Scholars Academic Journal of Biosciences (SAJB) ISSN 2321-6883 (Online) ISSN 2347-9515 (Print)

Scholars Academic Journal of Biosciences (SAJB) ISSN 2321-6883 (Online) ISSN 2347-9515 (Print)
Scholars Academic Journal of Biosciences (SAJB)
Sch. Acad. J. Biosci., 2015; 3(4):377-385
ISSN 2321-6883 (Online)
ISSN 2347-9515 (Print)
©Scholars Academic and Scientific Publisher
(An International Publisher for Academic and Scientific Resources)
www.saspublisher.com
Research Article
Production and Antiproliferative activity of Various Crude Extract from
Lentinus squarrosulus mycelium
Rahayu Ahmad, Siti Mariatul Usyida Alias, Aidil Abdul Hamid, Wan Mohtar Wan Yusoff, Endom Ismail,
Fauzi Daud*
School of Bioscience and Biotechnology, Faculty Science and Technology, National University of Malaysia,
43600 Bangi, Selangor, Malaysia.
*Corresponding author
Fauzi Daud
Email: fauzid@ukm.my
Abstract: Production of extracellular and intracellular polysaccharides (EPS, IPS) from Lentinus squarrosulus mycelium
was carried out through submerged fermentation. The one-factor-at-a-time method was adopted to investigate the effect
of medium components (carbon, nitrogen), and environmental factor (initial pH) on dry weight of mycelium and EPS
concentration. Sucrose and yeast extract gave the highest EPS concentration with 3.56 mg/mL and 1.23 mg/mL
respectively when carbon and nitrogen sources were employed. Combination of ammonium chloride and yeast extract
(30:70) ratio increased the mycelium dry weight up to 10.8 g/L and EPS concentration of 4.39 mg/mL. An initial pH of
7.5 showed maximum EPS concentration of 3.82 mg/mL with mycelium dry weight of 7 g/L. Crude extracts from hot
water and cold water extraction of Lentinus squarrosulus mycelium (IPS HWE, IPS CWE) and water extract from
supernatant (EPS WE) was proved to have antiproliferative activity against A549 lung carcinoma cell lines (ATCC) as
compared to ethanol extract.
Keywords: polysaccharides, Lentinus squarrosulus, mycelium, antiproliferative activity
INTRODUCTION
The global awareness of cancer as the second
largest cause of death in people of various ages and
background has led to so much research effort and
clinical studies in the fight against the disease [1].
Cancer is defined as a disease in which a group of
abnormal cells grow uncontrollably by disregarding the
normal rules of cell division[2]. There are several
treatments for cancer which includes surgery,
immunotherapy, radiotherapy and chemotherapy. Most
of these techniques are useful in particular situations,
but the combination of them offer a more efficient
treatment for cancer. Most cytotoxic drugs used in
cancer chemotherapy are also highly toxic to a wide
spectrum of normal tissues, such as those found in
gastrointestinal tract, bone marrow, heart, lungs, kidney
and brain. Iatrogenic failure of these organs is a
frequent cause of death from cancer[3]. The antitumor
activities of mushrooms polysaccharides have drawn
the most attention in recent years. The search for new
antitumor and other medicinal substances from the
higher Basidiomycetes and the study of medicinal value
of these edible mushrooms have become matters of
great interest.
Mushrooms are nutritionally functional food and a
source of physiologically beneficial and non-toxic
medicines[4]. A wide variety of mushrooms have been
used traditionally for the maintenance of health and for
prevention and treatment of diseases such as cancer,
inflammation, viral diseases, hypercholesterolemia,
blood platelet aggregation and hypertension[4-9]. The
use of medicinal mushrooms in anticancer activity has
been reported in China, Korea, Japan, Russia, United
States and Canada and it was effective against cancers
of the stomach, oesophagus, prostate and lung, belong
to the family of Polyporaceae[10]. Some species of
edible higher Basidiomycetes have been reported to
exhibit antitumor activity and approximately 200
species of higher Basidiomycetes have been reported to
exhibit antitumor activity[11-17].
Chihara and coworkers[18-20] were first to isolate
a water-soluble anti-tumor polysaccharide from the fruit
bodies of Lentinusedodes, which was name “Lentinan”
[β(1-3), β(1-6) glucan] after the generic name of this
mushroom. The molecular formula of Lentinan is
(C6H10O5)n, the mean molecular weight is about 1 x 105
– 5 x 105 Da, [α]D + 20°-22° (NaOH). It was confirmed
to be a β-D-glucan, as shown by electrophoresis and
ultracentrifugation, as well as by various techniques and
instrumental analysis[21]. The investigation of
antitumor properties of L. edodes proved that lentinan
was found to almost completely regressed the solid type
of tumors in synergic host-tumor system A. The
antitumor effect of Lentinan was originally confirmed
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Rahayu Ahmad et al., Sch. Acad. J. Biosci., 2015; 3(4):377-385
by using Sarcoma 180 transplanted in CD-1/ICS mice
[18]. Antitumor activity of lentinan was found to be
significantly higher than that of polysaccharides
isolated from many other fungi and higher vascular
plants[22].
was suspended in 1 mL distilled water and was assayed
by phenol-sulfuric acid method [25] using D-glucose as
a standard. The dry weight of mycelium was measured
after repeated washing of the mycelial pellet with
distilled water and drying at 70C to a constant weight.
To the best of our knowledge, there is no study
reported of the antiproliferative activity of
polysaccharides from Lentinus squarrosulus against
lung cancer cell, A549 (ATCC). Thus in this study,
various crude extracts from different extraction method
from mycelium and culture broth of Lentinus
squarrosulus was used to investigate their
antiproliferative activity against lung carcinoma cell,
A549 (ATCC cell line).
Production of polysaccharides
Submerged liquid fermentation was carried out
for production of mycelium using optimized culture
conditions (sucrose concentration 114.61 g/L, yeast
ectract 1.62 g/L, Mg.SO4.7H2O 0.5 g/L, KH2PO4 1 g/L
and initial pH 5.81) as described by[26]. A 5 mm plug
of Lentinus squarrosulus mycelia was removed with a
cork borer from 7 day-old-culture. The mycelia cut (10
plugs) was transferred into liquid medium, incubated in
orbital shaker for 7 days at 28°C, 150 rpm for mycelia
growth. Cultured mycelia were then dried using oven
until constant weight. Dried mycelia were used for
intracellular polysaccharide (IPS) extraction using
ethanol, cold water and hot water method. Culture
filtrates were used for extracellular polysaccharide
(EPS) extraction using ethanol and water extraction
method. EPS water extract were prepared by directly
subjecting the culture filtrate to freeze-dryer yielding
the exopolysaccharide water extract (EPS WE).
MATERIALS AND METHOD
Optimization of fermentation medium using onefactor-at-a-time method
Mushroom mycelium of Lentinus squarrosulus
were maintained on potato dextrose agar (PDA), 30 g/L
added with 2% yeast extract, stored at 4°C for a long
term use. The mycelium from stock culture was placed
in the centre on PDA plate and incubated for 7 days
prior to submerged liquid fermentation. Glucose yeast
extract (GYE) medium consists of (per liter) glucose 30
g, yeast extract 3 g, Mg.SO4.7H2O 0.5 g, KH2PO4 1 g
with initial pH of 5.5 [23] was used in this study. 10
plugs of Lentinus squarrosulus mycelium were
inoculated in each flask and fermentation was carried
out at 28C, 150 rpm for 7 days. The effect of different
carbon sources, nitrogen sources, manipulation of their
concentrations and various initial pH were investigated.
For carbon sources, glucose was substituted with
different carbon sources such as sucrose, fructose,
maltose, lactose, arabinose, sucrose and xylose. Initially
all carbon sources were screened at 30 g/L and after that
carbon source concentration were varied from 25 to 200
g/L. The effect of nitrogen sources was carried out by
replaced yeast extract with other organic nitrogen
sources such as peptone, beef extract, and inorganic
nitrogen sources like ammonium chloride, ammonium
nitrate, ammonium sulphate, sodium nitrate and urea.
Keeping total nitrogen source at 3 g/L, the ratio of
organic to inorganic nitrogen source was varied from
0:100 to 100:0. The effect of initial pH on growth of
mycelium and EPS production was carried out with
different pH value. pH values were varied from 4.5 to
8.0.
Analytical Methods
Samples collected were centrifuged at 10 000
rpm for 20 min, and the resulting supernatant (1 mL)
were subjected to the phenol-sulfuric acid assay. The
precipitation of EPS was conducted as followed the
methodology of [24]. The supernatant was mixed with 4
times (4:1 v/v) the volume of absolute ethanol, stirred
vigorously and kept overnight at 4C. The precipitated
extracts were centrifuged at 10 000 rpm for 20 min
discarding the supernatants. The insoluble component
Extraction of Polysaccharides
Cold Water Extraction
Approximately, 5 g of mycelia powder were
mixed with 500 mL of distilled water and stirred
vigorously for 3 h at room temperature. The extracts
were filtered using Whatman No 1 and the culture
filtrates
were
freeze
dry
for
intracellular
polysaccharides cold water extracts (IPS CWE).
Exopolysaccharide cold water extract were prepared by
directly subjected the culture filtrate (after separated
from mycelium) to freeze-dryer yielding EPS WE.
Hot Water Extraction
Intracellular polysaccharide hot water extracts
(IPS HWE) were extracted using the method as
described by [27]. Briefly, 40 g of dried mycelia
powder was extracted twice with distilled water (600
mL) at 100°C for 3 h in water bath. The extracts were
cooled, filtered and the filtrates were dried using freeze
dryer.
Ethanol Extraction
Ethanol extraction was conducted according to
the method used by[24]. Culture filtrates were mixed
with 4 times of absolute ethanol, stirred vigorously and
kept overnight at 4°C. Ethanol was removed using
rotary evaporator and crude extracts were dried using
freeze dryer yielding the extracellular polysaccharide
ethanol extracts (EPS EE). For intracellular
polysaccharide ethanol extracts (IPS EE), a modified
hot water extraction as described by[27] was used.
Briefly, the powders of dried mycelia (40 g) were
extracted twice with distilled water (600 mL) at 100°C
for 3 h in a water bath. The extracts were cooled,
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Rahayu Ahmad et al., Sch. Acad. J. Biosci., 2015; 3(4):377-385
filtered using filter paper (Whatmann No 1) and
precipitated using ethanol before dry using freeze dryer.
RESULT AND DISCUSSION
Optimization Using One-Factor-at-a-time
In this study, we demonstrated the effect of
medium components on the mycelia growth and
production of EPS through submerged fermentation.
The medium was supplemented with different carbon
sources such as glucose, sucrose, fructose, lactose,
maltose and xylose at a concentration of 30 g/L. Figure
1 shows the effect of different carbon sources on
mycelial growth and EPS production. Among the
carbon sources tested, glucose supported the mycelial
growth with the highest dry weight of 7.92 g/L followed
by maltose and fructose with 6.01 and 5.87 g/L
respectively. However glucose exhibited the lowest
EPS with 1.32 mg/ml. The production of EPS was high
when sucrose was used as carbon source with 3.56
mg/mL and 3.47 mg/mL when fructose was supplied in
the medium. Xylose showed lowest dry weight of
mycelium with 3.90 g/L. The lowest concentration of
EPS from glucose is contrast to some of previous
research, which reported that glucose supported the
mycelia growth as well as the production of EPS by
Paecilomyces tenuipes C240 [28]. The highest EPS
production obtained when sucrose was used as carbon
sources and this result was similar to[29] which
demonstrated
that
sucrose
showed
highest
schizophyllan
production
(1.62
g/L)
from
Schyzophyllum commune NRCM after 168 h of
fermentation. Previous research[30] also reported that
sucrose was the most suitable carbon source for both
cell growth (biomass concentration) and polysaccharide
production. During the microbial fermentations, carbon
source play a major role in building of cellular material
and also used in the synthesis of polysaccharide as
energy source[31-32].
Mycelium dry weight (g/L);
EPS (mg/mL)
In Vitro Cytotoxicity Assay
The A549 lung carcinoma cell lines and Chang
cell from ATCC were used for the cytotoxicity test. The
cells, A549 were grown in RPMI medium
supplemented with 20% fetal bovine serum (FBS) and
1% Penicillin-streptomycin. All cultures were
maintained at 37°C in humidified atmosphere of 5%
CO2. Cytotoxicity assay of five different crude extracts
particularly from mycelium (IPS CWE, IPS HWE, IPS
EE) and culture broth (EPS EE, EPS WE) of Lentinus
squarrosulus on A549 cell lines were evaluated in vitro
using the colorimetric 3-(4,5-dimethylthiazol-2-yl-2,5diphenyl tetrazolium bromide, MTT assay. Briefly, cell
suspension in culture medium were seeded in 96-well
plates with the concentration of 1 x 105 cells/well, and
incubated at 37°C in humidified atmosphere of 5%
CO2. After 24 h, cells were treated with different
concentration of crude extracts followed by incubation
until 72 h. The 100 µL of MTT (5 mg/mL) was added
to each well and incubated for 4 h. Finally the media
were removed and DMSO was added to the wells (150
µL/well) and absorbance was measured at 570 nm in a
micro plate reader. Tamoxifen drug (0.2 mg/mL) was
used as positive control. Tamoxifen was dissolved in
0.1 ml DMSO and added with 10 mL of complete
medium before diluted in different concentration range
from 10 to 100 ug/mL. Each measurement was
performed in triplicate. The half-maximal inhibitory
concentration (IC50) value was determined from the
percentage of the cell viability versus final
concentration of the extract curve.
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Mycelium dry weight (g/L)
Glucose Fructose Sucrose
Exopolysaccharides (mg/mL)
Lactose Arabinose Maltose
Xylose
Carbon sources
Fig-1: Effect of carbon sources on the mycelium dry weight and EPS concentration
In order to determine the suitable concentration of
carbon source for mycelia growth and EPS production,
sucrose concentration were varied from 25 to 200 g/L.
It was observed that increasing concentrations of
sucrose resulted in the increasing of mycelial growth up
to 33 g/L. The EPS production was increased from 1.36
mg/mL to 4.69 mg/mL when the concentration of
sucrose increased from 25 to 100 g/L. After that, the
EPS concentrations were decreased to 3.63 g/L when
the concentrations was further increased up to 200 g/L
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Rahayu Ahmad et al., Sch. Acad. J. Biosci., 2015; 3(4):377-385
as shown in Table 1. The decreased of EPS
concentration after further increased of the sucrose
concentration (100-200 g/L) might be due to the
substrate inhibition occurred when excess carbon source
was supplied to the culture medium. The result was
similar to the one reported by[33]. It was described that
the conversion rates of sucrose at high concentration
were found to be low and high carbon source (sucrose)
might have increased the osmotic pressure of the
medium and thus influenced exopolysaccharide
production.
Table- 1: Effect of different sucrose concentrations on the mycelium dry weight and EPS concentration.
Sucrose concentrations
Mycelium dry weight
Final pH
Exopolysaccharides
(g/L)
(g/L)
(mg/mL)
25
4.21 ± 1.71
4.9 ± 0.4
1.36 ± 0.16
30
4.45 ± 0.72
4.7 ± 0.1
3.52 ± 0.20
40
8.86 ± 0.68
4.8 ± 0.6
3.68 ± 1.11
60
13.47 ± 4.20
4.4 ± 0.3
3.75 ± 0.70
80
17.74 ± 4.27
4.3 ± 0.3
4.31 ± 0.43
100
20.41 ± 2.29
4.3 ± 0.3
4.69 ± 0.10
150
32.58 ± 3.61
4.4 ± 0.1
3.65 ± 0.03
200
33.0 ± 2.24
4.5 ± 0.4
3.63 ± 0.41
Fermentation was carried out for 7 days at 28C with initial pH 5.5.
Values are mean  S.D. of triplicate.
Mycelium dry weight (g/L); pH
Various inorganic and organic nitrogen
sources were tested out to enhance the mycelial growth
and EPS production. It was found that when organic
nitrogen sources were used, mycelial growth and EPS
production were high compared to inorganic nitrogen
sources. Figure 2 shows the effect of different organic
and inorganic nitrogen sources on EPS production.
Amongst eight kinds of nitrogen sources, yeast extract
as organic and ammonium chloride as inorganic
nitrogen source were favourable for the mycelial
growth and EPS production. Yeast extract gave highest
mycelium dry weight of 8.06 g/L with 1.23 mg/mL of
EPS whereas ammonium chloride showed 3.58 g/L
mycelium dry weight and 0.63 mg/L EPS. This suggests
that yeast extract and ammonium chloride might
contain the components necessary for mycelial growth
and EPS production. Comparing the influence of
different ammonium salts, the 𝑁𝐻4+ ion played a central
role in nitrogen metabolism as the form in which
nitrogen incorporated into organic cell components
(biomass)[34]. Amongst all inorganic nitrogen sources
tested, ammonium nitrate gave highest mycelium
biomass and EPS concentration. It was reported
previously by[34] that solely supplied of inorganic
nitrogen sources enhanced poor growth of mycelia.
10
9
8
7
6
5
4
3
2
1
0
Yeast
extract
Peptone
Beef
extract
Amm.
nitrate
Amm.
Amm.
sulphate chloride
Urea
Nitrogen sources
Mycelium dry weight (g/L)
Sodium
nitrate
Exopolysaccharides
Fig-2: Effect of different nitrogen sources on the mycelium dry weight and EPS concentration.
Combination of yeast extract and ammonium
nitrate was carried out in order to enhance the mycelia
growth and EPS production. The best combination
obtained was 30:70 (inorganic to organic nitrogen
source), which resulted in the increased of mycelium
dry weight up to 10.8 g/L and EPS concentration of
4.39 mg/mL as showed in Figure 3. Interestingly, result
obtained was contrast to the one reported by[29] where
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Rahayu Ahmad et al., Sch. Acad. J. Biosci., 2015; 3(4):377-385
combinations of yeast extract and ammonium nitrate at
various ratios have decreased schizophyllan and
mycelium dry weight. Taking into account that higher
fungi usually require long cultivation period for
successful submerged culture, introducing inorganic
nitrogen source to the medium might possibly reduce
the risk of culture contamination[28]. The influence of
initial pH on mycelial growth and EPS production was
examined in the range of 4.5 to 8.0. The optimal pH for
mycelial growth and EPS production was 7.5 as shown
in Figure 4. Initial pH of 7.5 supported the mycelial
growth of 7.0 g/L and maximum production of 3.82
mg/mL EPS. It was reported that a pH of 6.0 to be
optimum for schizophyllan production with 1.65
g/L[29] whereas [23] reported that pH 5.3 was optimum
for schizophyllan production. The end of pH values was
found in the range of 4.4 to 5.09 although different pH
value was applied in the initial of experiment. It has
been reported that many kinds of ascomycetes and
basidiomycetes have more acidic pH during submerged
culture[35]. It was also reported on previous study that
the effect of carbon, nitrogen, C:N ratio and initial pH
are amongst crucial factor on the production of
polysaccharide and lectin production in both mycelium
and culture medium[36] through submerged
fermentation. Submerged fermentation of mushroom
mycelium culture clearly provides a consistent mycelia
formed composition under controlled conditions with a
low risk of contamination[37-38].
Mycelium dry weight (g/L);
EPS (mg/mL)
Mycelium dry weight (g/L)
Exopolysaccharides (mg/mL)
12.0
10.0
8.0
6.0
4.0
2.0
0.0
0:100 10:90 20:80 30:70 40:60 50:50 60:40 70:30 80:20 90:10 100:0
Nitrogen ratio
Mycelium dry weight (g/L); pH
Fig-3: Effect of inorganic (ammonium chloride) to organic nitrogen sources (yeast extract) on mycelium dry
weight and EPS concentration.
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
4.5
5
5.5
6
6.5
Initial pH
Mycelium dry weight (g/L)
7
7.5
8
Exopolysaccharides
Fig-4: Effect of different initial pH value on the mycelium dry weight and EPS concentration.
Cytotoxicity assay
According to our previous study conducted
by[39], various crude extract from mycelium and
culture broth were determined their carbohydrate
concentration, total glucan, α-glucan and β-glucan
content. It was reported that IPS HWE and IPS CWE
were high in carbohydrate concentration, and this
amount was correlated with the high content of βglucan in these crude extract. In the present study,
various crude extract namely EPS EE, EPS WE, IPS
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Rahayu Ahmad et al., Sch. Acad. J. Biosci., 2015; 3(4):377-385
CWE, IPS HWE and IPS EE were further tested their
cytotoxicity effect towards lung carcinoma cell lines
(A549) and Chang cells as the normal cell lines. The
effect of different concentrations for 24, 48 and 72
hours incubation on the cell viability of lung carcinoma
cell lines (A549) were showed in Figure 5a,b and c
respectively.
Our results exhibited that out of five crude
extracts; only three crude extracts from water extraction
namely IPS HWE, IPS CWE and EPS WE showed
antiproliferative activity against lung carcinoma cell
lines (A549) as compared to ethanol extraction. For 24
hours incubation time, only IPS HWE inhibits the
growth of A549 lung carcinoma cells with 43% of cell
viability and IC50of 56 µg/mL. As the incubation time
prolonged to 48 hours, crude water extract from
mycelium (IPS HWE, IPS CWE) and from culture
broth (EPS WE) both were significantly decreased the
cell viability. IPS HWE showed the lowest IC50 with 26
µg/mL followed by IPS CWE and EPS WE with 43.5
µg/mL and 65 µg/mL respectively. The IC50 of all these
three crude extracts were further decreased as the
incubation time increased to 72 hours with 20.8 µg/mL
for IPS HWE, IPS CWE (26.3 µg/mL) and EPS WE (55
µg/mL). It is suggested that the antiproliferative activity
of IPS HWE, IPS CWE and EPS WE were time
dependent because of the significant decreased of IC50
as the incubation time increased to 72 hours and for IPS
CWE and EPS WE, the cell viability only starts to
decrease at 48 hours of incubation. It is also were
pronounced as a dosage-dependent due to the decreased
of cell viability with the increased of crude extract
concentration. In this study, we found out that crude
extract from hot water extraction (IPS HWE) exhibited
higher antiproliferative activities as compared to the
other crude extract. This might be due to the mushroom
extracted with hot water usually consists a
polysaccharide-protein complex which has been proved
to possess a strong antitumor effect[40]. It was also
suggested that the higher temperature applied in the hot
water extraction process managed to solubilize large
amount of cellular structural/storage proteins[41].
Previous studies also reported that hot water extract and
cold water extract from Lignosus rhinoceros exhibited
significant antipoliferative activity against different
kinds of leukemic cells, breast cancer cell (MCF-7) and
lung cancer cell (A549) respectively[41-42]. Our result
also showed that IPS HWE, IPS CWE and EPS WE
were essentially not cytotoxic against normal cell lines
(Chang). Tamoxifen drug (positive control) range from
10 to 100 ug/ml clearly inhibited the growth of normal
cell line. In contrast our water extract (IPS CWE and
IPS HWE) could support the growth of normal cell
lines (Chang) above 60% of cell viability. As far as it is
concern, this is the first report to demonstrate that
Lentinus squarrosulus water extract is not toxic to
normal cells and suggested as potentially bioactive
compound in anticancer treatment.
Cell viability (%)
a) 120
100
80
IPS CWE
60
IPS EE
40
EPS WE
20
IPS HWE
0
EPS EE
0
20
40
60
80
100
Concentration of crude extracts (ug/mL)
Cell viability (%)
b) 120
100
80
IPS CWE
60
IPS EE
40
EPS WE
20
IPS HWE
0
EPS EE
0
20
40
60
80
100
Concentration of crude extracts (ug/mL)
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Rahayu Ahmad et al., Sch. Acad. J. Biosci., 2015; 3(4):377-385
c) 120.0
Cell viability (%)
100.0
80.0
EPS EE
60.0
EPS WE
40.0
IPS HWE
20.0
IPS CWE
IPS EE
0.0
0
20
40
60
80
100
Concentration of crude extracts (ug/mL)
Fig- 5: Antiproliferative activities of various crude extract of Lentinus squarrosulus mycelial and culture broth.
Lung carcinoma cells, A549 (ATCC) were treated with different concentrations of crude extract ranging from 10
µg/mL to 100 µg/mL at 24 h (A), 48 h (B) and 72 h (C). Values are mean  S.D. of triplicate.
120
Cell viability (%)
100
EPS WE
80
EPS EE
60
IPS EE
IPS CWE
40
IPS HWE
Tamoxifen
20
0
0
10
20
30
40
50
60
70
80
90 100
Concentrations (ug/mL)
Fig-6: Antiproliferative activities of various crude extract of Lentinus squarrosulus mycelial and culture broth.
Normal cells (Chang), were treated with different concentrations of crude extract ranging from 10 µg/mL to 100
µg/mL at 72 h. Values are mean  S.D. of triplicate.
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Rahayu Ahmad et al., Sch. Acad. J. Biosci., 2015; 3(4):377-385
120
Cell Viability (%)
100
80
60
T-24H
T-48H
40
T-72H
20
0
-20
0
10
20
30
40
50
60
70
80
90 100
Concentration (ug/mL)
Fig-7: Antiproliferative activities of various crude extract of Lentinus squarrosulus mycelial and culture broth.
Lung cancer cells (A549) were treated with different concentrations of Tamoxifen ranging from 10 µg/mL to 100
µg/mL. Values are mean S.D. of triplicate.
CONCLUSION
Mycelium dry weight and EPS concentration
had increased when sucrose and yeast extract were
utilized as carbon and nitrogen sources respectively.
Combination of ammonium chloride and yeast extract
enhanced the mycelium dry weight up to 10.8 g/L with
4.4 mg/mL of EPS concentration. Crude water extract
from mycelium of Lentinus squarrosulus possess a
strong antiproliferative activity against A549 lung
carcinoma cell lines.
ACKNOWLEDGEMENT
This work was performed with financial
support from eScience fund grant (MOSTI), Project No:
02-01-02-SF09784
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