diterpenoids from Chromolaena odorata, their X-ray diffraction studies and potent
α-glycosidase inhibition of 16-kauren-19-oic acid.
Wang, G. J., Chen, Y. M., Wang, T. M., Lee, C. K., Chen, K. J., Lee, T. H., 2008. Flavonoids with iNOS inhibitory activity
from Pogonatherum crinitum. Journal of Ethnopharmacology 118, 71–78.
Wang, M., Takeda, K., Shiraishi, Y., Okamoto, M., Dakhama, A., Joetham, A., Gelfrand, E. W., 2010. Peanut-induced
intestinal allergy is mediated through a mast cell–IgE–Fc RI–IL-13 pathway. Journal of Allergy and Clinical
Immunology 126(2), 301-316.
Wanjala, C.C.W., Juma, B.F., Bojase, G., Gashe, B.A., Majinda, R.R.T., 2002. Erythrinaline alkaloids and antimicrobial
flavonoids from Erythrina latissima. Planta Medica 68, 640–642
Watt, J. M., Breyer-Brandwijk, M. G., 1962. The Medicinal and Poisonous Plants of Southern and Eastern Africa, second
edition, Livingstone, London.
174
Watt, J. M., Breyer-Brandwijk, M. G., 1996. The Medicinal and Poisonous Plants of Southern and Eastern Africa, 2nd ed. E.
& S. Livingstone Ltd., London.
Weigenand, O., Hussein, A. A., Lall, N., Meyer, J. J. M., 2004. Antibacterial activities of Naphthoquinones and Triterpenoids
from Euclea natalensis. Journal of Natural Product 67, 1936-1938.
Wernes, S. W., and Lucchesi, R., 1990. Free radicals and ischemic tissue injury. Trends in Pharmacological Sciences 11,
161-166.
Wettasinghe, M., Shahidi, F., Amarowicz, R., Abou-Zaid, M. M., 2001. Phenolic acids in defatted seeds of borage (Borago
officinalis L.). Food Chemistry 75, 49-56.
Wistrom, S. R., Norrby, E. B., Myhre, E. B., et al., 2001. Frequency of antibiotic-associated diarrhoea in 2462 antibiotic
treated hospitalized patients: a prospective study. Journal of Antimicrobial Chemotherapy 47, 43–50.
Wood, J. D., 2004. Enteric Neuroimmunophysiology and pathophysiology. Gastroenterology 127, 635-657.
World Health Organization (WHO) and United Nation Children Fund (UNICEF), 2004. Clinical World Health Organization
(WHO) and United Nation Children Fund (UNICEF), 2004: Clinical Management of Acute Diarrhea.
WHO/FCH/CAH/04.7 World Health Organization, Geneva.Management of Acute Diarrhea. WHO/FCH/CAH/04.7
World Health Organization, Geneva.
Wouters, M. M., Farrugia, G., Schemann, M., 2007. 5-HT receptors on interstitial cells of Cajal, smooth muscle and enteric
nerves. Neurogastroenterology Motility 19 (Suppl 2), 5–12.
Wu, S. W., Dornbusch, K., Kronval, G., 1999. Genetic characterization of resistance to extended-spectrum betalactams in
Klebsiella oxytoca isolates recovered from patients with septicemia at hospitals in the Stockholm area. Antimicrobial
Agents and Chemotherapy 43, 1294–1297.
Wynn, S. G., Fougere, B. J., 2007. A systems-based approach: Diarrhea-Therapeutic rationale. In: Veterinary Herbal
medicine. (Edited by Wynn GS and Fougere BJ) Mosby Elsevier 11830 Westline Industrial Drive, St Louis, Mussolini
63146: 33336-337.
Xu, Y., Oliverson, B. G., Simmons, D. L., 2007. Trifunctional inhibition of COX-2 by extracts of Lonicera japonica: Direct
inhibition, transcriptional and post-transcriptional down regulation. Journal of Ethnopharmacology 111:667-670.
Xue, C., Tada, Y., Dong, X., Heitman, J., 2007. The human fungal pathogen Cryptococcus can complete its sexual cycle
during a pathogenic association with plants. Cell Host and Microbes 1, 2633-273.
Yadav, A. K., Tangpu, V., 2009. Therapeutic efficacy of Biden pilosa L. Var. radiata and Galinsoga parviflora Cav. in
experimentally induced diarrhoea in mice. Phytopharmacology and Therapeutic values V, 35-45.
Yakubu, M.T., Afolayan, A.J., 2009. Reproductive toxicologic evaluations of Bulbine natalensis baker stem extract in albino
rats. Theriogenology 72, 322–332.
Yamagiwa, Y., Ohashi, K., Sakamota, Y., Hirakawa, S., Kamikawa, T., 1987. Syntheses of anacardic acids and ginkgoic
acid. Tetrahedron 43(15), 3387.
Yarnell, E., 2007. Plant chemistry in Veterinary medicine: Medicinal constituents and their mechanism of action. In:
Veterinary Herbal medicine. (Edited by Wynn GS and Fougere BJ) Mosby Elsevier 11830 Westline Industrial
Drive, St Louis, Mussolini 63146: 167-168.
Yff, B. T. S., Lindsey, K. L., Taylor, M. B., Erasmus, D. G., Jager, A. K., 2002. The pharmacological screening of Pentanisia
pruelloides and the isolation of the antibacterial acompound palmitic acid. Journal of Ethnopharmacology 79(1), 101107.
175
Yokozawa, T., Dong, E., Chung, H. Y., Oura, H., Nakagawa, H., 1997. Inhibitory effect of green tea on injury to a cultured
renal epithelial cell line, LLC-PK1. Bioscience Biotechnology and Biochemistry 61, 204-206.
Zschocke, S., Van Staden, J., Paulus, K., Bauer, R., Horn, M. M., Munro, O. Q., Brown, N, J., Drewes, S. E., 2000.
Stereostructure and anti-inflammatory activity of three diastereomers of Ocobullenone from Ocotea bullata.
Phytochemistry 54, 591-595.
Zwenger, S., Basu, C., 2008. Plant terpenoids: applications and future potentials. Biotechnology and Molecular Biology
Reviews 3, 001-007.
176
Appendix 2.1: ETHNOBOTANICAL AND LITERATURE INFORMATION OF MEDICINAL PLANT SPECIES USED TRADITIONALLY FOR TREATING DIARRHOEA IN SOUTH AFRICA
Family/Plant
Local names
Part used
Ethnopharmacological information
Biological activities investigated
Bioactive compound(s) isolated
species
Aizoaceae
Carpobrotus
perdevy
Leaf juice
Sore throat, dysentery, mouth infection
Antibactertial (Oskay et al., 2009)
2-descarboxy-betanidin (Dembitsky, 2005)
acinaciformis (L.) L.
(van Wyk, 2008)
Bolus
Carpobrotus edulis
Ikhambileaves
Diarrhoea, digestive problems,
Antibacterial (Van der Watt et al.,
Rutin, hyperoside, neohesperidin, catechin, ferulic acid (van
(L.) L. Bolus
lamabulawo.
allergy(Thring and Weitz, 2006); dysentery 2001)
der Watt et al., 2001)
Umgongozi
(van Wyk, 2008)
Carpobrotus muirii
Leaves
Dysentery, digestive problem, mouth
Antimicrobial (Springfield et al,
(L.) L. Bolus
ulcers, thrush (Thring and Weitz, 2006)
2003)
Alliaceae
Agapanthus
uMkhondo (X)
Roots
Diarrhoea in sheep and goat (Dold and
praecox Willd.
Cocks, 2001, McGaw and Eloff., 2008)
Tulbaghia alliacea
Umwelela X,
bulb
Stomach ache, fever, tuberculosis,
Antimycobacterial (Bamuamba et
L.f.
ivimba-mpunzi X,
influenza (Bisi-Johnson et al., 2010)
al., 2008), Mutagenicty and
Sikwa Z
antimutagenicity (Reid et al., 2006),
anticandidiasis (Thamburan et al.,
2006)
Amaranthaceae
Guilleminea densa
Sephatho (S)
Root
Decoction for diarrhoea (Mathabe et al.,
Moq
2006)
Hermbstaedtia
Ubuphuphu (X, Z)
leaves
Food and infusion for diarrhoea (Bisiodorata Wild
Johnson et al., 2010); Root cleansing
stomach wash alone or with Acaccia
xanthophloea and (Hutchings et al., 1996).
Amaryllidacceae
Scadoxus puniceus
UmphomphoBulb and
Stomach ache, diarrhoea, nausea (BisiAntimicrobial, anti-inflammatory,
(L.) Friis and Nordal wezinja,
root
Johnson et al., 2010)
acetylcholinesterase inhibition and
Isiphompho umgola
mutagenic activities (Ndhlala et al.,
Z
2010)
Anacardiaceae
Mangifera indica L.
Umango
Leaves,
Diarrhoea (de Wet et al., 2010)
Antidiarrhoeal (Sairam et al., 2003), Gallotannins (Engels et al., 2010), mangiferrin (Singh et al.,
bark
antidiabetic (Aderibigbe et al., 2001 2009).
Ozoroa insignis
Monoko
Stem bark
Decoction for diarrhoea (Mathabe et al.,
Antibacterial (Mathabe et al., 2006); 6-pentadecylsalicylic acid (antifouling), tirucallane triterpenes
Delile
2006), vinearal diseases, parasites, kidney antigardial (Johns et al., 1995),
(Liu and Abreu, 2006)
trouble (Liu and Abreu, 2006)
antimalarial (Asase et al., 2005),
Cytotoxicity (Rea et al., 2003),
antischistosomiasis (Molgaard et
al., 2001; Ndamba et al., 1994).
177
Ozoroa mucronata
(Bernh.ex C.Krauss)
R.fern & A. Fern
Ozoroa paniculosa
(Sond.) R. & A.
Fernandes
root
Diarrhoea, intestinal parasites and
stomach trouble (Yamagiwa et al., 1987)
LOX inhibition, PG synthase
inhibition (Kubo et al, 1987)
Anarcardic acid (LOX inhibition) (Ha and Kubo, 2005),
Moronic acid (Hotesttmann Kaldas and Nakanishi, 1979)
Mubandulakhali,
Mudumbula (V)
Bark, root
bark
Antioxidant (Motlhanka, 2008),
antimicrobial and antimycobacterial
-
Ozoroa
schaerocarpa R.
Fern & A. Fern
Protorhus longifolia
(Bernh.ex C.
Krauss) Engl.
Mudumbula (V)
Bark
Abdominal problems in animal (Hutching
et al., 1996), Diarrhoea, sweating sickness
(Van der Merwe et al., 2001; McGaw et
al., 2008)
Infusion for diarrhoea (Sibandze et al.,
2010)
Antiescherichial (Sibandze et al.,
2010)
-
i(u)Zntlwa, ikubalo,
umkupati X
Bark
Heartwater and diarrhoea in cows (Dold and
Cocks, 2001, McGaw and Eloff, 2008); Heart
burn and stomach bleeding (Hutchings et al.,
1996)
Antimicrobial (Suleiman et al.,
2010)
-
Sclerocarya birrea
(A. Rich.) Hochst.
subsp. caffra
(Sond.)
Mufula (V)
Leaves,
bark, roots
Diarrhoea and fractures (Van der Merwe
et al., 2001; McGaw et al., 2008)
Gallotannin, tannic, mallic, gallic and citric acid, triterpene,
flavonoid, coumarins (Ojewole et al., 2010)
Searsia gueinzii
Sond (Syn Rhus
gueinii Sond
Searsia incisa L.f.
Mushakaladza (V)
root
Gastrointestinal infections (Elgorashi et al.,
2003)
Mutagenicity, antimutagenicity
(Elgorashi et al., 2003),
Antibacterial, antihelmintic and
cytotoxicity (McGaw et al., 2007),
antidiarrhoea (Galvez et al., 1991),
antibacterial (Eloff, 2001), antiinflammation (Ojewole, 2010),
antioxidant (Braca et al., 2003),
anti-diabetic (Ojewole, 2004)
Mutagenicity , antimutagenicity
(Elgorashi et al., 2003)
uNongquthu
Root and
bark
decoction
Shock and diarrhoea (Dold and Cocks,
2001, McGaw et al., 2008)
Searsia lancea L.f.
Mushakaladza (V)
Searsia leptodictya
Diels
Mushakaladza (V)
leaves
Searsia pendulina
Jacq.
Searsia pentheri
Zahlbr.
Searsia rogersii
Schonland
-
Leaves
Muthasiri (V)
leaves
Muthasiri (V)
Bark
Pain, watery diarrhoea (Samie et al.,
2010)
Muembe (V)
Bark
Toothache, venereal, diarrhoea (Mabogo,
Annonaceae
Annona
Diarrhoea and gallsickness (Van der
Merwe et al., 2001; McGaw et al., 2008)
Browser, gall sickness in cattle, infectious
disease, chest and abdominal pain
(Sebothoma, 2010)
Stomach ailment, enema in children
(Coates-Palgrave, 2002)
Epilepsy (Svenningsen et al., 2006)
-
Antibacterial, antihelminttic and
cytotoxicity (McGaw et al., 2007)
Antimicrobial (Sebothhoma, 2010)
-
-
-
GABAA/benzodiazepine receptor
affinity (Svenningsen et al., 2006)
Antifungal (Samie et al., 2010),
Antimycobacterium (Green et al.,
2010)
Apeginin, agathisflavone (Svenningsen et al., 2006)
-
Antidiarrhoeal (Suleiman et al.,
Annnosenegalina (cytotoxic and antiparasitic), Annonacin
(-)-leucofisetinidin, (-)-epicatechin and [4,8]-( +)fisetinidol-(-)-epicatechin (Viviers et al., 1983)
178
senegalensis Pers.
Uvaria chamae P.
Beauv
Apiaceae
Alepidia
amatymbica Eckl. &
Zeyh.
Centella asiatica
(L.) Urb.
Iqwili, Ikhathazo (Z)
Centella glabrata L.
Foeniculum vulgare
Mill.
1990; More et al., 2008)
2008), antivenom (Adzu et al.,
2005), antimalaria (Okokon et al.,
2006)
Root
Catarrh, dysentery, fever, hematemesis,
inflammation, jaundice, wounds, yellow
fever (Reid et al., 2006)
Antimalaria (Okokon et al., 2006);
mutagenic and antimutagenic (Reid
et al., 2006)
Root
Decoction for diarrhoea (Appidi et al.,
2008)
Antimicrobial, anti-inflammatory and
genotoxicity (Mulaudzi et al., 2009)
Root
Chronic diarrhoea and dysentery;
diaphoretic (van Wyk, 2008)
Modulator of nitric oxide production
and TNF-α (Nhiem et al., 2011), ,
lipid peroxidation (Kumar and
Muller, 1999)
Root and
stalk
Leaf
Chronic diarrhoea and dysentery,
diaphoretic (van Wyk, 2008b)
Flatulence, cough, diuretic, digestive
problem, diarrhoea, stomach ache and
cramps (Watt and Breeyer-Brandwijk,
1962; van Wyk, 1997)
Antimicrobial (Bacillus cereus,
Clostridium botulinum, Salmonella
enteritidis, Staphylococcus aureus,
Yersinia enterocolitica) (Ceylan and
Fung, 2004)
Falcarindiol (antifungal, antibiotic and analgesic,
antinociceptive, DNA topoisomerase inhibitor, phytotoxic,
allelochemical, antimutagenic and antiproliferative agents), 1(4-hydroxyphenyl)-1,2-propanediol form, 4’ methyl ether
(phytotoxin, antiparasitic, nematocidal agent)
Severe gastrointestinal irritation
(Verschaeve and Van Staden, 2008),
Decoction for stomach ache, diarrhoea
(Bisi-Johnson et al., 2010)
Diarrhoea (de Wet et al., 2010)
Genotoxicity (Elgorashi et al.,
2003); Epilepsy and convulsion
(Risa et al., 2004)
Acolongifloroside K and H (antineoplastic agent)
Antimicrobial (van Vuuren and
Naidoo, 2010)
Infusion for diarrhoea (de Wet et al.,
2010), Increase livestock productivity
Antibacterial, anti-inflammatory and
mutagenic effects (Luseba et al,
Serpentine (antitumour activity);apparicine (cytotoxin, weak
antbacterial, antiviral agent active against Polio virus,
analeptic properties); β-carboline (induced mutagenicity,
antiparasitic, antitrypanosomal agent); Catharanthamine
(antitumour); Trichosetin (antibacteria); 16-Epi-2isositsirikine antineoplastic); Leurosine
(antihyperglycaemic); Lochnerinine (antitumour);
Pericyclivine (weak cytotoxic activity); 15’,20’anhydroviriblastine (antineoplastic agent); Vindoline
(antineoplastic); Vindolinine (antiglycaemic agent, antifungi);
Vingamine (cytotoxic); yohimbine (selective α2 –
adrenoceptor antagonist, antidepressant, antihypotensive, ,
antidiuretic activity, aphrodisiac, anxiogenic activity in rodent)
-
Apocynaceae
Acokanthera
oblongifolia
(Hochst.) Codd
inHlungunyembe
Intlungunyembe (X,
Z)
leaves
Catharanthus
roseus (L.) G.Don
Imbali, Ikhwinini,
Isishushlungu (Z)
Leaves,
stem and
root
Sarcostemma
viminale
Umbelebele,
Ingotshwa
Stem
(cytotoxic agent, insecticidal, mutagenic activity)
immunosuppressant), senegalene (cytotoxic agent), 17, 19kauranediol (ent-16β)-form. Dicarboxylic acid, 19-Methyl
ester (toxic to brime shrimp)
-
Rosmarinic acid, Dehydrokaurenoic aicd, Kaurenoic acid,
kaurene lactone, acetoxy kaurene lactone (Holzapfel et al.,
1995)
Asiaticoside G, asiaticoside, asiaticoside F, asiatic acid,
quadranoside IV, 2a,3b,6b-trihydroxyolean-12-en-28-oic acid
28-O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl] ester, kaempferol, quercetin,
astragalin, and isoquercetin (Nhiem et al., 2011)
-
179
(L) R. Br subsp.
viminale
Aquifoliaceae
Ilex mitis (L.) Radlk.
(Kunene and Fossey, 2006)
2007)
Root bark
Decoction for diarrhoea (Mathabe et al.,
2006)
Antimalaria and cytotoxicity
(Rasoanaivo et al., 2004)
-
Root, leaves
Decoction for diarrhoea (De Villiers et al.,
2010)
antimicrobial and antimalarial (De
Villiers et al., 2010)
Arboreaside A, Arboreaside B, Arboreaside C, Arboreaside
D, Arboreaside E, ciwujianoside C3 and 23-hydroxyursolic
acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl(1β6)-β-D-glucopyranosyl ester (Kougan et al., 2009)
iGwada (X),
Mutshulwa (V),
Lebegana (S)
Root, leaves
Diarrhoea and stomach pain in children
(Lewu and Afolayan, 2009)
-
iMbijela
Stem
Ishongwe (X, Z)
Roots
Diarrhoea in cattle (Dold and Cocks, 2001,
McGaw et al., 2008)
Diarrhoea , dysentery, stomach cramps,
headache, oedema, dysmenorrhoea (BisiJohnson et al., 2010)
Antimycobacterium (Green et al.,
2010), antifungal (Samie et al.,
2010), antimicrobial, antiinflammatory, anticholinesterase
and mutagenic activities (Ndhlala et
al., 2010)
Antihelminthic, antibacterial and
cytotoxicity (McGaw et al., 2007)
Antibacterial (Rabe and Van
Staden, 1997), PG inhibition (Jager
et al., 1996), Serotonin re-uptake
modulatory activity (Nielsen et al.,
2004), antidepression (Pedersen et
al., 2008)
Lefatshana (S)
Whole plant
Decoction for diarrhoea (Mathabe et al.,
2006)
Antibacterial (Mathabe et al., 2006)
-
Inhlaba,
Tshikhopha (V)
leaves
Diarrhoea and sore (Mlambo, 2008)
Ikhalana Inkalane
(X) Uphondonde
(Z)
Sekgopha (S)
leaves
Decoction for diarrhoea (Bisi-Johnson et
al., 2010)
Anti-inflammatory (Lindsey et al.,
2002); immunomodulator, antiinflammatory (Imanishi, 1993),
-
Aloctin A (Imanishi, 1993); aloenin, 2'-O-p-coumaroylaloesin,
2'-O-feruloylaloesin, isobarbaloin, and barbaloin (Beppu et
al., 2003)
-
Leaves
Decoction for diarrhoea (Mathabe et al.,
2006)
-
Bindamutsho,
Tshikhopha (V)
Leaves
Gallsickness, parasites, diarrhoea,
constipation, retain placenta, dystocia
maggots (Van der Merwe et al., 2001;
antioxidant (Botes et al., 2008),
antiplasmodial and cytotoxicity (Van
Dyk et al, 2009)
antimalaria (Pillay et al., 2008),
antibacterial, antihelminthic, antiamoebic (McGaw et al., 2000),
Monamane (S),
Mutanzwa-khamelo
(V)
Araliaceae
Cussonia arborea
Hochst ex A. Rich
Asclepiadaceae
Asclepias fruticosa
L.
Secamone filiformis
(L.f) J. H. Ross
Xysmalobium
undulatum (L.) W.T.
Aiton
Asparagaceae
Asparagus cooperi
Bak.
Asphodelaceae
Aloe arborescens
Miller
Aloe candelabrum
Berger
Aloe greatheadii
Schonl.
Aloe marlothii
Berger
-
-
180
Bulbine abyssinica
A. Rich
Bulbine
asphodeloides (L.)
Willd
Bulbine fructescen
Wild
Utswelana Intelezi
(X) Ibhucu (Z),
Incelwane (X)
Leaves,
tubers
tuber and
leaves
McGaw et al., 2008)
Vomiting, diarrhoea, tuberculosis (BisiJohnson et al., 2010)
antitick (Spicket et al., 2007)
Antileukemia, antiplasmodial,
cytotoxicity (Bringmann et al, 2002)
Chrysophanol, aloe-emodin, knipholone, isoknipholone,
Bulbine-knipholone (Bringmann et al, 2002)
rashes, sores wounds,
dysentery and diarrhoea (Iwalewa et al.,
2007)
Diarrhoea, burns, rashes, blisters, insect
bites, cracked lips and
mouth ulcers (Coopoosamy, 2011)
Decoction for diarrhoea (Appidi et al.,
2008)
Decoction for diarrhoea (Mathabe et al.,
2006); vomiting, diarrhoea,
convulsion,venereal diseases, diabetes
and rheumatism (Pujol, 1990)
-
-
Antibacterial (Coopoosamy, 2011),
antiplasmodial (Mutanyatta et al,
2005)
Antibacterial (Coopoosamy, 2011)
knipholone, 4-O-demethylknipholone-4-β-D-glucopyranoside
(Mutanyatta et al, 2005)
Sexuality behaviour (Yakubu and
Afolayan, 2008), Toxicity (Afolayan
and Yakubu 2008)
-
Intelezi (X)
leaf, root
and rhizome
Irooiwater
Root
Ibhucu (Z)
leaves
Inamathela
Whole plant
Diarrhoea (de Wet et al., 2010)
-
-
Umgwaqeni (Z)
Whole plant
Diarrhoea (Mlambo, 2008)
Antiherpesvirus and antipoliovirus
(Rocha Martin et al., 2010)
Acanthoaustralide, quercitin and chrysosplenol (Rocha
Martin et al., 2010)
leaves
Diarrhoea (Van Wyk et al., 2008)
-
Uvelemampo
ndweni uvelegoli
leaves
Bidens pilosa L.
iSanama,
Mushidzhi (V)
Root or
leaves,
flowers
Infusion for diarrhoea (Bisi-Johnson et al.,
2010), haemorrhage, reduce cancer, flu,
cold, fever (Pooley, 1998)
Stomach pain (Lewu and Afolayan, 2009);
diarrhoea, inflammation, female infertility,
excessive menstruation (Dold and Cocks,
2000)
Antimycobacterium (Gautam et al.,
2007)
Antidiarrhoea (Atta and Mouneir,
2005)
Centaurein, centaureidin, cytopiloyne (Chang et al., 2007,
Chiang et al., 2007)
Brachylaena ilicifolia
(Lam.) Phill. &
Schweick
Brachylaena
transvaalensis E.
Philips and
Schweick
Callilepis laureola
Hutch
Chromolaena
uMgqh
Leaves
Diarrhoea in lambs (Dold and Cocks,
2001; McGaw et al., 2008)
Antidiarrhoeal (Yadav and Tangpu,
2009), amoebicidal (Moundipa et
al., 2005), immunomodulator
(Chang et al., 2007, Chiang et al.,
2007)
-
Iphahlalehlathi
Leaves and
bark
Diarrhoea (de Wet et al., 2010)
--
-
Impila (Z)
Roots
Diarrhoea (Mlambo, 2008)
-
-
Usandanezwe (Z)
Leaves
Diarrhoea (Mlambo, 2008)
Anti-inflammatory, antipyretic
15-angeloyloxy-16,17-epoxy-19-kauronic acid, 16-kauren-19-
Bulbine latifolia (L.f)
Roem et Schult
Bulbine natalensis
(Bak. Cf. roowortel
Asteraceae
Acanthospermum
glabratum (DC) Wild
Acanthospermum
australe (Loefl.) O.
Kuntze
Artemisia
absinthium L.
Bidens bipinnata L.
Knipholone (antiplasmodial activity, cytotoxic agent)
-
-
181
odorata L.
Conyza scabrida
DC.
Dicoma anomala
Sond.
Herb
Umuna (Z),
Inyongana (X)
Roots
Dicoma capensis
Less.
Herb
Helichrysum
adenocarpum DC
Helichrysum
calophyllum Klatt
Helichrysum
ecklonis Sond
Helichrysum
odoratissimum (L.)
Pentzia incana
(Thunb.) Kuntze
Schkuhria pinnata
(Lam.) Thell.
Root
decoction
Root
Senecio
quinquelobus DC.
Vernonia glaberrima
Welw
Vernonia
kotschyana Sch.
Bip. ex Walp.
(Baccharoides
adoensis var.
kotschyana (Sch.
Bip. ex Walp.) M.A.
Isawumi, G.ElGhazaly & B.
Nordenstam)
Vernonia natalensis
Sch. Bip. ex Walp.
Imphepho (Z)
Root
decoction
Whole plant
Cold, influenza, inflammation, diarrhoea,
fever, diabetes, stomach affliction (Thring
et al., 2007)
Decoction for diarrhoea, stomach cramp
and skin lesion (Shale et al., 1999)
Bitter tonic and diuretic; kidney; bladder;
back pain; nausea; influenza; colds;
cancer; diarrhoea (van Wyk, 2008)
Diarrhoea and vomiting in children
(Lourens et al., 2008)
Hyperfunction of lower gastrointestinal
tract (Lourens et al., 2008)
Diarrhoea in children (Lourens et al.,
2008)
Diarrhoea (Mlambo, 2008)
Diarrhoea (Van Wyk et al., 2008)
Unsakansaka (Z)
Aerial parts
Usinini (Z)
Leaves
Leaves
Pneumonia, diarrhoea, eye infections,
heartwater (Van der Merwe et al., 2001;
McGaw et al., 2008)
Diarrhoea (Mlambo, 2008)
Inyathelo (Z)
leaves
Decoction for diarrhoea (De Villiers et al.,
2010)
Diarrhoea (Mlambo, 2008)
Uhlambihloshane,
Isibhaha
Leaves,
stem
Decoction for stomach cramps, nervous
spasms of the stomach (Fawole et al.,
antispasmodic (Taiwo et al., 2000),
antidiabetic (Wafo et al.,2011),
antimicrobial and cytotoxicity (Vital
and Rivera, 2009)
Antimicrobial (Thring et al., 2007)
oic acid, 6′-hydroxy-2′,3′,4,4′-tetramethoxychalcone,
isosakuranetin, acacetin, and kaempferide (Wafo et al. 2011)
Antibacterial, antioxidant, fibroblast
growth stimulant (Steenkamp et al.,
2004)
Cytotoxicity (Steenkamp and
Gouws, 2006)
-
-
-
-
-
-
-
Antimicrobial (Puyvelde et al.,
1989)
-
3,5-dihydroxy-6,7,8-trimethoxyflavone and 3-0methylquercetin, helichrysetin (Puyvelde et al., 1989)
-
Antibacterial , anti-inflammatory
mutagenicity (Luseba et al., 2007)
-
-
-
Antibacterial and antimalaria (De
Villiers et al., 2010)
-
Immunomodualting activity (Nergard et
al., 2004); antibacterial activity (Deeni
and Hussain, 1994)
pectic arabinogalactan (Nergard et al., 2004)
Anti-inflammatory (Fawole et al.,
2009a), antimicrobial, mutagenicity
-
182
2009)b; (Hutching et al., 1996)
Infusion for diarrhoea (Amusan et al.,
2007)
(Fawole et al., 2009b)
-
-
Roots
Diarrhoea, fever, flu, contraceptive
(Bessong et al., 2005; Obi et al., 2003)
-
-
Leaves
Diarrhoea (Mlambo, 2008)
-
-
leaves
Diarrhoea in cattle (Luseba and Van der
Merwe, 2006; McGaw et al., 2008)
Antiplasmodial and cytotoxicity
(Prozesky et al., 2001)
-
whole plant
Amenorrhoea, dysentery, diarrhoea and
swellings growth (Iwalewa et al., 2007)
Antibacterial and antifungal (Naidoo
et al., 1992)
Eriodictyol, naringenin, triandrin, n-pinitol (lD-4-O-methyl
chiroinositol), trans-p-coumaraldehyde, Exocarpic acid (13Eoctadecene-9,11-diynoic acid)
Leaves
Decoction for diarrhoea (De Villiers et al.,
2010)
-
Kigelia africana
(Lam.) Benth.
Bark
Dysentery and stomach ailments (van
Wyk, 2008b)
Tecomaria capensis
Spach
Bark
fever, diarrhea and dysentery, pains,
sleeplessness, stomach and chest pains
(Iwalewa et al., 2007)
Antiplasmodial and cytotoxicity
(Mbatchi et al., 2006), antimicrobial
and antimalaria (De Villiers et al.,
2010)
Antidiarrhoea (Akah, 1996),
analgesic and anti-inflammatory
(Owolabi and Omogbai, 2007),
antifungal and antibacterial
(Owolabi et al. 2007)
Antimicrobial (Saini et al., 2011)
Leaves,
bark, root
fruit
Fever, diarrhoea, haemoptysis, hiccup
remedy (van Wyk, 2008b)
Anti-inflammatory , antiviral
(Selvarani and Hudson, 2009),
antihyperglycermic and
hypolipidermic (Bhargav et al.,
2009), Antimicrobial (Mulaudzi et
al., 2011)
Epicatchin, procyanidin B2, procyanidin B5 (Kinghorn et al.,
2011)
Leaves
Disinfectant for wound, anthelmintic and
snakebite (Watt and Breyer-Brandwijk,
1962)
Antimicrobial (Suleiman et al.,
2010)
-
Vernonia
oligocephala Sch.
Bip
Vernonia myriantha
Hook. F
(synVernonia
stipulacea Klatt)
Vernonia tigna Klatt
syn V. corymbosa
lihlunguhlungu
Roots
Mululudza (V)
Uhlunguhlungu (Z),
Phathaphathane
(V)
Balanitaceae
Balanites
maughamii Sprague
Balanophoraceae
Sarcophyte
sanguine Sparrm
Bignoniaceae
Markhamia sessilis
Sprague
Bombacaceae
Adansonia digitata
L.
Bursareceae
Commiphora
harveyi (Engl.) Engl.
Muvhuyu (V)
Verminoside and Verbascoside (Picerno et al., 2005)
-
183
Capparaceae
Capparis tomentosa
Caricaceae
Carica papaya L.
Caryophyllaceae
Krauseola
mossambicina
(Moss.) Pax & K.
Hoffm.
Celastraceae
Elaeodendron
transvaalense (Burtt
Davy) R.H. Archer
syn Cassine
transvaalensis
Gomphocarpus
fruticosus Dryand.
Umqoqolo (Z),
Muoba-dali (V)
Root
infusions
and
decoctions
Diarrhoea in cattle, stomach ailments in
animals (Watt and Breyer-Brandwijk,
1962, Pujol, 1990, McGaw et al., 2008)
Antimicrobial (Ramalivhana et al.,
2010), antifungal (Samie et al.,
2010)
Papawe (V)
Leaves,
seed
Amoebic dysentery, fever, gastric
problems, asthma, immune-stimulant
(Green et al., 2010; Aruoma et al., 2006)
Antiamoebic (Tona et al., 1998),
anthelmintic (Kermanshai et al., 2001)
Alternariol Carpamine (cardiotonic agent, CNS
depressant), Chymopapain; Glycerol triacetate
(antifunal and adjuvant); Papain ; 2,4’-Dihydroxy-3’,5’dimethoxyacetophenone (antifungal), Benyl
isothiocyanate (Kermanshai et al., 2001)
Diarrhoea (de Wet et al., 2010)
-
-
Bark
Cough, piles, venereal diseases,
diarrhoea, stomach ache, laxative (Samie
et al., 2010)
Antimicrobial (Tshikalange et al.,
2005), hypoglycaermic (Deutschlander
et al., 2009), Cytotoxicity (Tshikalange
and Hussein, 2010)
lup-20(30)-ene-3,29-diol , lup-20(29)-ene-30-hydroxy-3one-(2), ψ– taraxastanonol, β-sitosterol and 4’ –Omethylepigallocatechin (Tshikalange and Hussein, 2010)
Leaf infusion
Diarrhoea and stomach ache in children
(Hutchings et al., 1996; Fouche et al.,
2008)
Diarrhoea (de Wet et al., 2010)
-
Gomphoside (cardiotonic agent)
Isihlaza, Isihlazi
Mulumanamana
Mukuhazwhi,
Umgugudo (Z)
Stachydrine L-form (Systolic depressant, rheumatism)
Gymnosporia
senegalensis (Lam.)
Loes
Maytenus
heterophylla Eckl. &
Zeyh.) Robson
Ubuhlangwe
-
Isibhubu (Z),
Tshiphandwa (V)
Bark and
leaf
infusions
Diarrhoea in stock animals (Watt and
Breyer-Brandwijk, 1962; McGaw et al.,
2008)
Antimicrobial (Orabi et al., 2001), antiinflammatory and cytotoxicity (Da Silva
et al., 2010), anticytomegalovirus
(Murayama et al.,2007)
Maytenus
peduncularis
(Sond.) Loes.
Maytenus
procumbens (L.f.)
Loes.
Mukwatule (V)
root
Backache, pain (Gonzalez et al., 2000)
-
1β-acetoxy-9α-benzoyloxy-2β,6α-dinicotinoyloxy-βdihydroagarofuran, β-amyrin, maytenfolic acid, 3αhydroxy-2-oxofriedelane-20α-carboxylic acid, lup-20(29)ene-1β,3β-diol, (−)-4′-methylepigallocatechin, and (−)epicatechin (Da Silva et al., 2010) , pristimerin, lupeol and
2-acetylphenol-1-β-D-glucopyranosyl (1→6)-β-Dxylpyranoside (acetophenol glycoside) (Murayama et
al.,2007)
-
-
-
-
-
-
184
Maytenus
senegalensis (Lam.)
Exell
Tshiphandwa (V)
Maytenus undata
(Thunb.) Blakelock
Tshinembane (V)
Leaves
Diarrhoea (Van Wyk et al, 2008)
Unakani, Ikhambi
Laeves,
flower
Aerial part
Chenopodiaceae
Atriplex nummularia
Lindl.
Chenopodium
ambrosioides L.
Clusiaceae
Garcinia livingstonei
T. Anderson
Combretaceae
Combretum
bracteosum
(Hochst.) Brandis ex
Engl.
Combretum
imberbe Wawra
Combretum molle
R. Br.ex G. Don
Antimicrobial and anti-inflammatory
(Matu and van Staden, 2003); antiinflammatory and cytotoxicity (Da Silva
et al., 2010)
Antimicrobial, anti-inflammatory and
antioxidant (Muhammed et al., 2000),
antimalaria (Muthaura et al., 2007)
Wilforine (insecticidal), β-amyrin, lupenone, maytenoic
acid, β-sitosterol, pristimerin (Da Silva et al., 2010)
-
Diarrhoea (de Wet et al., 2010)
Antitumorigenic activity (Amara et al.,
2008)
Antisecretory against cholera toxin
(Velazquez et al., 2006), antiamoeba
and antigardia (Calzada et al., 2006)
leaves
Diarrhoea (de Wet et al., 2010)
Antibacterial (Kaikabo, 2008)
Amentoflavone (Bradykinin antagonist, anti-HIV activity,
inhibitor of human cathepsin B, antiiflammatory
properties), amentoflavone and 4″-methoxy
amentoflavone (Kaikabo, 2008)
leaves
-
anti-inflammatory, anthelmintic and
antischistosomal (McGaw et al., 2001)
-
Mudzwiri (V)
Root
Decoction for diarrhoea (Mathabe et al.,
2006)
anti-inflammatory, anthelmintic and
antischistosomal (McGaw et al., 2001),
antimicrobial (Angeh et al., 2007)
Mugwiti (V)
Roots
Abdominal pain, fever, snake bite, leprosy
and convulsions (Bessong et al. ,2005;
Mabogo, 1990)
anti-inflammatory, anthelmintic and
antischistosomal (McGaw et al., 2001)
1α, 23β-Dihydroxyl-12-Oleanen-29-oic acid-23β-O-α-4acetylrhamnopyranoside; 1, 22-Dihydroxyl-12-Oleanen30-oic acid; Ethyl cholesta-7, 22,25-trien-O-β-Dglucopyranoside (Angeh et al., 2007), imberbic acid
(Katerere et al., 2003)
Punicalgin, 4-epi-sericoside, sericoside (Asres et al.,
2001), β-D-glucopyranosyl 2α,3β,6β-trihydroxy-23galloylolean-12-en-28-oate, combregenin, arjungenin,
arjunglucoside I, combreglucoside (Ponou et al., 2008),
mollic acid glucoside (Oyewole, 2008)
1α,23β-dihydroxy-12-oleanen-29-oic-acid-23β-O-α-4acetylrhamnopyranoside, 1,22-dihydroxy-12-oleanen-30oic acid, 24-ethylcholesta-7,22,25-trien-O-β-Dglucopyranoside (Angeh et al., 2007)
apigenin (Eloff et al., 2008)
Umphimbi,
Muphiphi (V)
Root used for chest pain, rheumatism,
snakebites, diarrhoea and fever. Leaves
for eye infection (Matu and van Staden,
2003)
Combretum
padoides Engl. &
Diels
Combretum vendae
A.E. van Wyk
Leaves
Leprosy, ophthalmic remedy, and blood
purification (Watt and Breyer-Brandwijk,
anti-inflammatory, anthelmintic and
antischistosomal (McGaw et al., 2001);
antifungal ( Masoko et al., 2007);
Antibacterial (Angeh et al., 2007)
Antimicrobial (Ahmed.et al, 2008;
Suleiman et al., 2010)
3-oxo-11α-methoxyolean-12-ene-30-oic acid, 3-oxo-11αhydroxyolean-12-ene-30-oic acid, 3-oxo-olean-9(11),12diene-30-oic acid, 3,4-seco-olean-4(23),12-diene-3,29dioic acid (20-epikoetjapic acid), 3,11-dioxoolean-12-ene30-oic acid (3-oxo-18β-glycyrrhetinic acid), koetjapic acid,
12-oleanene artifact 3-oxo-11α-ethoxyolean-12-ene-30oic acid (Muhammed et al., 2000)
Ascaridole, quercetin, kaempferol, isorhamnetin,
ambroside, malic acid, succinic acid
185
1962)
Combretum woodii
Dummer
Combretum zeyheri
Sond
Terminalia laxiflora
Engl.
Terminalia
phanerophlebia
Engl.
Terminalia sericea
Burch. ex DC.
Convolvulaceae
Ipomoea batatas
(L.) Lam.
Mufhatela-thundu,
Mufhatela (V)
Root
infusion
Leaves
Root bark
Bloody diarrhoea (Hutchings et al., 1996;
Fouche et al., 2008)
Decoction for diarrhoea (De Villiers et al.,
2010)
Diarrhoea and colic (Iwalewa et al., 2007)
anti-inflammatory, anthelmintic and
antischistosomal (McGaw et al., 2001);
antifungal ( Masoko et al., 2007)
Antibacterial (Breytenbach and Malan,
1989)
Antifungal (Batawila et al., 2005)
Combretastatin B5 (Eloff et al., 2005)
Antimicrobial (Shai et al., 2008a)
Mususu (V),
Ikonono
Leaves
roots
Wound (Luseba and Van der Merwe,
2006); diarrhoea (Van der Merwe et al.,
2001; McGaw et al., 2008)
Antimicrobial, antidiabetic, cytotoxicity
(Moshi and Mbwambo, 2005), COX-1
and COX-2 assays (Eldeen et al.,
2006).
Anolignan B (Eldeen et al., 2006), Termilignan B, Arjunic
acid (Eldeen et al., 2008), 3′5′-dihydroxy-4-(2-hydroxyethoxy) resveratrol-3-O-β-rutinoside, resveratrol-3-βrutinoside glycoside, 3′,4,5′-Trihydroxystilbene
(resveratrol), arjungenin (Joseph et al., 2007)
Sweet potato
Leaves
Decoction for diarrhoea (De Villiers et al.,
2010)
Umlahleni (X, Z)
Unsirayi (X),
Umgxina
Bark, root
Diarrhoea, stomach ailments (BisiJohnson et al., 2010)
Antimicrobial (Shai et al , 2008a, Shai
et al., 2009)
Lupeol, betulinic acid, ursolic acid, and 2α-hydroxyursolic
acid (Shai et al, 2008b)
Karkay, karkey (K)
Fresh leave
Diarrhoea (van Wyk, 2008)
-
-
Karkay, karkey (K)
Fresh leave
Diarrhoea (van Wyk, 2008)
-
-
Leaves, root
Decoction for abdominal pains, diarrhoea
(Fawole et al., 2009); Hutching et al.,
1996)
Diabetes, childhood diarrhoea (Samie et
al., 2009)
-
-
Shigellocidal (Iwalokun et al. 2001);
Cytotoxicity and antiamoebic (Samie et
al, 2009)
Balsaminapentaol A, Balsaminol A, Balsaminol B,
Cucurbalsaminol A, Cucurbalsaminol B, cucurbita5,23(E)-diene-3β,7β,25-triol, karavilagenin E (Ramalhete
et al.,(2009)
Decoction for bloody faeces and dysentery
(Fawole et al., 2009); (Hutching et al.,
-
Hydroxyisodispyrin (cytotoxic agent)
4,5-Di-transcaffeoyldenoic acid (antioxidant), 6-Ocaffeoylsophorose (α-glucosidase inhibitor, antioxidant);
3,5-Di-O-caffeoylquinic acid (active against HIV-1
integrase, antiviral, antihepatotoxic activity); Petrovin B
(antibacterial and antitumour)
Cornaceae
Curtisia dentata
(Burm.f.) C.A.Sm.= C.
faginea Assegaai
Crassulaceae
Crassula ovata
(Mill.) Druce
Crassula tetragona
L.
Cucurbitaceae
Cucumis hirsutus
Sond.
Mormodica
balsamina L.
Ebenaceae
Diospyros lycioides
Desf.
Lubavhe (V)
Whole plant
Umbulwa (Z)
Bark, root
186
Diospyros
mespiliformis
Diospyros pallens
(Thunb.) F. White
Euclea crispa
Thunb Gurke
Euclea natalensis A.
DC
Musuma
Ungwali (Z),
Bark and
leaves
Root and
stem
leaves
1996)
Dysentery, fever, ringworm, skin infection,
wound healing (Samie et al., 2010)
Stomach arch; diarrhoea (van Wyk, 2008)
Diosquinone, plumbagin (Lajubutu et al.,1995)
-
-
Dysmenorrhoea (Steenkamp, 2003)
Mutangule-thavha
(V), Umzimane (Z)
root
oral health care, for chest complains,
bronchitis, pleurisy, chronic asthma,
urinary tract infections, venearal diseases
(Lall and Meyer, 2001) , Infertility and
abortifacient (Arnold and Gulumian, 1984)
Antibacterial (Weigenand et al., 2004),
antimycobacterium (Lall and Meyer,
2001)
Octahydroeuclein, 20(29)-lupene-3β-isoferulate,
shinanolone, lupeol, betulin (Weigenand et al., 2004);
diospyrin (Lall and Meyer, 2001)
Mupalakhwali (V)
Leaf
Antimicrobial (Fawole et al., 2009)
-
Bridelia micrantha
(Hochst.) Baill
Munzere (V)
Bark,
leaves, roots
Antidiarrhoea (Lin et al., 2002), betalactamase inhibition (Gangoue-Pieboji
et al., 2007); antimalarial (Abo and
Ashidi (1999). n-butanol fraction of
methanolextract has IC50 of 7.3_g/ml
against the RNA-dependent DNA
polymerization (RDDP) function of
HIV-1 RT (Bessong et al., 2006)
Taraxerol, gallic and ellagic acid, friedelin,
delphinidin, methyl salicylate (Ngueyem et al., 2009)
Bridelia mollis
Mukumbakumba
Leaves
Euphorbia cooperi
N. B. Br. Ex. Berger
Euphorbia hirta L.
Umhlonhlo (X)
Root bark
Jatropha zeyheri
Sond.
Xidomeja
Decoction for abdominal cramps and
dysentery (Fawole et al., 2009; Hutching
et al., 1996)
Stomach ache, diarrhoea, abortifacient
(Bessong et al., 2005; Lin et al., 2002),
Gastro-intestinal ailments, painful joints,
retained placenta, diabetes mellitus,
syphilis prehepatic jaundice, tape worm
abdominal pain, conjunctivitis, headache,
scabies, bloody diarrhoea, dysentery,
emetic, wound infection, coughs,
threadworms, tonic for children, sore eyes,
epigastric pain, relief of headache,
purgative (Ngueyem et al., 2009), diabetes
mellitus (Abo et al., 2008)
Dysentery, burning and itching (Samie et
al., 2010)
Diarrhoea, stomach disorder (Bisi-Johnson
et al., 2010)
Decoction for diarrhoea (De Villiers et al.,
2010); dysentery, gonorrhoea, jaundice,
pimples, digestive problems and tumours,
antibacterial, anti-inflammatory,
antimalarial, galactogenic, antiasthmatic,
antidiarrheal, anticancer, antioxidant,
antiferlity, antiamoebic, and antifungal
activities (Kumar et al., 2010)
General ailments, diarrhoea (Luseba and
Van der Merwe, 2006; McGaw et al.,
2008)
Euphorbiaceae
Antidesma venosum
E. Mey. ex Tul.
Leaves
Roots
Antifungal (Samie et al., 2010)
-
-
Antiamoebic , spasmolytic (Tona et al.,
2000), Antidiarrhoeal (Galvez et al.,
1993)
β-amyrin, 24-methylenecycloartenol, β-Sitosterol,
Quercitrin (Galvez et al., 1993). Quercitol, gallic acid,
afzelin, quercitrin, myricitrin, rutin, gallic acid, quercitin,
euphorbin-A and ephorbin-B, euphorbin-C, euphorbin-D,
β-amyrin, 24-methylenecycloartenol, β-sitosterol,
heptacosane, n-nonacosane, shikmic acid, tinyatoxin,
choline, camphol, and quercitol (Kumar et al., 2010)
Antimicrobial and Antifungal (Dekker et
al, 1987)
Jaherin (Dekker et al, 1987)
187
Ricinus communis
L.
Mupfure (T)
leaves
Spirostachys
africana Sond
Morekhure (S)
Wood
umkhaya
uMnga (X),
Umunga (Z)
Bark, leaves
Fabaceae
Acacia burkei Benth
Acacia karoo Hayne
Acacia mearnsii De
Wild Blackwood
Acacia robusta E.
Meyer
Acacia sieberiana
DC.var woodii (Burtt
Davy) Keay &
Brenan
Acacia tortilis
(Forssk.) Hayne
Bauhinia bowkeri
Harv
Bauhinia galpinii N.
E. Br
Bauhinia petersiana
Bolle
Bauhinia variegata
Linn
Wound and sores, asthma arthritis, flu,
fever, tuberculosis, toothache, diarrhoea,
antihelmentic (Bessong et al., 2005;
Grierson and Afolayan, 1999)
Stomach ulcers, acute gastritis, headache,
rashes, boil, emetic, purgative, diarrhoea,
dysentery (Verschaeve and Van Staden,
2008)
-
-
Antibacterial and cytotoxicity (Mathabe
et al., 2008)
-
Diarrhoea (de Wet et al., 2010)
Diarrhoea, intestinal parasites in goats,
sheep, poultry and pig (Dold and Cocks,
2001; McGaw et al., 2008) fractures and
diarrhoea (Van der Merwe et al., 2001)
Infusion for diarrhoea and dysentery (BisiJohnson et al., 2010)
Anti-inflammatory (Adedapo et al.,
2008); Acute toxicity (Adedapo et al,
2008)
-
Protective against acrolein-induced
oxidative damage (Huang et al., 2010)
Robinetinidol-(4β→8)-epigallocatechin 3-O-gallate
(Huang et al., 2010)
Ublekwana (X)
Udywabasi (X, Z)
Indywabasi
Umngamanzi (Z)
Bark
leaves
Diarrhoea (Mlambo, 2008)
Antifungal (Hamza et al., 2006)
Musaunga,
Muunga-luselo (V)
Bark
Enemas, antiseptic, fever, stomach ache,
tapeworm, astringent, haemostatic,
diarrhoea (Verschaeve and Van Staden,
2008)
Diarrhoea (Van der Merwe et al., 2001;
McGaw et al., 2008)
Induce vomiting (Ndawonde et al., 2007)
Mutagenicity , antimutagenicity;
antibacterial, antiinflammatory, anticholinesterase and mutagenic effects
(Eldeen et al., 2005)
-
-
Diarrhoea, infertility (Samie et al., 2010),
infertility (Arnold and Gulumian, 1984),
amenorrhoea (Van Wyk and Gericke,
2000)
Antimutagenic (Reid et al., 2006);
antioxidant and cytotoxicity of leaf
extracts (Aderogba et al., 2007);
Anticampylobacterial, antiamoebic and
cytotoxicity of root extract(Samie et
al.,2009)
-
Quercetin-3-O-β-glactopyranoside, Myricetin-3-O-βglactopyranoside, 2″-O-rhamnosylvitexin (Aderogba et al.,
2007)
Anti-inflammatory (Rao et al., 2008);
Immunomodulator (Ghaisas et al.,
2009)
kaempferol, ombuin, kaempferol 7,4′-dimethyl ether 3-O-β
-D-glucopyranoside , kaempferol 3-O-β -Dglucopyranoside (4), isorhamnetin 3-O-β -Dglucopyranoside, hesperidin, 3β-trans(3,4-dihydroxycinnamoyloxy)olean-12-en-28-oic acid (Rao
et al., 2008)
Muunga-khanga,
Muswu (V)
uMdandlovu
Mutswiriri (V),
Umhuwa (Z)
Mushakule (V)
Leaves,
bark
Bark, leaves
root
Cold (Coates-Palgrave, 2002); infertility
and dysmenorrhoea (Van Wyk and
Gericke, 2000)
Leaves,
bark
Diabetes, goiter, dysentery, diarrhoea
(Parekh and Chanda, 2007)
-
-
-
188
Dichrostachys
cinerea (L.) Wight
and Am.
Elephantorrhiza
burkei Benth.
Elephantorrhiza
evoluta (Burch.)
Skeels
Elephantorrhiza
elephantina (Burch.)
Skeels
Eriosema
psoraleoides (Lam.)
G. Don
Erythrina latissima
E. Mey
Indigofera daleoides
Benth. ex Harv &
Sond
Indigofera jucunda
Schrire syn
Indigofera
cylindrical sensu E.
Mey
Indigofera
sessilifolia DC.
Mucuna coriacea
Baker
Peltophorum
africanum Sond.
Rhynchosia
adenoids E. & Z.
Senna italic Mill.
Murenzhe (V)
Bark
Diarrhoea and steaming to get ride of acne
(Mlambo, 2008)
spasmodic in guinea-pig isolated
trachea (Aworet-Samseny et al., 2011)
dichrostachines A-R (Long et al., 2009)
Umdabu (Z),
Tshisese-thavha,
Tshisesevhufa (V)
iNtololwane (X, Z)
root
abdominal pains, diarrhoea, coughs,
bacterial infections (Iwalewa et al., 2007)
Antimicrobial (Mathabe et al., 2006)
Triterpenoids, α-amyrim, β-sitosterol, alkaloids and
saponin
Roots, aerial
part and
bulb
Stem
rhizome
Diarrhoea and dysentery in cattle, horse
and humans (Watt and Breyer-Brandwijk,
1962; McGaw et al., 2008)
Decoction for diarrhoea (Mathabe et al.,
2006)
-
-
Leaves
Decoction for diarrhoea (De Villiers et al.,
2010)
Leshitsana
Muvhale (V)
Antimicrobial (Mathabe et al., 2006),
antiparasitic (Naidoo et al., 2006),
antibabesia (Naidoo et al., 2005
Antimicrobial (Khan et al., 2000)
Sores (Coates-Palgrave, 2002)
Whole plant
Decoction for diarrhoea (Mathabe et al.,
2006)
Antimicrobial (Mathabe et al., 2006)
Root
Intestinal worm (Coates-Palgrave, 2002)
-
erysotrine, erysodine, syringaresinol, vanillic acid, (+)10,11-dioxoerysotrine, 2-(5′-hydroxy-3′-methoxy phenyl)6-hydroxy-5-methoxybenzofuran, 7,3′-dihydroxy-4′methoxy-5′-(γ,γ-dimethylallyl)isoflavone (erylatissin A)
(Wanjala et al., 2002), 7,3′-dihydroxy-6″,6″-dimethyl-4″,5″dehydropyrano [2″,3″: 4′,5′]isoflavone (erylatissin B), (−)7,3′-dihydroxy-4′-methoxy-5′-(γ,γ
dimethylallyl)flavanone (erylatissin C) (Chacha et al.,
2004)
(6,2-O-[3-nitropropanoyl-β-D-glucopyranose]), (6,3′,4′trihydroxyflavan 5′-O-glucopyranoside) (Mathabe et al.,
2009)
-
iKhubalo
Roots
-
-
Vhaulada
Roots
Diarrhoea in calves (Dold and Cocks,
2001; McGaw et al., 2008)
Fever, diarrhoea (Bessong et al., 2005)
Antimicorbial (Samie et al., 2009)
N.A
Musese (T)
Bark , root
bark
Roots
Bark, roots
Anti parasitic (Bizimenyera et al.,
2006), anti HIV (Bessong et al., 2005)
Cyclooxygenase inhibitory (Jager and
Van Staden, 2005)
-
Catechin, gallotannin, bergenin (Bessong et al., 2005)
Ximbangam
bangana
Tonic, diarrhoea (Van der Merwe et al.,
2001; McGaw et al., 2008)
Decoction for rheumatic pains, menstrual
pains and dysentery (Shale et al., 1999)
Diarrhoea and gallsickness diarrhoea,
(Luseba and Van der Merwe, 2006;
McGaw et al., 2008)
--
189
Senna occidentalis
(L) Link
Schotia
brachypetala Sond.
Ikhoshokhosho
Leaves, root
Diarrhoea (de Wet et al., 2010)
Mulubi (V)
Bark
Schotia latifolia
Jacq.
Zornia milneana
Umgxam
bark
Lukandulula (V)
Whole
plant
Diarrhoea (Mathabe et al., 2006), root for
dysentery and diarrhoea (Hutching et al.,
1996)
Decoction for diarrhoea (Appidi et al.,
2008)
Dysentery and diarrhoea (Samie et al.,
2005)
Antibacterial (McGaw etal., 2002)
Linolenic acid and methyl-5,11,14,17-eicosatetraenoate
(McGaw etal., 2002)
Antibacterial (Masika et al., 2004)
Epicathechin and catechin (Masika et al., 2004)
Anticampylobacterial and antiamoebic
(Samie et al.,2009)
-
Dysentery bladder problem (Verschaeve
and Van Staden, 2008)
Mutagenicity , antimutagenicity,
Epilepsy and convulsion (Risa et al.,
2004)
-
treat acne, sores and diarrhoea (Watt and
Breyer-Brandwijk, 1962; van Wyk et al.,
1997)
Antibacterial (Thring et al., 2007)
-
leaves
Diarrhoea (Amabeoku, 2009; Van Wyk et
al. 1997)
-
Herb and
root
Diarrhoea, dysentery, cold and
inflammation (van Wyk, 2008)
Antidiarrhoearic (Amabeoku, 2009);
antimicrobial and cytotoxicity (Babajide
et al., 2010)
-
Herb and
root
Tubers
Diarrhoea, dysentery, cold and
inflammation (van Wyk, 2008)
Used as astringent, diarrhoea, dysenteric
fever (Brendler and van Wyk, 2008)
Antioxidant (Mamphiswana et al.,
2010)
-
-
Umsongelo (X)
ishwaqa
Leaf, root
Diarrhoea, dysentery, fever and colic
(Brendler and van Wyk, 2008)
-
-
iNtololwana,
uVendle
Tuberous
root
Diarrhoea and dysentery (van Wyk, 2008)
Antibacterial, antifungal and
antioxidant (Adewusi and Afolayan,
2009a), Acute toxicity (Adewusi and
Afolayan, 2009b)
Immunomodulatory (Kayser et al.,
2001), antibacterial, antifunfal and
antitubercular (Mativandlela et al.,
2006)
-
scopoletin, umckalin, 5,6,7-trimethoxycoumarin, 6,8dihydroxy-5,7-dimethoxycoumarin, (+)-catechin, gallic
acid (Kayser and Kolodziej, 1997)
Flacourtiaceae
Oncoba spinosa
Lam
root
Gentianaceae
Chironia baccifera
L.
Geraniaceae
Geranium incanum
Burm. f.
Monsonia
emarginata (L.f.)
L’Hèr.
Monsonia burkeana
Planch. Ex Harv.
Pelargonium
antidysentericum
(Eckl.& Zeyh.)
Kostel
Pelargonium
luridum (Andr.)
Sweet
Pelargonium
reniforme Curtis
Pelargonium
sidoides DC.
Pelargonium triste
Isikhwali (Z)
Umsongelo (X)
Tuberculosis, diarrhoea (Brendler and van
Wyk, 2008)
Tuberous
Diarrhoea and dysentery (van Wyk, 2008)
-
-
scopoletin, umckalin, 5,6,7-trimethoxycoumarin, 6,8dihydroxy-5,7-dimethoxycoumarin, (+)-catechin, gallic
acid (Kayser and Kolodziej, 1997)
-
190
(L.) L’Hèr.
Hyacinthaceae
Eucomis autumnalis
(Mill.) Chitt.
root
Ubuhlungu becanti
Isithithibala (X)
Umathunga (Z)
Bulb
E. regia (L.) L’Herit
Ledebouria revoluta
(L.f.) Jessop
iKreketsana (X)
Bulb
Schizocarphus
rigidofolius
Scilla nervosa
(Burch.) Jessop
Ingcino (S)
leaves
Umagaqana,
magaqana (X)
Imbizankulu
ingema (Z)
Root, bulb
Ubuklunga (X)
Umavumbuka (Z)
Umafumbuka (X)
Hydnoraceae
Hydnora africana
Hypoxidaceae
Hypoxis latifolia
Hook.
Hypoxis
hemerocallidea
Fisch. & C. A. Mey
Iridaceae
Crocosmia
paniculata (Klatt.)
Goldbl.
Gladiolus dalenii
van Geel
Stomach ache, diarrhoea, back pain,
healing of fractures (Bisi-Johnson et al.,
2010)
Veneral diseases, lumbago, diarrhoea,
respiratory conditions especially coughs,
biliousness and to prevent premature
childbirth (Watt and Breyer-Brandwijk,
1962)
Bulb infusion for diarrhoea in goat, leavf
decoction for gallsickness (Dold and
Cocks, 2001; McGaw et al., 2008)
Anti-inflammatory (Zschocke et al.,
2000)
-
COX-1 assay (Taylor and van Staden,
2001)
-
Infusion for diarrhoea (Amusan et al.,
2007)
All purpose herb. Diarrhoea, tuberculosis
(Bisi-Johnson et al., 2010)
-
(3R)-5,7-dihydroxy-3-(4′-methoxybenzyl)-4-chromanone,
(3R)-5,7-dihydroxy-3-(4′-hydroxybenzyl)-4-chromanone,
3R)-5-hydroxy-7,8-dimethoxy-3-(4′-hydroxybenzyl)-4chromanone, (3R)-5,7-dihydroxy8-methoxy-3-(4′-hydroxybenzyl)-4-chromanone (Moodley
et al., 2006)
-
-
-
Fruits, tuber
leaves
Diarrhoea, dysentery (Bisi-Johnson et al.,
2010)
-
-
Inongwe Ilabateka
(X)
Inongwe Ilabateka
(X)
Tuber
Decoction for diarrhoea (Bisi-Johnson et
al., 2011)
Decoction for diarrhoea (Ojewole et al,
2009)
Antibacterial, antifungal (Buwa and
Van Staden, 2006)
Antinociceptive, anti-inflammatory and
antidiabetic (Ojewole, 2006),
Antidiarrhoeal, acute toxicity test
(Ojewole et al, 2009)
Undwendweni (Z)
corms
Diarrhoea in bovine (Watt and BreyerBrandwijk, 1962; McGaw et al., 2008)
-
-
corm
Dysentery, diarrhoea, stomach cramps
(Fawole et al., 2009; Hutching et al., 1996)
Anti-inflammatory (Fawole et al.,
2009Ndhalala), amoebicidal
(Moundipa et al., 2005), Antimicrobial
and mutagenicity (Fawole et al., 2009
Finnie)
-
Tuber
191
Gladiolus sericeovillosus Hook. F
Watsonia densiflora
Bak.
Watsonia tabularis
Bak
Lamiaceae
Ballota africana (L.)
Berth.
Cissus
quandrangularis
(Linn)
Leonotis leonurus
(L.) R.Br
Leucas capensis
(Benth.) Engl.
Umnunge (X),
Umlunge (Z)
Corm
Corm
Decoction fro dysentery, cold, tuberculosis
diarrhoea (Bisi-Johnson et al., 2010)
Diarrhoea in calves (Watt and BreyerBrandwijk, 1962; McGaw et al., 2008)
-
-
Antibacterial, antifungal,
acetylcholinestarase inhition,
mutagenicity, COX 1and 2 (Ndhala et
al., 2010)
Antimicrobial and mutagenicity
(Fawole et al., 2009 Finnie)
corm
Diarrhoea in human and calves (Fawole et
al., 2009; Hutching et al., 1996)
Isinwasi (Z),
Nyangala (T)
Root, stem
Burns, wounds, gastrointestinal complaints,
backache, body- and febrile pain, malaria (Lin et
al., 1999; Hutchings et al., 1996)
Antibacterial, anti-inflammatory and
mutagenicity (Luseba, et al., 2007)
Imunyamunya (Z)
leaves and
stem bark
feverish headache, dysentery, coughs and
colds, and haemorrhoids (Iwalewa et al.,
2007)
1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecan-1-ylpalmitate, succinic acid, uracil, luteolin 7-O-glucoside,
acteoside, geniposidic acid (Agnihotri et al., 2009)
uPhiphiyo
leaves
Decoction with Aloe forex and
Brachylaena ilicifolia for diarrhoea in
lambs (Dold and Cocks, 2001)
Anti-diarrhoea (Naseri et al., 2008)
Anticonvulsant (Bienvenu et al., 2002),
antinociceptive, anti-inflammatory and
hypoglycaemic activities (Ojewole,
2005)
Spasmolyte (Naseri et al., 2008)
-
-
Mentha longifera
(L.) L.
Rotheca myricoides
(Hochst.) Steane &
Mabb.
Leaf
Root bark
Fever and diarrhoea in cattle (Verschaeve
and Van Staden, 2008)
Mutagenicity and antimutagenicity
(Verschaeve and Van Staden, 2008)
-
Salvia africanacaerulea L.
Leaf
Coughs, colds, women ailments; diarrhoea
(van Wyk, 2008)
-
Salvia repens
Burch. Ex. Benth
Roots,
leaves,
whole plant
Sores on the body, stomach problems,
diarrhoea (Kamatou et al., 2008)
Antimicrobial, antioxidant, antiinflammatory, antiplasmodial,
cytotoxicity and antituberculosis
(Kamatou et al., 2006)
Antimicrobial, antioxidant, antiinflammatory, antiplasmodial,
cytotoxicity and antituberculosis
(Kamatou et al., 2006)
Mutagenicity , antimutagenicity
(Verschaeve and Van Staden, 2008);
Antibacterial, antifungal,
acetylcholinestarase inhition,
mutagenicity, COX 1and 2 (Ndhala et
al., 2010)
-
Tetradenia riparia
(Hochst.) Codd
Iboza (Z)
leaves
Cough, sore throats, malaria, dengue,
dropsy, fever, diarrhoea, haemoptysis,
boils, mumps, induce drowsiness
(Verschaeve and Van Staden, 2008)
Teucrium riparium
Hochst
umnunu
Root
Infusion for diarrhoea (Amusan et al.,
2007)
-
-
-
192
Lauraceae
Ocotea bullata
(Burch.) Baill.
Loganiaceae
Strychnos
henningsii Gilg.
Loganiaceae
Sida alba Forrsk
Malva parviflora L.
uMnonono,
Umqalothi (Z)
Ujongilanga
Bark
Headache, infantile diarrhoea, stomach
problems, emetic for emotional and
nervous disorder (Verschaeve and Van
Staden, 2008)
Mutagenicity , antimutagenicity
(Verschaeve and Van Staden, 2008),
anti-inflammatory (Zschocke et al.,
2000)
Ocobullenone, iso-ocobullenone, sibyllenone (Zschocke
et al., 2000)
Bark
infusion
Heartwater and diarrhoea in cattle (Dold
and Cocks, 2001; McGaw et al., 2008)
-
-
Leaves
Diarrhoea and dysentery (Samie et al.,
2005)
Decoction for diarrhoea (Appidi et al.,
2008)
Antibacterial (Samie et al., 2005)
-
Antibacterial and anti-inflammatory
(Shale et al., 2005)
-
leaves
Melastomataceae
Dissotis princeps
(Kunth) Triana
Leaves
Infusion for diarrhoea and dysentery
(Fawole et al., 2009; Hutching et al., 1996)
Anti-inflammatory (Fawole et al.,
2009), Antimicrobial and mutagenicity
(Fawole et al., 2009)
-
Meliaceae
Ekebergia capensis
Sparrm
Root, bark
Stomach and intestinal complaints,
dysentery, heart burn, purgative, kidney
problem, indigestion (Verschaeve and Van
Staden, 2008)
Mutagenicity , antimutagenicity
(Verschaeve and Van Staden, 2008),
antiplasmodial (Murata et al., 2008)
Ekersenin, 4,6-dimethoxy-5-methylcoumarin, oleanolic
acid, 3-epioleanolic acid, oleanoic acid (15), 3,11dioxoolean-12-en-28-oic acid, melliferone, 3-oxo11,13(18)-oleandien-28-oic acid, ekeberin A, (Z)volkendousin, ekeberin B, 7-deacetoxy-7-oxogedunin, 7acetylneotrichilenone, proceranolide, mexicanolide,
swietenolide, methylangolensate, ekeberins C1, C2, and
C3, 2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl6,10,14,18-tetracosatetraene (3R,22R), 2-hydroxymethyl2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl6,10,14,18-tetracosatetraene (2R,3R,22R), ekeberins D1,
D2, D3, D4, and D5 (Murata et al., 2008)
antibacterial, antiinflammatory, anticholinesterase and mutagenic effects
(Eldeen et al., 2005)
Antimicrobial, antioxidant, antiinflammatory, antimalarial, cytotoxicity
(Komane et al., 2011)
cycloart-23-ene-3,25-diol (Eldeen et al., 2007)
Melia azedarach L.
Trichilia dregeana
Sond.
Umsilinga (Z)
Umkhuhlu (Z)
Leaves
Leaves
Diarrhoea (de Wet et al., 2010)
Trichilia emetica
Vahl.
Umkhuhlu (Z)
Leaves
Diarrhoea (de Wet et al., 2010)
Umgandanganda
,ungandingandi
Root
Diarrhoea , dysentery, cough, colic, bloody
stool (De Wet and van Wyk, 2008)
Menispermaceae
Albertisia
delagoensis (N.E.
sendanin, trichilinin, trichilin A, trichilin B, trichilin C,
trichilin D, trichilin E, dregeana, nymania 1,
rohituka, rohituka, rohituka, Trichilia substance Tr-A,
Trichilia substance Tr-B, Trichilia substance Tr-C and
seco-A-protolimonoid (Komane et al., 2011)
Antiplasmodial and cytotoxicity (De wet
et al., 2007)
193
Br.) Forman
Antizoma
angustifolia (Burch.)
Miers ex Harv
Cissampelos
capensis (L.f.) Diels
Cissamperos hirta
Klotzch
Cissampelos
mucronata A. Rich.
Cissampelos
torulosa E.Mey
Moraceae
Ficus capensis
Thunb.
Ficus craterostoma
Mildbr. & Burret
Root
Diarrhoea , dysentery, cough, colic, bloody
stool (De Wet and van Wyk, 2008)
-
-
Root,
rhizome
Purgative, tincture for dysentery (van Wyk,
2008)
Diarrhoea (de Wet et al., 2010)
-
-
-
-
Root
Diarrhoea (Giess and Snyman, 1986; Von
Koenen, 2001)
Bisbenzylisoquinone alkaloid (Tshinbagu et al., 2003)
Lukandulula (V)
Leaves
Diarrhoea and dysentery, sore throat
(Mabogo, 1990; Samie et al., 2005)
Anti-ulcer (Akah and Nwafor, 1999),
sedative (Akah et al., 2002),
Antiplasmodial (Tshinbagu et al.,
2003)
Antiamoebic (Samie et al., 2009),
antibacterial (Samie et al., 2005)
Infusion
Fruit
Diarrhoea (Pallant and Steenkamp, 2008)
intestinal motility modulation (Ayinde
and Owolabi, 2009)
-
-
Decoction for diarrhoea (Venter and
Venter , 2002)
Diarrhoea (Mlambo, 2008)
-
-
Spasmolytic and gastrointestinal
protection (Kunle et al., 1999)
-
Limonene, copaene, Asiatic acid, β-carotene, morin-3-Oα-L-arabinopyranoside, avicularin, gauijaverin, quercitin
ellargic acid (Gutierrez et al., 2008)
Umbombo (Z)
Umanyokane,
khalimelo (Z)
Muumo (V),
inTendekwane,
umThombe(X)
Stomach-ache (Bhats and Jacobs , 1995)
Ficus glumosa
Delile
Ficus sur Forssk
Bark
Umkhiwane (Z)
Leaves
Myrtaceae
Psidium guajava L.
Ugwava (X, Z)
Leaves
Infusion for bloody diarrhoea (BisiJohnson et al., 2010)
Syzygium cordatum
Hochst. Ex. C.
Krauss
Umdoni, Mutu (V)
Leaves,
bark
Respiratory disorders, tuberculosis,
stomach complaints, emetics, diarrhoea,
cold, fever (Verschaeve and Van Staden,
2008)
Syzygium
paniculatum
Gaertner
Olacaceae
Ximenia caffra Sond
-
-
-
Antidiarrhoeal Tona et al., 1999;
Lutterodt, 1992); antispasmolytic
(Conde et al., 2003), antirotavirus
(Goncalves et al., 2005), antimicrobial
intestinal adhesion (Coutino et al.,
2001)
Mutagenicity , antimutagenicity
(Verschaeve and Van Staden, 2008);
antimycobacterium (Mativandlela et al.,
2008); Antiescherichia (Sibandze et
al., 2010)
--
Mutswili (V)
leaves
Diarrhoea and dysentery (Green et al.,
2010; Fabry et al., 1996)
Antigardial (John et al., 1995),
Antiamoebic (Samie et al., 2009),
-
-
-
-
194
Punica granatum L.
Oleaceae
Olea europaea L.
Subsp africana
(Mill.) P.S.Green
Orchidaceae
Polystachya
ottoniana Rchb.f.
Pedaliaceae
Ceratotheca triloba
(Bernh.) Hook
iRharnathi (X)
Uzintlwa (X),
uMnquma (X)
Portulaceae
Portulacaria afra
Jacq.
Proteaceae
Protea caffra Meisn.
Diarrhoea and dysentery (van Wyk, 2008;
Dold and Cocks, 2000)
antifungal (Samie et al., 2010)
Antidiarrhoeal (Pillai, 1992; Qnais et
al., 2007), Anti-inflammatory (Lansky
and Newman, 2007)
Ellagitannins, anthocyanins, flavone glucosides, flavones,
flavonol, flavonol glucosides, hydroxycinnamic acid,
hydroxybenzoic acid, flavan-3-ols, alkaloids, sterol,
triterpenoids (Lansky and Newman, 2007)
Anti-hypertensive, diuretic, tonic,
diarrhoea, sore throat (Amabeoku and
Bamuamba, 2010)
Antidiarrhoeal (Amabeoku and
Bamuamba, 2010)
-
Diarrhoea (Chinsamy et al., 2010)
-
-
Leaf
Infusion for diarrhoea and gastrointestinal
cramps (Watt and Breyer-Brandwijk, 1962;
Roberts, 1990)
5-lipooxygenase inhibitory and
antioxidant (Akula and Odhav, 2008),
α-amylase inhibitory (Odhav et al.,
2010).
-
Seed, root
Diarrhoea (van Wyk, 2008)
Indicain, plantagonin, baicalein, hispidulin, plantaginin,
aucubin, fumaric acid, syringic acid, vanillic acid, phydroxy benzoic acid, ferulic acid, p-coumaric acid,
gentisic acid, salicylic acid, benzoic acid, cinnamic acid
oleanolic acid, ursolic acid, 18β-glycyrrhetinic acid and
sitosterol (Samuelsen, 2000)
Seed
Diarrhoea (van Wyk, 2008)
Antidiarrhoeal (Atta and Mouneir,
2005), Hepatoprotective and antiinflammatory (Turel et al., 2007),
wound healing activity, antiinflammatory, analgesic, antioxidant,
weak antibiotic, immuno modulating
and antiulcerogenic activity
(Samuelsen, 2000)
-
uTshintshini
Roots
Diarrhoea in cow (Dold and Cocks, 2001;
McGaw et al., 2008)
Antibacterial and anti cancer (BisiJohnson et al., 2011)
-
Idololenkonyane
(Z), Idolonyana (X)
Idololenkonyane
(X, Z)
Roots
-
-
leaves
Infantile diarrhoea, tapeworm, wound and
sores (Dold and Cocks, 2000)
Diarrhoea (Bisi-Johnson et al., 2010)
-
-
Umndibili (Z)
Leaves
Diarrhoea (Mlambo, 20080
-
-
Tshidzungu (V)
Root bark
decoction
Calves with bloody diarrhoea (Hutching et
al., 1996)
-
-
Udonqabathwa (Z)
Plantaginaceae
Plantago major L.
Plantago lanceolata
L.
Plumbaginaceae
Plumbago
auriculata
Polygonaceae
Rumex lanceolatus
Thunb
Rumex obtusifolius
Fruit rind,
roots
-
195
Protea nitida Mill.
Protea simplex
Bark
Root, bark
Protea welwitschii
Engl.
Astringent for diarrhoea (van Wyk, 2008)
Decoction and infusion for diarrhoea,
dysentery, stomach pain in human
(Fawole et al., 2009; Hutching et al., 1996)
Dysentery, diarrhoea in calves and
humans (Watt and Breyer-Brandwijk,
1962; McGaw et al., 2008)
Anti-inflammatory (Fawole et al.,
2009Ndhalala), Antimicrobial and
mutagenicity (Fawole et al., 2009
Finnie)
-
-
-
Punicaceae
Punica granatum L.
Mokgranata
Root
Decoction for diarrhoea (Mathabe et al.,
2006)
-
-
Rhamnaceae
Ziziphus mucronata
Willd.
Mukhalu,
Mutshetshete (V)
Leaves,
bark, roots
Boils, sores, grandular swelling, diarrhoea,
dysentery, cough (Verschaeve and Van
Staden, 2008; Green et al., 2010)
-
Root-stock
Diarrhoea, internal parasites, general
ailments (Van der Merwe et al., 2001;
McGaw et al., 2008)
Anti-inflammatory (Fawole et al.,
2009Ndhalala), Antimicrobial and
mutagenicity (Fawole et al., 2009
Finnie
-
Umkhakhazi (X),
Umkakase (X)
Root
Diarrhoea, abdominal ailments (BisiJohnson et al., 2010)
Ipesika
Leaf
decoctions
Diarrhoea in lamb and kid goats (Dold and
Cocks, 2001; McGaw et al., 2008)
Bark
decoctions
Diarrhoea, bloody stool, colic (Neuwinger,
1996; Venter and Venter, 2002)
Antiescherichial (Sibandze et al.,
2010)
Root
Dysentry, dyspepsia, fever, gastritis,
gonorrhea, malaria,leprosy, measles,
piles, toothache (Reid et al., 2006)
Vomiting, diarrhoea in children((BisiJohnson et al., 2010)
Antiamoebic (Tona et al., 1998;
Moundipa et al., 2005); antidiarrhoeal
(Owolabi et al., 2010)
Antibacterial (Yff et al., 2002)
Palmitic acid (Yff et al., 2002)
Diarrhoea and vomiting (Bisi-Johnson et
al., 2010)
Diarrhoea and dysentery (van Wyk, 2008)
Diarrhoea, haemorrhoids, epilepsy (van
Wyk, 2008)
Diarrhoea and dysentery, toothache,
-
-
-
-
-
-
Ziziphus zeyheriana
Sond.
Rosaceae
Prunus africana
(Hook.f) Kalkman
Red Stinkwood
Prunus persica (L.)
Batsch.
Rubiaceae
Breonadia salicina
(Vahl) Hepper & J.
R. I. Wood
Nauclea latifolia
Smith
Pentanisia
prunelloides
(Klotzsch exEckl. &
Zeyh) Walp
Psychotria capensis
(Eckl.) Vatke
Rubia petiolaris DC.
Rubus pinnatus
Willd.
Rubus rigidus
Icishamlilo,
Icimamilo (X, Z)
Root,
leaves, bulb
Ishithitibala (Z),
UmGono-gono (X)
Fruits
iQunube
Root
Roots
Root
-
196
Vangueria infausta
Burch. subsp.
infausta
Rutaceae
Agathosma betulina
(Bergius) Pillans
Agathosma
crenulata (L.)
Pillans
Rutaceae
Clausena anisata
(Willd.) Hook.F. Ex
Benth.
Ruta graveolens L.
Sapindaceae
Hippobromus
pauciflorus (L.f.)
Radlk.
coughs and colds (Iwalewa et al., 2007).
Diarrhoea (de Wet et al., 2010)
Umviyo
Antispasmodic, antipyretic, cough, Kidney
and urinary tract infection, cholera and
stomach ailment (Molla and Viljoen, 2008)
Antispasmodic, antipyretic, cough, Kidney
and urinary tract infection, cholera and
stomach ailment (Molla and Viljoen, 2008)
Antibacterial and antifungal (de Boer et
al., 2005)
-
Antidiarrhoea and antibacterial (LisBalchin et al., 2001); anti-inflammatory
and antioxidant (Steenkamp et al.,
2006)
Antidiarrhoea and antibacterial (LisBalchin et al., 2001)
-
-
Bark
infusion
Dysentery in cattle (Hutching et al., 1996)
-
-
iVendrit (X)
Leaves
Fever, convulsion, epilepsy, diarrhoea,
cardiac asthma, jaundice (Dold and
Cocks, 2000)
-
-
Ulwathile, iLathile
(X)
Bark, root ,
leaves
Heartwater and diarrhoea in cattle (Dold
and Cocks, 2001; McGaw et al., 2008),
Diarrhoea and dysentery (Bisi-Johnson et
al., 2010)
Acute toxicity (Pendota et al., 2010),
anti inflammatory, analgesic antipyretic
(Pendota et al., 2009)
-
Igquzu (X)
Leaves
-
-
Umqumqumu (Z)
Leaves
Herb
Stomach disorder (Bisi-Johnson et al.,
2010)
Diarrhoea (Mlambo, 2008)
Antispasmodic, stimulant; convulsions;
cough; bronchitis (van Wyk, 2008)
Antibacterial (Ovenden et al., 2004)
-
Physaloside A (Ovenden et al., 2004)
-
Root
Cancer, dysentery, catarrh, leprosy (Watt
and Breyer-Brandwijk, 1996; Fouche et al.,
2008)
Anti-inflammatory and analgesic (Han
et al., 2007)
1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid,
chlorogenic acid, 4-O-caffeoylquinic acid, cynarin, 1,4-Odicaffeoylquinic acid, 1,5-O-dicaffeoylquinic acid, 1,5-Odicaffeoylquinic acid, 1,3,5-O-tricaffeoylquinic acid, 3,4,5O-tricaffeoylquinic acid (Han et al., 2007)
Fruit decoction for haemorrhoids,
dysentery, fruit as enema for diarrhoea
(Bisi-Johnson et al., 2010)
Root infusion for back ache (Amusan,
2007)
Antimicrobial (Koduru et al., 2006);
Anticancer (Koduru et al., 2007)
tomatidine and solasodine (Koduru et al., 2007)
-
-
Scrophulariaceae
Physalis peruviana
L.
Physalis viscose L.
Jamesbrittenia
atropurpurea
(Benth.) Hilliard
Xanthium
strumarium L.
Solanaceae
Solanum
aculeastrum Dun
umthuma (X, Z)
Fruit, root,
leaves
Solanum incanum
L.
uMthuma, intfuma
(S)
Root
197
Solanum
mauritianum
Solanum
panduriforme E.
Mey
Solanum supinum
Dun.
Sterculiaceae
Withania somnifera
(L.) Dun
Umtotovane (Z)
Leaf
Thuthula
Fruit sap
Thola (S)
Root
Dombeya
rotundifolia
(Hochst.) Planch.
Hermannia incana
Cav
Tshiluvhari (V)
Root, bark,
wood
Mavulakuvaliwe
leaves
uBuvimba
Waltheria indica L.
Strychnaceae
Strychnos
madagascariensis
Pior.
Ulticaceae
Pouzolzia mixta
solms
Verbenaceae
Clerodendrum
glabrum E. Mey
Viscaceae
Viscum capense L.
F.
Vitaceae
Lippia javanica
(Burm.f.) Spreng
Whole plant
Umkwakwa,
Mukwakwa (V)
Infusion for dysentery and diarrhoea (Watt
and Breyer-Brandwijk, 1962)
Diarrhoea (Van der Merwe et al., 2001;
McGaw et al., 2008)
-
-
-
-
Decoction for diarrhoea (Mathabe et al.,
2006)
-
-
Fever, cold and flu, abdominal discomfort,
diarrhoea, worms sedative and hypnotic
(van Wyk and Gericke, 2000; Fouche et
al., 2008)
Internal ulcers, haemorrhoids, diarrhoea,
stomach problems, nausea, chest pain
(Verschaeve and Van Staden, 2008)
Crushed with cold water and taken orally
for diarrhoea (Appidi et al., 2008)
Anti-inflammatory, antitumor,
immunomodulatory (Mishra et al.,
2000); antichorelae (Acharya et al.,
2009)
Mutagenicity , antimutagenicity
Isopelletierine, anferine, withanolides, withaferins,
sitoindosides (Mishra et al., 2000)
-
Decoction for diarrhoea (Mathabe et al.,
2006)
Toxicological assay (Appidi et al.,
2009); antimicrobial, anti-inflammatory,
antioxidant and cytotoxicity (Essop et
al., 2008)
Antibacterial, antifungal and antiviral
(Maregesi et al., 2008)
Diarrhoea (de Wet et al., 2010)
-
-
-
-
Muthanzwa
Root, leaves
Dysentery (Verschaeve and Van Staden,
2008); diarrhoea (Samie et al., 2010)
-
-
Umqangazani
Uqangazana (X),
iNunkisiqaqa (X)
Umqangazane
leaves
Bloody stool, chest infections (BisiJohnson et al., 2010)
-
--
Diarrhoea (Forbes, 1986; Van Wyk et al.,
2008)
-
-
Prophylactics against dysentery, diarrhoea
and malaria (Mabogo, 1990; Fouche et al.,
2008)
-
-
Iphakama (Z)
Musudzungwane
(V)
Leaf infusion
198
Rhoicissus
tridentata (L.F.) Wild
& Drums.
Cyphostemma
cirrhosum (Thunb.)
Zingiberaceae
Aframomum
latifolium (Afzel.) K.
Schum
Elytropappus
rhinocerotis (L.f)
Less.
Umthwazi (Z),
Murumbulambudzana (V)
Udekane (Z)
Tuber
decoction
Diarrhoea in goat and sheep (Dold and
Cocks, 2001; McGaw et al., 2008)
Antispasmolytic (Katsoulis et al., 2000)
-
Leaves
Diarrhoea (Mlambo, 2008)
-
-
Leaves
Decoction for diarhhoea (De Villiers et al.,
2010)
-
-
Twigs
Bitter for dyspepsia, indigestion, diarrhoea
(van Wyk, 2008)
-
ent-15β-senecioyloxy-16,17-epoxy-kauran-18-oic acid
V=Vhavenda, Z=Zulu, X=Xhosa, S=Swazi
199
Appendix 9.1: 1D and 2D NMR spectra data of Ursolic acid
Peak
Hydrogen
13C/DEPT
HSQC
number
1
1.46-1.6
CH2
37
HMBC
LITERATURE
15(C25), 27(C2), 56(C5), 78(C3)
39.2
2
1.38-1.5
CH2
27
37(C1), 56(C5), 78(C3)
28.1
3
3.0
CH
78
16(C23), 27(C2)
78.2
4
-
C
39
-
39.6
5
0.64
CH
56
16(C23), 18 (C6), 37(C1)
55.9
6
1.26, 1.44
CH2
18
56(C5)
18.8
7
1.24, 1.4
CH2
33
56(C5)
33.7
8
-
C
39
-
40.1
9
1.42
CH
48
15(C25), 23(C11), 37 (C1), 38.8(C10), 39(C8)
48.1
10
-
C
37
-
37.5
11
1.76-1.88
CH2
23
39 (C8), 125(C12), 140(C13)
23.7
12
5.2
CH
125
42(C14), 48(C9), 53(C18)
125.7
13
-
C
140
-
139.3
14
-
C
42
-
42.6
15
0.95, 1.4
CH2
28
24(C16), 48(C17)
28.8
16
1.5, 1.9
CH2
24
28 (C15), 42(C14), 48(C17), 53(C18), 178(C28)
25
17
-
C
48
-
48.1
18
2.08
CH
53
53.6
19
0.9
CH
38.6
17.5(29), 24(16), 38.8(19), 42(14), 37(C20), 125(12),
140(13), 178(28)
39(C19), 37(C20)
20
1.29
CH
37
39.4
21
1.48-1.58
CH2
30
31
22
0.87, 1.48
CH2
38.9
24(C16), 53(C18)
37.4
23
0.64
CH3
16
29(C24), 39(C4), 56(C5), 78(C3)
16.5
24
0.87
CH3
29
16(C23), 78(C3)
28.8
25
0.85
CH3
15
37(C1), 56(C5)
15.7
26
0.72
CH3
17.3
33(C7), 39(C8), 42(C14), 48(C9)
17.5
27
1.02
CH3
23.4
28(C15), 39(C8), 42(C14), 140(C13)
24
28
-
C
178
-
179.7
29
0.78
CH3
17.5
38.8(C19), 53(C18)
17.5
30
0.90
CH3
22
31(C21), 48(C20)
21.4
39.5
200
Appendix 9.2: 1D and 2D NMR spectra data of mixture of corosolic acid and maslinic acid
1H
13C/DEPT
Pea1k
HSQC
HSQC
HMBC (H→C)
number
(Corosolic
(Maslinic
acid)
acid)
1
1.7-1.8, 0.73- CH2
47.75
47.75
16.92(C25), 38.27(C10), 55.35(C5),
0.79
68(C2), 83.49(C3)
2
3.4
CH
68.41
68.41
83.49(C3)
3
2.7
CH
83.49
83.49
68.41(C2), 29.87(C24)
4
C
39.61
39.61
5
0.68-0.75
CH
55.35
55.35
39.61(C4), 38.25(C10), 29.43(C24)
6
1.41-1.47,
CH2
18.71
18.71
38.12(C10), 39.61(C4)
1.26-1.33
7
1.36-1.45,
CH2
33
33
17.49(C26), 55.35(C5), 18.71(C6)
1.56-1.61
8
C
39.75
39.75
9
1.5
CH
47.53
47.53
23.74 (11) (24.01)
10
C
38.25
38.25
11
1.8-1.9, 1.43- CH2
23.74
24.01
47.53(C9), 125.55(C12) (122.72),
1.47
139.43(C13) (145.09)
12
5.11
CH
125.55
122.72
23.74(C11) (24.01), 42.19(C14) (41.92),
47.53(C9), 53.05(C18) (41)
13
C
139.42
145.09
14
C
42.19
41.92
15
0.92-0.99,
CH2
28.21
28.21
39.57(C8), 23.60(C27) (26.8), 42.19(C14)
1.75-1.79
(41.92)
16
1.26-1.33,
CH2
24.51
27.94
178 (C28) (179.24), 28.21(C15),
1.47-1.52
53.05(C18), (41), 48.13(C17)
17
C
48.33
48.33
18
2.09 (2.7)
CH
53.05
41
17.75(C29), 24.51(C16) (27.94),
39.10(C19) (27.94), 42.19(C14) (41.92),
48.13(C17), 37(C22),125.55(C12)
(122.72), 139.42(C13) (145.09), 178
(C28) (179.24)
19
0.88-0.92
CH
39.07
27.94 (CH2)
20
1.25-1.31
CH
39.02 (CH) 46.13 (C)
21
1.48-1.58
CH2
30.6
37
24, 31
22
1.58, 1.00CH
37
47.53
178(C28) (179.24) , 24.51(C16) (27.94)
1.09
23
0.90
CH3
29.2
29.2
17.76(C24), 55.35(C5), 39.61(C4)
24
0.68
CH3
17.76
17.76
29.43(C23), 39.61(C4), 55.35(C5)
25
0.88
CH3
16.94
16.94
38.25(C10), 47.53(C9)
26
0.72
CH3
17.4
17.4
47.53(C9), 42.19(C14) (41.92), 33.01(C7)
27
1.00
CH3
23.60
26.8
28.21(C15), 39.75(C8), 42.19(C14)
(41.92), 139.42(C13) (145.09)
28
C
178
179.70
29
0.79
CH3
17.5
17.5
39.07(C19) (27.94), 53.05(C18) (41)
30
0.88
CH3
21.6
21.6
39.02(C20) (46.13)
LITERAT
URE
46.8
68.9
83.8
39.1
55.4
18.4
32.9
39.6
47.5
38.3
23.4
125.3
138.1
42.1
28.0
24.3
48.1
52.8
39.1
38.9
30.7
36.7
28.7
17.0
17.0
17.0
23.7
177.9
17.0
21.2
201
Appendix 9.3: 1D and 2D NMR spectra data of mixture of asiatic aicd and arjunolic acid
1H
13C/DEPT
Peak
HSQC
HSQC
HMBC (H→C)
number
(Asiatic acid)
(Arjunolic
acid)
1
0.69, 1.73
CH2
48.31
48.31
18.04(C25), 68.67(C2),
76.50(C3)
2
3.45
CH
68.67
68.67
76.50(C3)
3
3.13
CH
76.50
76.50
68.67(C2), 14.30(C24),
65.01(C23), 43.62(5)
4
C
43.62
43.62
5
1.14
CH
47.27
47.27
76.50(C3), 47.27(C4), 65.01
(C23), 14.30(C24), 33.40 (C7),
18.04 (C25)
6
1.33, 1.18
CH2
18.03
18.03
38.12(C10), 39.61(C4)
7
1.44, 1.20
CH2
33.40
33.40
40.49 (C8)
8
C
40.49
40.49
9
1.5
CH
47.51
47.54
23.74 (11) (24.01)
10
C
38.40
38.40
11
1.78, 1.42
CH2
23.00
24.01
125.19(C12) (122.72),
138.92(C13) (145.09)
12
5.09 (5.12)
CH
125.19
122.28
42.21(C14) (41.92), 47.51(C9)
(47.54), 52.62(C18) (41.73)
13
C
138.71
144.68
14
C
42.21
41.92
15
1.73, 1.58
CH2
28.07
28.21
39.57(C8), 23.60(C27) (26.8),
42.19(C14) (41.92)
16
1.88, 1.48
CH2
24.31
27.94
179.13 (C28) (179.24),
17
C
47.99
48.33
18
2.05 (2.72)
CH
52.62
41.73
18.16(C29), 24.31(C16)
39.44(C19) (27.94), 42.21 (C14)
(42.52), 47.99(C17) (47.01),
125.19(C12) (122.28),
138.92(C13) (144.68), 179.13
(C28) (179.24)
19
0.88-0.92
CH
39.44
24.31(CH2)
20
1.25-1.31
CH
38.90(CH)
47.27 (C)
21
1.38, 1.20
CH2
31.42
31
24, 31
22
1.55. 1.39
CH2
33
33
24.31(C16), 31.61 (C21)
23
3.25, 3.00
CH2
65.01
65.01
14.91(C24), 43.62(C5),
47.27(C4), 76.50 (C3)
24
0.50
CH3
14.91
14.91
65.01(C23), 47.27(C4),
43.62(C5)
25
0.65
CH3
18.04
18.04
40.49(C10), 43.62(C5),
48.31(C1)
26
0.68
CH3
17.04
17.04
47.53(C9), 42.19(C14) (41.92),
33.18 (C7)
27
0.99 (1.04)
CH3
23.40
26.8
28.48(C15), 40.49(C8) (39.96),
42.21(C14) (42.57), 138.92(C13)
(144.68)
28
C
179.13
179.24
29
0.77 (0.82)
CH3
18.16
26.0
39.44(C19) (27.94), 53.05(C18)
(41)
30
0.87 (0.82)
CH3
23.0
33
39.02(C20) (46.13)
LITERATUR
E
46.8
68.9
83.8
39.1
55.4
18.4
32.9
39.6
47.5
38.3
23.4
125.3
138.1
42.1
28.0
24.3
48.1
52.8
39.1
38.9
30.7
36.7
28.7
17.0
17.0
17.0
23.7
177.9
17.0
21.2
202
Appendix 9.4: 1D and 2D NMR spectra data of combretastatin B5-2’-O- glucopyranoside
13C/DEPT
Peak number 1H
HSQCa
HMBC (H→C)
1
2
3
4
5
6
1a
1a’
1’
2’
3’
4’
5’
6’
1’’
2’’
3’’
4’’
5’’
6’
3-OCH3
4-OCH3
5-OCH3
4’-OCH3
6.5
6.5
2.7
2.9, 3.0
6.7
6.6
4.5
3.3
3.2
3.2
3.2
3.5, 3.7
3.5
3.5
3.5
C
CH
C
C
C
CH
CH2
CH2
C
C
C
c
CH
CH
CH2
CH
CH
C
CH
CH2
CH3
CH3
CH3
CH3
132.72
106.26
144.35
133.79
148.25
106.26
36.96
31.73
128.55
144.35
139.71
147.26
109.40
118.95
106.16
74.45
76.62
70.15
77.85
61.34
56.34
56.34
56.30
C1, C3, C6, C1a
C1, C5, C2, C1a
C1, C2, C1a’, C1’
C1, C1’, C2’, C6’, C1a
C1’, C3’, C4’, C6’
C2’, C4’, C1a’
C2’, C2”, C5’’,
C1”, C3”
C4”, C5”
C3”, C5”
C3”
C4”, C5”
C3
C5
C4’
LITERATURE DATA HSQCa’
132.43
105.96
144.77
133.44
147.77
105.96
36.58
31.35
128.22
143.95
139.29
146.90
109.10
118.68
105.79
74.04
76.14
69.71
77.44
60.89
56.0
56.0
56.0
b Pelizzoni Francesca, 1994: Combretastatin derivatives with antitumoral activity and process for the preparation thereof. Patent Cooperation Treaty (PCT), WO 94/05682, CO7H 15/203,
CO7C 43/23, A61K 31/70, 31/085
Appendix 9.5: 1D and 2D NMR spectra data of combretastatin B1-2’-O- glucopyranoside
13C/DEPT
Peak number 1H
HSQCb
HMBC (H→C)
1
2
3
4
5
6
1a
1a’
1’
2’
3’
4’
5’
6’
1’’
2’’
3’’
4’’
5’’
6’
3-OCH3
4-OCH3
5-OCH3
4’-OCH3
6.5
6.5
2.69, 2.79
2.92, 3.20
6.7
6.6
4.5
3.3
3.2
3.5, 3.7
3.5
3.5
3.5
3.5
C
CH
C
C
C
CH
CH2
CH2
C
C
C
c
CH
CH
CH2
CH
CH
C
CH
CH2
CH3
CH3
CH3
CH3
138.54
105.60
152.69
135.53
152.72
105.60
36.92
31.34
128.27
143.78
139.13
147.02
108.54
119.24
105.69
74.21
76.63
69.72
77.03
60.97
55.14
59.69
55.34
55.42
C1, C1a, C3, C4
C1, C1a, C4, C5
C1, C1’, C2, C6, C1a’
C1a, C1’, C2’, C6’
C1’, C3’, C4’,
C1a’, C2’, C4’
C3
C4
C5
C4’
LITERATURE DATA HSQCb’
138.10
105.63
152.65
135.45
152.65
105.63
36.91
31.18
128.03
143.93
139.35
146.95
108.99
118.56
105.77
74.09
76.26
69.76
77.51
60.96
55.78
60.04
55.78
55.94
b Pelizzoni Francesca, 1994: Combretastatin derivatives with antitumoral activity and process for the preparation thereof. Patent Cooperation Treaty (PCT), WO 94/05682, CO7H 15/203,
CO7C 43/23, A61K 31/70, 31/085
203
Appendix 9.6: 1D and 2D NMR spectra data of 3β-ethoxy sitosterol
1H
13C/DEPT
Peak number
HSQC
1
1.036, 1.817
CH2
37.47
2
2.233
CH2
31.8
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
3.502
2.194
1.058
0.988
1.234
1.058
0.988
CH
CH2
C
CH
CH2
CH
CH
C
CH2
CH2
C
CH
CH2
CH2
CH
CH3
72.04
40.00
140.97
121.93
32.12
29.91
50.35
36.73
21.30
40
42.54
56.99
24.52
28.47
56.27
12.08
19
0.659
CH3
20.03
20
21
1.326
0.904
CH
CH3
36.36
19.24
0.988
0.796
CH2
CH2
CH
CH
CH3
CH3
34.16
26.29
46.05
29.37
19.61
19.00
CH2
CH3
CH3
CH2
23.28
12.20
22
23
24
25
26
27
28
29
CH3CH2
CH3CH2
5.33
1.427
-
0.822
7.87
6.88
7.72
C
C
C
CH
C
C
CH
CH
LITERATURE
37.3
31.9
36.73 (C10), 72.04 (C3),
121.93 (C6), 140.97 (C5)
32.12 (C7), 36.73 (C10)
140.97 (C5), 37.47 (C1), 50.35
(C9)
56.99 (C14), 56.23 (C17),
42.52 (C12), 42.54 (C13),
56.27 (C17), 36.36 (C20),
34.16 (C22)
29.37 (C25), 46.05 (C24),
19.61 (C26)
23.25 (C28)
71.8
40.5
140.7
121.7
31.9
31.6
50.2
36.5
21.1
39.8
42.3
56.8
24.3
28.3
56.1
11.9
19.5
36.2
18.9
33.9
26.1
45.8
29.1
19.4
19.1
23.1
12.0
76.86
Appendix 9.7: 1D and 2D NMR spectra data of Quercetin
13C/DEPT
Peak number 1H
HSQC
2
C
147.96
3
C
137.23
4
C
177.31
5
C
162.50
6
6.17
CH
99.28
7
C
165.55
8
6.37
CH
94.39
9
10
1’
2’
3’
4’
5’
6’
HMBC (H→C)
158.21
104.51
124.13
116.30
147.96
148.75
116.78
121.65
Appendix 9.8: 1D and 2D NMR spectra data of Myricetin
1H
13C/DEPT
HSQC
Peak
number
HMBC (H→C)
162.50 (C5), 104.51 (C10), 94.39 (C8)
165.55 (C7), 158.21 (C9), 104.51 (10),
99.28 (C6)
147.94 (C2), 144 (C3’), 121 (C6’)
144 (C3’), 121 (C6’)
HMBC (H→C)
LITERATURE
146.8
135.8
175.9
160.8
98.2
163.9
93.4
156.2
1103.0
122.0
115.1
145.1
147.7
115.6
120.6
LITERATURE
204
2
3
4
5
6
7
8
9
10
1’
2’
3’
4’
5’
6’
6.17
C
C
C
C
CH
146.57
135.92
175.86
161.05
97.80
6.37
C
CH
164.17
92.95
7.38
C
C
C
CH
156.76
103.06
121.65
107.10
7.38
C
C
C
CH
145.29
135.51
145.29
107.10
164.17 (C7), 161.05 (C5),
103.06 (C10), 99 (C6)
164.17 (C7), 158 (C9), 103.06
(C10), 99 (C6)
145.29 (C3’), 135.51 (C4’),
121.65 (C1’), 107.10 (C6’)
145.29 (C5’), 135.51 (C4’),
121.65 (C1’), 107.10 (C2’)
Appendix 9.9: 1D and 2D NMR spectra data of Isoetin 2’ methyl ether/ Isoetin 4’ methyl ether
1H
13C/DEPT
HMBC (H→C)
Peak
number
2
C
163.09
C3, C6’
3
7.2(s)
CH
108.85
C6’
4
C
184.24
5
C
163.67
6
6.2(d, J=)
CH
99.97
7
C
166.22
8
6.4 (d, J=)
CH
94.86
C6
9
C
159.43
10
C
105.09
C3, C8
1’
C
110.17
C3’
2’
C
153.27
OCH3, C3’, C6’
3’
6.65 (s)
CH
101.36
4’
C
153.27
OCH3, C3’, C6’
5’
C
140.86
C3’, C6’
6’
7.38 (s)
CH
114.44
OCH3
3.8 (s)
56.34 at C2’ or C4’
aIsoetin 5’methyl ester (AbdurRahman and Moon, 2007), b isoetin (Gluchoff-Fiasson et al., 1991)
148.2
137.5
177.5
162.6
99.5
165.8
94.6
158.4
104.7
123.3
108.8
146.9
137.1
146.9
108.8
LITERATURE
163.2a
108.7
183.5
162.7
99.8
165.3
95.1
159
105.1
108.9
154.2
105.4
152.8
142.8
112.6
57.6 at C5’
161.70b
106.77
181.74
161.41
98.48
163.88
93.48
157.19
103.46
106.97
150.50
104.20
151.60
138.73
113.44
-
205
Appendix 9.10: 1D and 2D NMR spectra data of Quercetin-3-O-β-galactopyranoside
1H
13C/DEPT
Peak
HSQC
HMBC (H→C)
number
2
C
156.76
3
C
133.85
4
C
177.83
5
C
161.42
6
CH
99.17
162.50 (C5), 104.51 (C10),
94.39 (C8)
7
C
164.58
8
CH
94.09
165.55 (C7), 158.21 (C9),
104.51 (10), 99.28 (C6)
9
C
156.80
10
C
102.36
1’
C
122.14
2’
CH
115.66
147.94 (C2), 144 (C3’),
121 (C6’)
3’
C
144.97
4’
C
148.75
5’
CH
116.47
6’
CH
121.42
1’’
5.18 (d)
CH
104.24
133.85(C2)
2’’
3.83 (t)
CH
71.55
104.12 (C1”), 73.70 (C3”)
3’’
3.56 (m)
CH
73.40
104.12 (C1”), 71.87 (C2”),
4’’
3.87 (s)
CH
68.30
144 (C3’), 121 (C6’)
5’’
3.94 (t)
CH
75.96
6’’
3.66 (dd), 3.5 (m)
CH2
60.45
75.80 (5”), 68.61 (C4”)
Appendix 9.11: 1D and 2D NMR spectra data of Myricetin-3-O-β-galactopyranoside
1H
13C/DEPT
HSQC
HMBC (H→C)
Peak number
2
C
156.95
3
C
134.56
4
C
177.97
5
C
161.54
6
6.19
CH
98.47
164.70 (C7), 161.54 (C5), 104.12
(C10), 93.25 (C8)
7
C
164.70
8
6.38
CH
93.25
164.70 (C7),157.24(C9), 104.12
(C10), 98.47 (C6)
9
C
157.24
10
C
104.14
1’
C
120.26
2’
7.37
CH
108.52
156.95 (C2), 144.95 (C3’), 136.71
(C4’), 120.26 (C1’), 108.52 (C6’)
3’
C
144.95
4’
C
136.71
5’
C
144.95
6’
7.37
CH
108.52
156.95 (C2), 144.95 (C5’), 136.71
(C4’), 120.26 (C1’), 108.52 (2’)
1’’
5.18 (d)
CH
104.12
134.56 (C2)
2’’
3.83 (t)
CH
71.87
104.12 (C1”), 73.70 (C3”)
3’’
3.56 (m)
CH
73.70
104.12 (C1”), 71.87 (C2”),
4’’
3.87 (s)
CH
68.61
5’’
3.94 (t)
CH
75.80
6’’
3.66 (dd), CH2
60.52
75.80 (5”), 68.61 (C4”)
3.5 (m)
LITERATURE
158.3
135.8
179.4
163.0
99.8
166.0
94.7
158.8
104.2
123.2
117.8
145.8
149.9
116.1
122.9
105.4
73.2
75.1
70.0
77.2
61.9
LITERATURE
156.4
135.4
177.6
161.4
98.7
164.4
93.4
156.4
103.9
120.0
108.6
145.5
136.8
145.5
108.6
105.4
73.2
75.1
70.0
77.2
61.9
206
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement