phytochemical analysis and antimicrobial activity of roasted beans of

International Journal of Pharmacy and Biological Sciences
ISSN: 2321-3272 (Print), ISSN: 2230-7605 (Online)
IJPBS | Volume 8 | Issue 1 | JAN-MAR| 2018 | 89-95
Research Article | Biological Sciences | Open Access | MCI Approved|
|UGC Approved Journal |
PHYTOCHEMICAL ANALYSIS AND ANTIMICROBIAL ACTIVITY
OF ROASTED BEANS OF COFFEA ROBUSTA
Ritu Mahajan* and Nisha Kapoor
School of Biotechnology, University of Jammu, Jammu (J&K)
*Corresponding Author Email: ritufeb@gmail.com
ABSTRACT
Coffee consumption has been growing globally due to its pleasant aroma and health benefits. The present study
investigated the presence of phytochemicals along with its antioxidant activity. Three different extracts were
prepared from the roasted beans of Coffea robusta and the preliminary phytochemical screening for the crude
extracts showed results for phenolics, flavonoids, steroids, flavonoids, saponins and terpenoids compounds in each
of the extracts. High antioxidant activity was observed in methanolic extracts using DPPH and FRAP assay.
Regarding the antimicrobial activity, the highest zone of inhibition was observed in methanol
extract against Alcalygens denitrificans. Thus, roasted beans of Coffea robusta could be good source of natural
antioxidants and antimicrobial agents.
KEY WORDS
Coffea robusta; Phytochemicals; Antifungal; Antibacterial; Antioxidants
INTRODUCTION
Human beings have been using bioactive compounds
produced by plants as remedies to improve their health
and cure illness. Coffee is a popular beverage that is
widely consumed around the globe due to its
physiological effects as well as its pleasant taste and
aroma. Coffea robusta is an important class of coffee
belonging to the family rubiaceae. Caffeine is the most
important constituent of coffee which is widely used as
stimulant. Various studies have revealed a number of
beneficial health properties of coffee diuretic,
antimicrobial and antioxidant activities [1, 2, 3].
Phytochemicals are natural bioactive compounds
present in plants that have defense role. The plants
produce these chemicals to protect themselves, but
recent research exhibits their importance in human
health [4, 5]. Antioxidant acts as radical scavengers and
reduction of oxidative damage to macromolecules thus
protecting the human body from various diseases [6].
Coffee is also rich source of the alkaloids, especially
caffeine which stimulates the central nervous system
International Journal of Pharmacy and Biological Sciences
and has role in diuretic and peripheral vasoconstriction.
Caffeine also contributes to the brew bitterness [7].
Therefore, the present study focuses on the evaluation
of phytochemicals, antioxidant and antimicrobial
properties of three different extracts of the roasted
beans of Coffea robusta.
MATERIALS AND METHODS
Sample Collection
Roasted beans of Coffea robusta were collected in a
sterilized bag from Coorg district of Karnataka. The
sample was brought to laboratory. The coffee beans
were ground into fine powder and three extracts viz.
methanol, aqueous and chloroform were prepared. 15
g of powdered coffee sample was mixed in 75 ml of each
solvent and the mixture was stirred for 24
hours. The suspended mixture was filtered through
whatman’s filter paper and filtrate was collected. This
procedure was repeated thrice to get three filtrates and
residue. The filtrates were then dried at room
temperature. Each of the solvents was evaporated and
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the gummy solid thus obtained was labeled and stored
for further use.
Phytochemical Qualitative Analysis
Methanol, aqueous and chloroform plant extracts were
assessed for the existence of the phytochemicals by
using the following standard methods [8, 9].
Test for carbohydrates
To1 ml of extract, added few drops of Molisch’s reagent
and then added 1 ml of concentrated sulphuric acid at
the side of the tubes. The mixture was then allowed to
stand for 2 to 3 minutes. Formation of violet coloured
ring indicated the presence of carbohydrates in the
sample extract
Test for proteins
About 1.0 ml of each crude extract was taken and two
drops of freshly prepared 0.2% Ninhydrin reagent was
added and heated. The appearance of pink or purple
colour indicated that the presence of proteins, peptides
or amino acids.
Test for phenolics
To 1.0 ml of cude extract, few drops of 5% aqueous
ferric chloride solution was added. Formation of bluish
black colour indicated that the presence of phenolics.
Test for Saponins
5.0 ml of distilled water was mixed with 1.0ml aqueous
crude plant extract in a test tube and it was mixed
vigorously. The frothing was mixed with few drops of
olive oil and mixed vigorously, and the foam appearance
showed the presence of saponins.
Tests for Flavonoids
2 ml of each extract was added to few drops of 20%
sodium hydroxide. Formation of intense yellow colour
was observed. To this, few drops of 70% dilute
hydrochloric acid were added and yellow colour
disappeared. Formation and disappearance of yellow
colour indicated the presence of flavonoids in the
sample extract.
Test for Terpenoids
2.0 ml of chloroform was added with the 5 ml aqueous
plant extract and evaporated on the water bath and
then boiled with 3 ml of concentrated H2SO4. Formation
of grey color showed the presence of terpenoids.
Test for Steroids
2 ml of chloroform and concentrated H2SO4 were added
with the 5 ml aqueous crude plant extract. In the lower
chloroform layer red color appeared that indicated the
presence of steroids.
International Journal of Pharmacy and Biological Sciences
Quantitative estimation of total phenolics and
flavonoids
Total phenols were estimated using Folin-Ciocalteu
method [10]. To 0.5mL of each extract solution 0.5ml of
1N Folin-Ciocalteu reagent was added. The mixture was
kept at room temperature for 5 min, followed by the
addition of 1ml of 20% Na2CO3. After incubating for 10
min at room temperature, the absorbance was
measured at 750nm. Gallic acid was used as standard.
The concentration of phenolics was calculated as per
following equation.
Absorbance = 0.0364 gallic acid (μg) + 0.009.
Total flavonoid content was estimated using a
colorimetric method [11]. The extracts were diluted
with distilled water to a volume of 3.5ml, followed by
the addition of 150μl of 5% sodium nitrite solution. After
incubation of 5 min, 300 μl of 10% of aluminium chloride
solution was added to each extract mixture. This was
followed by addition of 550µl of the distilled water after
incubation of 6 min at room temperature. The
absorbance was measured at 510nm. Querticin was
used as standard.
Determination of Antioxidant Activity
DPPH Radical Scavenging Assay
Free radical scavenging ability of the extracts was tested
by DPPH radical scavenging assay as described by Blois
[12]. The hydrogen atom donating ability of the plant
extractives was determined by the decolorization of
methanol solution of 2,2-diphenyl-1-picrylhydrazyl
(DPPH). A solution of 0.1 mM DPPH in methanol was
prepared, and 2.4 ml of this solution was mixed with
1.6 m of each plant crude extract in methanol at
different concentrations (12.5–150 μg/ml). The reaction
mixture was vortexed thoroughly and incubated in the
dark for 30 min at 37oC. The absorbance of the mixture
was measured spectrophotometrically at 517 nm. BHT
was used as reference. Decrease in absorbance
indicated increased radical scavenging activity which
was determined by the following equation:
Inhibition (%) = (abs. of control - abs. of test solution) X
100/ abs. of control
Where, absorbance of control = total radical activity
without inhibitor and absorbance of test = activity in the
presence of test compounds.
Ferric reducing antioxidant power (FRAP Assay)
Total antioxidant capacity was determined using FRAP
assay [13]. The FRAP reagent included 10 mM TPTZ
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solution in 40 mM HCl, 20 mM FeCl3 solution and 0.3 M
acetate buffer (pH 3.6) in ratio of 1:1:10 (v/v). 100 μl of
all three pre-prepared extracts of coffee beans were
mixed with 900 μl of freshly prepared FRAP reagent (pH
3.6) and incubated for 4 minutes at room temperature.
Absorbance of the colored product (ferrous
tripyridyltriazine complex) was measured at 593 nm
using trolox as the standard solution.
Antimicrobial activity
Five bacterium strains i.e. Bacillus subtilis, Alcalygens
denitrificans and Klebsiella pneumonia, Camphylobacter
jejni, Micrococcus luteus were incubated for 24 hours at
37°C on nutrient agar medium and then maintained at
4°C. Two fungal strains Fusarium oxysporum and
Bipolaris specifera were grown on PDA medium at 28oC
and maintained at 4°C.
In vitro antibacterial activity was observed for
chloroform, methanol, and aqueous extracts using agar
well diffusion assay [14]. The agar plates were seeded
with indicator test strains (bacterial). Stock solution of
each plant extract was prepared at a concentration of 1
μg / μl in different solvents viz. Methanol, chloroform
% Mycelial inhibition =
and aqueous. 100 μl of each plant solvent extracts was
added into wells using sterile syringe and allowed to
diffuse at room temperature for 3 hrs. The plates were
incubated at 37°C for 18- 24 hours for bacteria growth.
The effect was compared with that of positive reference
(ampicillin) to determine the sensitivity of bacterial
growth.
The antifungal activity was determined using poisoned
food technique [15]. Five-day old fungal cultures of
Fusarium oxysporum and Bipolaris specifera were
punched aseptically with a sterile cork borer of 7mm
diameter. The fungal discs were then put on the gelled
agar plates that were prepared by impregnating desired
concentration of plant extract. The plates were then
incubated at 26±1°C temperature. Colony diameter was
recorded by measuring the two-opposite circumference
of the colony growth. Percentage inhibition of mycelial
growth was evaluated by comparing the colony
diameter of poisoned plate (with plant extract) and nonpoisoned plate (with distilled water) and calculated
using the formula given below [16].
Mycelial growth(control)-Mycelial growth(treated)
Mycelial growth(control)
RESULTS AND DISCUSSION
Qualitative analysis of different extracts of Coffea robusta L.
The results for the qualitative estimation of different crude extracts of coffee are given below in table 1
Table 1: Phytochemical analysis for the methanolic, aqueous and chloroform extracts of C. robusta
Phytochemical Tests
Methanol extract
Chloroform extract
Water extract
Carbohydrates
++
+
Proteins
++
+
Flavanoids
++
+
Phenolics
++
+
Terpenoids
++
+
Saponins
++
+
Steriods
++
+
++: Present in high amount +: Present in traces -: Absent
Quantitative phytochemical Analysis
compounds in test samples was calculated from the
Antioxidant and antimicrobial properties of various Standard Gallic acid curve.
extracts from many plants have recently been of great The total phenolic content in different solvent extracts
interest in both research and the food industry. The is shown in Table 2. The highest amount of phenolics
presence of phytochemicals as natural additives have were
observed
in
methanolic
extract
tendency to replace synthetic antioxidants and (16.84±0.12µgGAE/ g dry wt), while the aqueous extract
antimicrobials with natural ones.
has only 4.857±0.23µg GAE/ g dry wt (Table 2). The total
Total Phenolic and flavonoid Content
phenolic content in plant depends on the type of
Total phenolic content was determined according to extract, i.e. the polarity of solvent used in extraction.
Folin- Ciocalteau method. The concentration of phenolic High solubility of phenols in polar solvents provides high
International Journal of Pharmacy and Biological Sciences
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Int J Pharm Biol Sci.
concentration of these compounds in the extracts
obtained using polar solvents for the extraction [17].
The results of the present study showed that much
different between the amounts of flavonoids was not
recorded in different solvent extracts. The crude
methanolic extract had highest amount of flavonoids
(76.04±0.23 mgQEs/g) as compared to chloroform and
aqueous extracts which had 68.89±0.87 mgQEs/g and
51.65±0.45 mgQEs/g of flavonoids respectively (Table
2). Hecimovic et al. [18] observed a good correlation in
the content of total flavonoids and phenolics. Their
results indicated that roasting affects the polyphenolic
compounds of coffee, and confirmed that light and
medium roasting are more favourable in terms of
preserving these beneficial compounds during coffee
roasting.
Table 2: Total phenol and flavonoid content in different solvent extracts of C. robusta
Extracts
Aqueous
Methanol
Chloroform
Total Phenolics (µg GAE/g dry wt)
4.857±12
16.84±23
10.91±23
Antioxidant activity
DPPH Assay
DPPH assay is a simple and quick method for
determining the ability of anti-oxidants to scavenge the
free radicals. Among all the solvent extracts, methanolic
extract of C. robusta was found to be the most potent
antioxidant (IC50 89.50± 1.1µg/ml), followed by
aqueous extract (IC50 241.66± 0.5µg/ml). Chloroform
extract showed poor radical scavenging activity (IC50
425.92± 0.3µg/ml), whereas BHT was taken as reference
antioxidant (IC50 23.5±.0.5µg/ml) (Table 3).
Pérez-Hernández et al. [19] observed a high antioxidant
activity in Green Arabica and robusta coffee beans. But
our studies show lower value as compared that of green
coffee beans. These changes in the antioxidative
capacity of coffee upon roasting are associated with the
degradation of chlorogenic acid [20]. A linear
correlation has been obtained by comparing the
antioxidant activity and polyphenols and flavonoid
content of the extracts. The methanol extract contained
Total flavonoids (mg QE/g dry wt)
68.98±0.87
76.0±0.23
51.65±0.45
good amount of phenols and flavonoids and thus
possess potential antioxidant activity. Previous studies
have also reported positive correlation between
phenolic and flavonoid content and DPPH scavenging
activity of plant extracts
FRAP assay
The ferric reducing antioxidant activity is shown in Table
3. The FRAP assay showed difference in all the three
crude extracts. The results showed that the FRAP
activity was high in methanol extract (58.02 mg Fe2+/g
of extract) whereas both chloroform and aqueous
extracts showed poor FRAP activity viz. 16.79±34 mg
Fe2+/g of extract and 12.79±0.76 mg Fe2+/g of extract
respectively. Budryn and Nebesny [21] observed higher
antioxidative efficacy in the extracts of robusta coffee
had than those from arabica coffee beans. This is due to
Progressive reduction in the antioxidant during brewing
of coffee beans that causes degradation of phenolic
compounds and the generation of Maillard’s reaction
products [22].
Table 3: Antioxidant activity of extracts of Coffea robusta using DPPH and FRAP assay
S.No.
Extracts
1
2
3
4
Aqueous
Methanol
Chloroform
BHT
DPPH assay
IC50 in g/ml
241.665± 0.52
89.502± 1.1
425.92± 0.3
23.5±.0.5
Antimicrobial Assay
Antibacterial activity
The results of antibacterial activity assay of three
extracts of Coffea robusta were obtained by agar
International Journal of Pharmacy and Biological Sciences
FRAP assay
(mg Fe2+/g of extract)
12.79± 0.76
58.02± 0.12
16.79± 0.34
------
diffusion method. Zone of inhibitions of three extracts
that is aqueous, chloroform and methanol against five
bacterial strains is shown in the table 4. The highest
zone of inhibition was shown against Alcaligenes
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Int J Pharm Biol Sci.
denitrificans which was 22.5mm while with
Camphylobacter jejni, the zone of inhibition was
10.2mm. However, no antibacterial activity was
observed against Bacillus subtilis, Micrococcus luteus
and Klebsiella pneumonia.
Huck et al., 2005 confirmed the presence of an alkaloid
caffeine in roasted coffee and the rough green beans in
both Arabica and Robusta coffee and observed that the
antimicrobial activity of coffee against several
microorganisms is due to presence of caffeine which
inhibits the bacterial growth [23]. Murthy and
Manonmani [24] observed antimicrobial activity against
the growth of E.coli, S. enterica and S. aureus, using the
disc diffusion method. Almeida et al. [25] observed that
the high amount of phenolics in coffee also exert
antimicrobial activity by altering the structure of the
cytoplasmatic membrane, disrupting the proton motive
force and electron flow.
Table 4: Antibacterial activity of the aqueous, chloroform and methanolic extracts of Coffea robusta
Bacterial strains
Diameter of zone of inhibition in (mm)
Concentration Aqueous
Methanol
Chloroform Standard
mg/ml
extract
extract
extract
(Chloramphenicol)
Bacillus subtilis
20.0
39.0
Micrococcus luteus
20.0
35.0
Alcaligenes
20.0
22.5
27.5
35.0
denitrificans
Camphylobacter jejni
20.0
10.2
19.0
39.0
Klebsiella pneumonia 20.0
31.0
Antifungal activity
The results of antifungal activity of methanolic, aqueous
extracts at different concentrations of Coffea robusta
against pathogenic fungus Bipolaris specifera and
Fusarium oxysporum was studied using poisoned food
technique and the percentage inhibition is given in
table 5. It was observed that as the extract
concentration was increased to 0.9ml, the colony
diameter decreased, and the percentage inhibition
increased in all the three extracts. However maximum
inhibition was obtained in aqueous extracts (Table 5).
Table 5: Antifungal activity of Coffea robusta against different fungal strains at different concentrations
S. No extract concentration
Bipolaris specifera
Fusarium oxysporoum
(ml)
Aqueous extract
Growth diameter
%age inhibition
Growth diameter
%age inhibition
(cms)
(cms)
1.
Control
3.5
0
3.0
0
2.
0.3
2.5
28.5
2.2
26.7
3.
0.6
2.0
42.8
1.9
36.6
4.
0.9
1.3
62.9
1.6
46.6
Methanol extract
1.
Control
3.5
0
3.0
0
2.
0.3
2.8
20.0
2.4
20.2
3.
0.6
2.5
28.5
2.1
30.0
4.
0.9
1.9
45.7
1.7
40.1
Chloroform extract
1.
Control
3.5
0
3.0
0
2.
0.3
2.2
16.1
2.1
18.6
3.
0.6
1.5
23.8
1.4
22.5
4.
0.9
1.1
35.1
0.8
33.1
CONCLUSIONS AND FUTURE PROSPECTS
The presence of high amounts of phenolic and
flavonoids in Coffea robusta revealed that it could be
International Journal of Pharmacy and Biological Sciences
used as a potential source of natural antioxidants. Since
the present study involves crude plant extract to study
the partial presence of active compounds in the total
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extract, further investigations are still needed for the
presence of bioactive molecules and their structural
elucidation.
Acknowledgement
The authors are thankful to School of Biotechnology,
University of Jammu, Jammu for providing basic
facilities to carry out this present research work. The
authors are also thankful to Ms Shivani Koul for helping
in phytochemical assays.
11.
12.
13.
14.
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*Corresponding Author:
Ritu Mahajan*
Email: ritufeb@gmail.com
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