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Gyanoderma
lucidum is an oriental
fungus commonly known as lingzhi or reishi mushroom in parts of China and
Nepal. The fungi is well known for its numerous nutritional and
ethnopharmacological properties. In the present study, the hexane, methanol and
chloroform crude extracts of local Nepalese G.
lucidum were evaluated for their phytochemical content and biological
activities. The anti-oxidant activities of crude fungal extracts were studied
using the standard DPPH radical scavenging assay. For anti-microbial tests,
clinically important bacteria such as Pesudomonas
aeruginosa, Staphylococcus aureus
and Salmonella typhimurium were
selected and anti-bacterial screening
was conducted using theagar
well diffusion technique. Anti-cancer effect of medicinal fungi was
studied by evaluating its cytotoxic effects on HeLa cells. Murine (BALB/6)
peritoneal macrophages were utilized as control cells. Anti-inflammatory
activity was studied by evaluating the inhibitory effect of the fungal crude
extracts on nitric oxide (NO) production by heat-killed S. typhimurium (HKST)-stimulated peritoneal macrophages.
Phytochemical screening indicated that chloroform and methanol crude extracts
were rich in active phytoconstituents, namely, flavonoids, glycosides and
alkaloids. Anti-oxidant tests indicated that the chloroform and methanol
extracts of G.lucidum exhibited significant (P<0.05) levels of
anti-oxidant activity with the
concentration bringing about 50% inhibition (IC50) in the range of
1.62-2.21 mg/ml. Likewise, the anti-microbial assay showed that chloroform and
methanol crude extracts of G. lucidum
were most effective against P. aeruginosa
and S. aureus. The methanol extract
of G. lucidum exhibited the highest
selective cytotoxicity (P<0.05) against HeLa cells with an IC50
value of 125±7.27 µM. None of the extracts exhibited significant cytotoxic
effect on the cell viability of peritoneal macrophages at given concentration
(0-300 μg/ml). Pre-treatment of G.
lucidum methanol extract caused significant (P<0.05) inhibition
of NO production in HKST-stimulated peritoneal macrophages. Taken together, the
results suggest that G. lucidum has a wide-spectrum therapeutic
potential which requires further in-depth investigation.
Keywords: Cancer, Gyanoderma,
Immunology, Inflammation
INTRODUCTION
Indigenous medicinal plants and fungi have been
traditionally used for their anti-analgesic and anti-inflammatory properties to
cure multiple diseases and disorders including fever, headache, edema,
parasitic infestation, skin allergy and diarrhea [1,2]. Ganoderma lucidum is
the species name for edible mushroom called “Rato Chau” in Nepalese, “Lingzhi”
in Chinese, “Reishi” in Japanese, “Yeongji” in Korean, “Glossy ganoderma” or
“shiny polyporus” in English. The fungus has been reported to grow on logs of
dead woods and tree stumps in shady places [3]. In Chinese folklore, the
fruiting bodies of G. lucidumare perceived as a panacea for all types of
illnesses including chronic hepatitis, arthritis, hypertension, hyperlipidemia,
neoplasia, gastric ulcer, diabetes and atherosclerosis [4,5]. Due to the
numerous medicinal properties attributed to G. lucidum, it is regarded as a
‘mushroom of immortality’ in China, Japan and Korea [4]. In these regions the
fungus is cultivated and utilized as a useful source of feed supplement in
animals and as food-based medicine for promoting health and immune functions in
humans [6,7]. Recent studies have revealed increasing trend in fungus-derived
drug development. Thus, G. lucidum being a highly significant mushroom with a
rich history of ethno traditional uses makes it a luring target for
researchers. Previous studies have indicated that G. lucidum has antioxidant
properties attributed to its ability to inhibit H2O2-induced
cell death [8]. Similarly, the anti-microbial properties of the fungal extract
against pathogenic strains of Escherichia coli, Staphylococcus aureus, Bacillus
subtilis, Salmonella typhi, and Pseudomonas. aeruginosa have been reported [9].
In addition, the fungus has also shown its promising therapeutic potentials as
an effective anticancer and anti-inflammatory agent. Studies have shown that
certain triterpenes from G. lucidum can induce apoptosis in human cancer cell
lines via mitochondria-dependent pathway [5].A Chinese herbal medicine
preparation containing G. lucidum as the main ingredient has been reported to
reduce the expression of interleukin (IL)-8, vascular endothelial growth factor
and platelet-derived growth factor thus indicating potent anti-inflammatory and
antitumor activities [10]. Nonetheless, the phytochemical constituents,
climatic variations and genetic diversity within floral species can have
substantial effect on the overall quality and efficiency of the fungal extract.
The hilly regions of Nepal are known for their rich biodiversity and contain a
large variety of Gyanoderma species [11]. Thus, in our present study, we
investigated the local species of G. lucidum for its phytochemical constituents
and biological activities with the main focus on antioxidant, antimicrobial,
anticancer and antiinflammation properties.
MATERIALS AND METHODS
Plant material
The G. lucidum under investigation was identified
and collected from the hilly region of Nepal with the help of a professional
botanist. Fruiting body of the mushroom was selected for further
experimentation. In brief, the fungal materials were air dried in the shade.
The dried materials were crushed and ground into fine powder. The resulting powder
was filtered through a sieve filter and subjected to extraction by three
different solvents, namely, hexane, chloroform, and methanol using percolation
with intermittent sonication. After extraction, the resulting filtrate
containing phytochemicals were concentrated under reduced pressure using a
rotatory vacuum evaporator. Finally, the condensed crude extract was
transferred to clean, dry glass vials and allowed to dry at room temperature.
The tentative yield percentage of extract fraction was calculated by dividing
the weight of final extract fraction by the weight of starting fungal materials
used.
Cancer cell Line
The human cervical cancer cell line (HeLa ATCC
CCL-2) was used for evaluation of anti-cancer effect of plant extracts. These
cells were obtained from Everest Biotech™, Khumaltar, Lalitpur, Nepal. The
cells were cultured and maintained using RPMI complete medium containing 90%
RPMI, 10% heat-inactivated fetal bovine serum (FBS), 12.5
mM HEPES (pH 7.3) and 1× working concentration of Antibiotic-Antimycotic
commercially supplied as 100× stock containing 10,000 U/ml of penicillin,
10,000 µg/mL of streptomycin, and 25 µg/mL of amphotericin B.. All reagents
used for cell culture were purchased from Gibco®(Thermofisher, USA).
The cell culture condition was maintained at 37οC, 5% CO2
using an animal cell incubator (Sheldon Labs™, USA).
Mice and
generation of peritoneal macrophages
BALB/C mice were obtained from Natural Development
Center, Thapathali, Kathmandu. Mice were maintained under specific pathogen-free
conditions. All animal procedures were humanely performed according to the
animal safety and ethics guidelines of the Institutional Animal Care and Use
Committee. Peritoneal macrophages were obtained as previously described with
minor modifications [12,13]. Specific-pathogen-free, typically 6 to 8 weeks old
BALB/c mice were selected and injected intraperitoneally with 3 ml of 3% starch
solution in PBS. On day-3, mice were euthanized using carbon dioxide. Resident
peritoneal cells were obtained by washing the peritoneal cavity with sterile
warm PBS (37°C). The peritoneal lavages were collected in a chilled sterile
test tube since macrophages can adhere to the glass at room temperature.
Macrophages were washed once with cold PBS and re-suspended in cold RPMI 1640.
Cell viability was determined with the Trypan blue (Sigma-Aldrich, USA)
exclusion method [14]. The number of viable macrophages in this suspension was
determined using a haemocytometer chamber. The macrophages were adjusted to 4 ×
105 cells/ml in complete RPMI medium containing 90% RPMI, 10%
heat-inactivated fetal bovine serum (FBS) 12.5
mM HEPES (pH 7.3) and 1× working concentration of Gibco® Antibiotic-Antimycotic
(Thermofisher, USA) commercially supplied as 100× stock containing 10,000 U/ml
of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of amphotericin B.
Bacteria and generation of heat-killed Salmonella
typhimurium
Standard strains of gram negative bacteria (S.
typhimurium ATCC 14028, and P. aeruginosa ATCC 27853) and a single standard
strain of gram positive bacterium (Staphylococcus aureus ATCC 25923) were used
for anti-bacterial assay. These strains of bacteria were obtained from
Institute of Medicine, Maharajgunj, Nepal and National Public Health
Laboratory, Kathmandu, Nepal. Heat-killed S. typhimurium (HKST) were generated
and used as previously described [15]. Briefly, S. typhimurium was cultured in
Luria-Bertani (LB) broth and then heat-killed by incubating at 75ο C
for 30 mins. The resulting heat-killed status of bacteria was confirmed by
culturing the inoculum in LB agar. To stimulate nitric oxide (NO) production by
peritoneal macrophages the HKST with multiplicity of infection (MOI) 5 was used
as an adequate source of lipopolysaccharide (LPS).
Phytochemical screening
The phytochemical screening was conducted as
previously described methods with minor modifications [16-20]. The hexane,
chloroform and methanol extracts were screened for the presence of alkaloids,
carbohydrates, resins, phenols, flavinoids, proteins, lipids, glycosides,
saponin, diterpenes, tannins and phytosterols.
Assay of inhibition of DPPH free radical scavenging
activity
Antioxidant activities of the extracts were
measured using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging
activity as previously described with minor modifications [21]. The stable form
of DPPH radical was utilized to evaluate the antioxidant potential of the G.
lucidum crude hexane, chloroform, and methanol extracts. The samples were
tested at various concentrations (0-10 mg/ml). In brief, 50 µl of the crude
extract was added to 450µl of Tris-HCl buffer (0.05 M, pH 7.4) and 1ml of 0.1mM
DPPH was added to the resulting mixture. Solution in the test tubes were then
shaken well and incubated in dark for an additional 30 mins at the ambient
temperature. Absorbance of the solutions in each tube was measured
spectrophotometrically (Thermo Scientific, USA) at 517nm. Gallic acid
(Sigma-Aldrich, USA) was used as the positive control. Blank sample containing
only buffer and DPPH was taken as the negative control for the experiment. The
radical scavenging activity (RSA) was measured in terms of percentage for the
difference in absorbance between the sample and the control divided by the absorbance
of the control.
Screening for anti-bacterial activity
The antimicrobial activity of the crude extracts
was studied by the agar well diffusion method with minor modifications [22].
This is the qualitative method for testing anti-bacterial efficacy. Briefly,
bacteria were carpet cultured on the agar maintaining 0.5 McFarland turbidity
standard. A fixed volume of extract solutions was placedin wells of equal size
bored on the preset agar plates along with the positive and negative controls.
Then the zone of inhibition were marked as a halo zone was measured. After 24 h
incubation, the diameters of clear zones were measured to evaluate
anti-bacterial efficacy of the extract being tested. Chloremphenicol
(50cg/disc) was used as a control antibiotic.
Cytotoxicity test for HeLa cells and peritoneal
macrophages
The cytotoxicity test was conducted as previously
described with minor modification [23]. Briefly, to determine cytotoxicity,
HeLa cells or peritoneal macrophages were washed three times with PBS , re-suspended
in complete RPMI medium, and then seeded on sterile 96-well tissue culture
plate (0.7 × 104 cells/well) and allowed to incubate overnight.
5-flurouracil (5-FU, MW:130.07) was or G. lucidum extracts were dissolved in
DMSO, added at a final concentration of 0-300μg/ml and incubated for additional
48 h at 37οC, 5% CO2. The final concentration of compound
diluent (DMSO) was maintained below 0.1% in the culture medium. Cell viability
was evaluated in each well by the addition of 50μl of
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; 2.5
mg/ml in PBS). After 4 h of incubation, the cell-free supernatants were removed
and 100 μl of dimethyl sulfoxide (DMSO) was added. The optical density of
formazan crystals formed were measured using a multiwall spectrophotometer
(Thermo scientific, USA) reader at wavelength of 540 nm. For visual evidence of
cytotoxicity, images of selected cultured cells were taken using an inverted
microscope (Olympus, USA). The IC50 values for the growth inhibition
were calculated using CalcuSyn v2.11 software (Biosoft, Cambridge, UK).
Measurement of nitric oxide
The measurement of NO was done using a previously
described method with minor modification [24]. Peritoneal macrophages were
seeded on 96 well plates (1.5 x 104 cells / well) and incubated for
2 h. Then the cells were pre-incubated for 1 h with various concentrations of
extract and stimulated with HKST at multiplicity of infection (MOI) 5 for 24 h
in complete RPMI medium. The culture supernatants were utilized for measurement
of NO production. Fifty microliters of the culture supernatant were mixed with
50 μl of the Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine
dihydrochloride and 2.5% phosphoric acid) and the absorbance was measured at
570 nm (Kim et al. 2008). The NO concentrations in medium were determined by
measuring the nitrite levels in the culture media supernatant. Nitrite levels
in the samples were calculated from a standard curve with known concentrations
of sodium nitrite.
STATISTICAL ANALYSIS
Statistical analyses were carried out using
independent t-test or one way ANOVA whenever appropriate. Values were expressed
as Mean ± standard error in mean (SEM). Data with P <0.05 were
considered as statistically significant. Unless specified, all assays were
performed in triplicate and all images are representative of three independent
experiments.
RESULTS AND DISCUSSION
The secondary metabolites of several ethnopharmacologically
important plants and fungi possess medicinal properties [6,25]. These secondary
metabolites comprise a broad range of phoytochemicals such as flavonoids,
alkaloids, polysaccharides, saponins, glycosides, steroids, and tannins which
have medically importance bioactivities [26, 27]. Such bioactive constituents
can exert anti-microbial, anti-inflammatory or suppressive effects and can also
stimulate the immune system to neutralize toxins orpathogens [28]. The
solubility and bioactivity of different metabolites can differ depending on the
nature of the solvent, thus, we investigated the crude extract of G. lucidum
using a variety of organic solvents namely, hexane, chloroform and methanol.
During solvent extraction, we observed a considerable yield from each of the
solvents (Table 1). Phytochemical
screening indicated that proteins, carbohydrates and tannins were detected in
all fractions of crude extract while fats and saponins were undetectable (Table 2). The chloroform and methanol
extracts of G. lucidum showed moderately positive results for the
presence of resins. Remarkably, the methanol and chloroform extracts showed
moderate to high levels of diterpenes, alkanoids, phenolics, flavinoid and
glycosides in different fractions indicating the presence of these biologically
active phytochemicals (Table 2).
The antioxidant property enables neutralization of excess reactive free radicals and thus protects from cellular damage. This assists healthy physiology and leads to a delay of cellular senescence which is commonly termed as anti-ageing effect [29]. Similarly, free radicals can cause DNA damage and eventually lead to cancer. Many types of phenolics, diterpenes, phytosterols and flavonoids are known to possess free radical scavenging activity which makes them a potential target for the development of antioxidant and anticancer drugs [30,31]. DPPH assay is a well-established method for determining the antioxidant activity of phytochemical extracts. Thus, we utilized the percentage inhibition values for DPPH oxidation to calculate the antioxidant effect. The 50% inhibitory concentration (IC50) value for the DPPH standard gallic acid was found to be 0.047 mg/ml. The chloroform extract of G. lucidum showed the highest anti-oxidant effects with the IC50 value of 1.62 ± 0.34 mg/ml (Figure 1). The presence of multiple biologically active phytochemicals namely, alkanoids, phenolics, flavinoid and glycosides in the G. lucidum crude extract may have contributed to the observed antioxidant effect. Phytoconstituents and mainly the secondary metabolites such as flavonoids, alkaloids, tannins, polyphenols, and terpenoids are known to be responsible for the antimicrobial effects of several medicinal flora [32,33]. Certain aromatic phytochemicals including quinines are known for their antimicrobial effect by rendering the substrate unusable for microbial consumption [34]. During our investigation the hexane, chloroform, and methanol extracts of G. lucidum exhibited significant inhibition against gram negative bacteria S. typhimurium (Table 3). Hexane and chloroform extracts of G. lucidum were able to exhibit considerable inhibition toward the gram- positive bacteria S. aureus. Likely, the inhibitory effect of G. lucidum methanol extract on growth of P. aeruginosa was found to be comparable to that of the standard drug chloramphenicol (50 mcg), which indicates a therapeutic potential of the extract.
Screening for the cytotoxicity of the G. lucidum
extract was conducted in the human cervical cancer cell line ‘HeLa’ cells using
the standard MTT assay based on cell metabolic activity. These cancer cells
provide a suitable in vitro model for preliminary screening of potential
anticancer activities. The standard conventional drug for treatment of cervical
cancer (5-FU) was used as the positive control [35].
Representative images from light microscopy provided visual evidence of the
dose-dependent cytotoxic effect exerted by G. lucidum methanol extract
on HeLa cells (Figure 2 a). Similar dose-dependent cytotoxic
effects were observed for other G. lucidum extracts with varying
potencies of cell death induction (data not shown).
The cell viability assay indicated that treatment
with DMSO alone had no significant effect on cell death (Figure 2b). In
contrast, 5-FU treatment exerted significant (P<0.05) dose-dependent
cytotoxicity on HeLa cells with an IC50 of 12 ± 2.23 µM (Figure
2c). The G. lucidum chloroform and hexane extracts induced moderate
dose-dependent cell death of the cancer cells with the IC50 values
218 ± 7.45 and 190 ± 15.45 µg/ml, respectively (Figure 2b). Among the
crude extracts, the methanol fraction of G. lucidum exhibited highly
significant (P<0.05) dose-dependent cytotoxic effect on HeLa cells
with an IC50 value of 125 ±7.27 µM µg/ml, which was considerable for
a crude extract compared to treatment
with the standard 5-FU drug (Figure 2b and 2c). In contrast, the
morphological evidence from light microscopy indicated little or no effect of G.
lucidum methanol extract on the growth of peritoneal macrophages (Figure
3a).
Similarly, results from the MTT assay also indicated
that treatment with G. lucidum extract or 5-FU had little or no effect
on the viability of peritoneal macrophages (Figure 3b and 3c). Only the
treatment with 5-FU above 50 µM exhibited considerable effect on the viability
of peritoneal macrophages (Figure 3c). Taken together, the results
suggests that G. lucidum extract exerts selective cytotoxicity towards
HeLa cells which is noteworthy for its therapeutic potential. Phytochemicals
such as polysaccharide, triterpenoids, phenloics, flavinoides and other
phytochemicals alone or in combination may be responsible for the observed
effects [36,37]. Such bioactive chemicals are reported to possess anti-cancer
and anti-angiogenesis properties indicating their possible contribution to the
observed effects [38,39].
Macrophages are one of the principal immune cells
bridging adaptive and innate immunity. Macrophages and dendritic cells
stimulated with LPS can induce production of significant levels of NO via
Toll-like receptors (TLR)-4 signaling pathways [40]. Under normal conditions,
this leads to activation of immune response with secretion of pro-inflammatory
cytokines and inflammatory mediators like NO to protect the host from infection
or cell damage. However, excessive production of NO can be associated with
tissue damage and organ dysfunction which include vasodilation, hypotension and
septic shock [41,42]. In addition, over secretion of NO is known to be involved
in several inflammation-associated diseases such as inflammatory bowel disease
and Crohn’s disease [43, 44]. Previous study has indicated that heat-killed S.
typhimurium provides a suitable source of LPS for TLR-4-mediated immune
response in macrophages and can be utilized to assess the anti-inflammatory
effects of drugs [45]. In our present study, peritoneal macrophages were
cultured and treated with HKST as a source of LPS to stimulate macrophages for
NO production. The HKST served as a source of LPS and a ligand for TLR-4
activation and further minimized the complication of infections by live
pathogens. The HKST was added at MOI 5 for maintaining the optimum amount of
LPS to stimulate macrophages [15]. During our investigation, in the absence of
HKST stimulation a very little amount of NO was produced by peritoneal
macrophages. In contrast, HKST stimulation significantly (P<0.05) increased
NO production by peritoneal macrophages (Figure 4). Pretreatment with G. lucidum chloroform and methanol
extracts caused significant (P <0.05) dose-dependent inhibition of
HKST-induced NO production by peritoneal macrophages (Figure 4). The
results indicate that G. lucidum
chloroform and methanol extracts have promising anti-inflammatory activity. The
presence of flavonoids, phenolic acids and phenolic diterpenes may have
collectively contributed to the observed anti-inflammatory effect.
CONCLUSION
The results from our
phytochemical screening and biological activity evaluation collectively
indicate that G. lucidum extract has promising therapeutic potentials. Our
study suggests that the local variety of G. lucidum can be a potent source for
development of chemotherapeutic drugs and it calls for further in-depth
investigation for its medicinal properties.
ACKNOWLEDGEMENT
The research was a part
of an independent study for evaluating biological properties of locally
available flora of Nepal. The study was supported by Central Department of
Biotechnology, Tribhuvan University, Nepal.
CONFLICT OF INTEREST
Authors declare no
conflict of interest.
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