Anticancer and Antibacterial Activity of Ganoderma lucidum

19 Pages·2016·1.17 MB·English

Int.J.Curr.Microbiol.App.Sci (2016) 5(10): 891-909
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 5 Number 10 (2016) pp. 891-909

Journal homepage: http://www.ijcmas.com

Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.510.097

Anticancer and Antibacterial Activity of Ganoderma lucidum

A. Thameem Fathima* and M. Reenaa

Department of Microbiology, Ethiraj College for Women, Chennai- 600008, Tamil Nadu, India
*Corresponding author

A B S T R A C T

Ganoderma lucidum is considered as one of the richest sources of natural

antibiotics and various species of them inhibit the growth of a wide diversity of

micro organi sms. The pharmacological activities of Ganoderma lucidum,
especially its intrinsic immunomodulating and anti- tumor properties have been

well documented. Ganoderma has been used in the folk medicine of China and

Japan for 4,000 years to treat the liver diseases such as Hepatitis, Nephritis,

Keywords
Arthritis, Gast ric ulcers. It is also believed that the Ganoderma lucidum acts as an

immunologica l agent and has been as a natural adjuvant for Immunotherapy. Based
Anticancer,
upon its effe ctive components, this mushroom was selected for studying
Antibacterial,

Ganoderma lucidum, the anticancer as well as antibacterial study. The present study is to check

Hep G2, E.coli, the anticancer and antibacterial activities of ethanolic, methanolic and aqueous
Micrococ cus luteus, extracts of G. lucidum. The dried mushroom sample was powdered and extracted
Bacillus t huringiensis, with ethanol, methanol and aqueous extracts using Soxhelet apparatus in the
Pseudom onas laboratory. Ve ro and Hep G2 liver cancer cell lines were used for determining the
flouresce ns.
cytotoxic and anticancer activity (MTT ASSAY). From the MTT Assay , it was

Article I nfo observed that 53.96% of inhibition of cancer cells from the dilution of 15.6 μg/ml
in methanolic extracts of G.lucidum, 52.38% of inhibition was obtained from the
dilution of 31. 2 μg/ml in ethanol extracts of G.lucidum, 50.75% of inhibition was
Accepted:
obtained from the dilution of 62.5 μg/ml in aqeous extracts of G.lucidum, It was
25 Septem ber 2016
Available Online: concluded tha t the methanolic extracts showed inhibition(IC 50) of 53.96% at the
10 Octobe r 2016 least dilution of 15.6μg/ml, when compared to the ethanolic and aqueous extracts

of G.lucidum. For checking the antibacterial activities of G.lucidum, Bacillus

thuringiensis, Micrococcus luteus, Pseudomonas flourescens, and E.coli were

selected rando mly. The methanolic extract of G.lucidum showed about 20mm of

zone of inhibi tion in the Pseudomonas flourescens (MTCC 103) from the dilution
of 25 μg/ml and 10mm of zone of inhibition was observed in Escherichia coli,

which was isolated from UTI patients from the dilution of 25 μg/ml. The ethanolic
and aqueous e xtracts were least effective against all the strains. From these studies

the methanolic extracts of Ganoderma lucidum can be fruitfully applied and used
as the more effective and competent antimicrobial agent. Thus this edible
mushroom can be used as antimicrobial agent in the expansion of new drug therapy

for the liver diseases.

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Introduction concentrate nutrients, such as phosphorus
and water. Then when the mycelia wrap
Mushrooms are the macro fungi with the
around the plant roots, even entering the
distinctive basidiomata or ascomata which
plant‟s cells, the plants can absorb these
can be either hypogeus or epigeous, large
concentrated nutrients straight from the
enough to be seen with the naked eye and to
fungus. Thus the plant‟s capabilities are
be picked up by the hand. The number of
absorbing greatly increased. In return, the
different kinds of mushrooms in the earth is
fungus can absorb complex compounds that
estimated around 1, 40,000. Around 2,000 of
the plant makes, such as sugars and amino
them are edible and 25 species are largely
acids. Thus, both the plant and fungi gets
used as foods, being produced in
benefited.
commercial scale. Mushrooms resemble

plants in that they cannot move on their own
The fruit of this complex process are
and they have cell walls. Compared to
mushroom, which can benefit the immune
Fungi, the cell wall of fungi composed of
system and provide many health benefits for
Chitin and not cellulose, and fungi do not
humans. In particular, compounds called
carry out the photosynthesis. Instead fungi
hetero polysaccharides – large, complex,
absorb food and nutrients from their
branched, chain like molecules built from
surroundings (Andreia et al., 2013).
many smaller units of sugar molecules have

been intensively studied for their anti- tumor
A mushroom is merely the fruiting body of
and immune stimulating properties. Beta
a much larger fungal colony. Fruiting bodies
glucans are the polysaccharides found in
are reproductive structures that grove above
mushrooms that are known as “biological
the ground to release spores. Fungal
response modifiers” because of their ability
colonies composed of mycelium, a thread-
to activate the immune system. They also
like network that grow underground or in
reportedly lower cholesterol, aid in wound
the wood. It is through the mycelium that
healing, help prevent infections and are an
the fungus absorbs nutrients from its
adjuvant in cancer treatments.
environment. Nutrient absorption takes

place in two stages: first the mycelium
Smaller compounds such as terpenes and
secretes enzymes that break down plants,
steroids have also been found. A great
animal matter and components in the soil
number of polysaccharides and protein
into usable food for the fungal colony, as
bound polysaccharides have been shown to
well as for the surrounding trees and plants.
have antibiotic and antiviral properties, as
Next, the mycelium absorbs the ensuing
well as the ability to lower blood pressure
nutritional matter it needs to fuel growth.
and reduced blood levels of lipids and sugar.

These active compounds make many fungi
Mushrooms are the nature‟s recyclers,
useful for treating infections, flu, and
breaking down plant and animal matter to
diabetes and heart conditions.
release the carbon and other nutrients locked

inside. In the early spring the fungi invades
The mushrooms are poor in calories and rich
the composted leaves and break them down
in protein fibers, carbohydrates, and
into food for the new spring growth of plants
important vitamins such as Thiamin,
and trees. Many fungi are symbionts,
Riboflavin, Ascorbic acid and minerals.
helping the plants to obtain vital nutrients.
They are usually considered as functional
As the mycelia of the fungi grow through
foods or nutraceutical products. Mushrooms
the soil like a net of fingers, they absorb and
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Int.J.Curr.Microbiol.App.Sci (2016) 5(10): 891-909
contains many substances and many of them shiny or brilliant which refers to the
could have some biological activity and appearance of the fungus fruiting body,
those are such as antioxidants, which has a modeled, varnished look.
antitumor/anticancer, antimicrobial, immuno Ganoderma has been used in the folk
modulatory, anti inflammatory actions, anti medicine of China and Japan for 4,000 years
atherogenic, and hypoglycemic actions. to treat the liver diseases such as Hepatitis,
Nephritis, Arthritis, Gastric ulcers. The
Among them, the Chinese mushroom called extracts of G.lucidum have potent anti
as Ganoderma lucidum is an excellent oxidant and radical – scavenging effects,
medicinal mushroom which is also called as which contribute to the hepatoprotection.
„Spirit plant‟. It is commonly known as When compared to other species of
„Reishi‟ in Japan and „Zing-Zhi‟ or „Spirit Ganoderma, G.lucidum has more efficacies
plant‟ in China, where it is also referred to to treat the hepatic injuries caused by
the “Plant of Mortality”. Lucidum means various factors.

Ganoderma lucidum

The liver is a large, complex organ that is taking up and storing glucose as glycogen
well designed for its central role in (glycogenesis), breaking it down to glucose
carbohydrates, proteins and fat metabolism. when needed (glycogenolysis) and forming
It is the site where the waste products of glucose from non carbohydrate sources such
metabolism, such as ammonia, are as amino acids (gluconeogenesis). The liver
detoxified. In conjunction with the spleen it plays an important role in drug elimination
is involved in the destruction of remnants of and detoxification and liver damage may be
the red blood cells and with the recycling of caused by many xenobiotics, such as alcohol
their constituents. It is responsible for and many medicines, malnutrition, infection
synthesizing and secreting bile and for and anemia.
synthesizing lipoproteins and plasma
proteins, including clotting factors. It Liver damage is a wide spread disease
maintains a stable blood glucose level by which, in most cases, involves oxidative
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Int.J.Curr.Microbiol.App.Sci (2016) 5(10): 891-909
stress and is characterized by a progressive oxidase component of an electron transport
evolution from steatosis to chronic hepatitis, chain. In modern medicines, corticosteroids
fibrosis, cirrhosis and hepatocellular and immunosuppressant are commonly used
carcinoma. Although almost all tissues in to treat liver disease in allopathic form of
the body have some ability to metabolize medicines. But these drugs are associated
chemicals, smooth endoplasmic reticulum in with adverse effects such as
liver is the principal “metabolic clearing immunosuppressant and bone marrow
house” for both endogenous substances. A depression. However there are number of
group of enzyme located in the endoplasmic drugs employed in traditional system of
reticulum known as Cytochrome P-450, is medicine for the liver infections. (Manju
the most important family of metabolizing Gowda et al., 2014).
enzymes in the liver. It is the terminal

Cancerous Growth in Liver

In this study, the aqueous, methanolic and The MTT Cell viability Assay provides
ethanolic extracts of G.lucidum is used to convenient, sensitive, quantitative and
check out the anticancer activity of the reliable assay for determining the number of
mushroom by using the Liver cancer cell viable cells in the cell culture. This
lines such as HepG2 and Vero cell lines. homogeneous colorimetric assay is based on
The hepatoma cell lines are frequently used the principle of the conversion of
as in vitro alternatives to primary human tetrazolium salt, 3- (4, 5-dimethyl-2-
hepatocytes. Cell lines are characterized by thiazolyl)-2, 5-diphenyl-tetrazolium
their unlimited life span, stable phenotype, bromide(MTT), a pale yellow substrate to
high availability, and easy handling. formazon, a purple dye. This cellular
However, their major limitation is the lower reduction reaction involves the pyridine
expression of some metabolic activities nucleotide cofactors NADH/ NADPH and is
compared with the hepatocytes. The only catalyzed by living cells. The formazon
anticancer and cytotoxic activities of product has the low aqueous solubility and
methanol, ethanol & aqueous extracts of is present as purple crystals. Dissolving the
Ganoderma lucidum was carried out by resulting formazon with solubilization buffer
using the MTT Assay. permits the convenient quantification of
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Int.J.Curr.Microbiol.App.Sci (2016) 5(10): 891-909
product formation. The intensity of product purpose.
colour measured at 550- 620 nm is directly
proportional to the number of living cells in Ethanolic extracts of Ganoderma lucidum
the culture.
In the laboratory, the dried mushroom
The main aim of this study includes to sample was powdered and stored in an
prepare the aqueous, methanolic and airtight container for further use. The
ethanolic extracts of Ganoderma lucidum. powder of Ganoderma lucidum (25gms) was
To perform the Cytotoxic activity of the extracted separately with the 250ml of
mushroom extracts by using the Vero cell ethanol in a Soxhlet extractor for 6 hours.
lines. To perform the Anticancer activity of The extractions were repeated twice. The
the Ganoderma lucidum extracts introducing total extractions were filtered and the
to the Liver Cancer Cell line such as HepG2 extracts were concentrated under the
and also to perform the Antibacterial activity reduced pressure to dryness. The
of G. lucidum extracts by Well diffusion concentrated extracts were stored in the
method. To study the anticancer activity of refrigerated conditions for further use.
methanolic, ethanolic and aqueous extracts
from Ganoderma lucidum using invitro Aqueous extracts of Ganoderma lucidum
(Tissue culture). And also to study the
antibacterial activity of G.lucidum extracts In the laboratory, the dried mushroom
by well diffusion method. sample was powdered and stored in an
airtight container for further use. The
Materials and Methods powder of Ganoderma lucidum (25gms)
was extracted separately with the 250ml of
Collection of mushroom sample distilled water in a Soxhlet extractor for 6
hours. The extractions were repeated twice.
250gms of the dried mycelia fruiting body The total extractions were filtered and the
of G.lucidum were collected from the extracts were concentrated under the
Camillotek Private Ltd, Chennai. reduced pressure to dryness. The
concentrated extracts were stored in the
Preparation of mushroom extracts refrigerated conditions for further use.

Methanolic extracts of Ganoderma Cell lines and culture
lucidum
Vero and Hep G2 cell lines were obtained
In the laboratory, the dried mushroom from Veterinary College, Vepery, Chennai.
sample was powdered and stored in an air The cells were maintained in Minimal
tight container for further use. The powder Essential Medium supplemented with 10%
of Ganoderma lucidum (25gms) was FBS, Penicillin (100U/ml) and Streptomycin
extracted separately with the 250ml of (100 μg/ml) in a humidified atmosphere of
methanol in a Soxhlet extractor for 6 hours. 50μg/ml CO at 37°C.
2
The extractions were repeated twice. The
total extractions were filtered and the In vitro assay for cytotoxicity/anti cancer
extracts were concentrated under the activity (mtt assay) (mossman, 1983)
reduced pressure to dryness. The
concentrated extracts were stored in the Cells (1×105 / well) were placed in 24- plates
refrigerated condition for future research
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Int.J.Curr.Microbiol.App.Sci (2016) 5(10): 891-909
and incubated at 37° C with the 5% CO Antibacterial activity
2
condition. After the cell reaches the
confluence, the sample was removed from For determining the antibacterial activity of
the well and washed with phosphate- Ganoderma lucidum, Bacillus thuringiensis,
buffered saline (pH 7.4) or MEM without Micrococcus luteus, Pseudomonas
serum 100µl/well (5mg/ml) of 0.5% 3- (4, 5- flourescens, and E.coli were selected
dimethyl-2-thiazolyl)-2, 5-diphenyl-tetra- randomly. The isolated bacterial colonies
zolium Bromide (MTT) was added and from the agar plates were inoculated into
incubated for 4 hours. After incubation, 1ml the sterile saline. The saline test tubes with
of DMSO was added in all the wells. The the bacterial cultures were incubated at 37°
absorbance at 570nm was measured with C for 2 hours. After 2 hours of incubation,
UV- Spectrophotometer using DMSO as the the saline test tubes with the bacterial
blank. Measurements were performed and cultures were taken and compared with
the concentration required for a 50% 0.5% Mac Farland‟s standard for the
inhibition (IC50) was determined standardization of bacterial cultures. 200 ml
graphically. The percentage of cell viability of Muller Hinton Agar was prepared by
was calculated using the following formula: dissolving 7.6 gms of Muller Hinton agar in
200ml of sterile distilled water. The agar
% cell viability = A570 of treated cells / was sterilized by autoclaving at 121°C for
A570 of control cells ×100 15 minutes. After the sterilization, the
Muller Hinton Agar was poured onto the
Graphs are plotted using the % of Cell sterile petriplates aseptically. The agar was
viability at Y- axis and Concentration of the allowed to solidify for few minutes. With
sample in X-axis. Cell control and sample the help of sterile swabs, the each bacterial
control is included in each assay to compare culture was swabbed onto the surface of the
the full cell viability assessments. Muller Hinton Agar aseptically and allowed
them to dry. The three wells (1cm) were cut
Antibacterial activity of Ganoderma by using the sterile micropipette tips in
lucidum equal intervals in each petriplate and
marked. Then, the ethanol, methanol, and
Cultures aqueous extracts of Ganoderma lucidum
were taken. 25µl of various extracts of
Bacillus thuringiensis- MCC 2095 – MCC, mushroom was inoculated onto the wells
NCCS, Pune, which causes hospital oriented aseptically. After the inoculation, the plates
infections and acts as commensals. were kept for incubation at 37°C for 24
hours. After 24 hours of incubation, the
Pseudomonas flourescens- MTC 103- plates were checked for the zone of
MTCC Gene Bank, Chandigarh, which inhibition.
causes hospital oriented infections.
Results and Discussion
E.coli – Isolated from Urinary Tract
Infection(UTI) patients MTT assay/ anticancer activity

Micrococcus luteus – Isolated from the From the 3- (4, 5-dimethyl-2-thiazolyl)-2, 5-
environmental source by open plate method. diphenyl-tetrazolium bromide (MTT) Assay,
cell viability (IC50) of 53.96% was
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Int.J.Curr.Microbiol.App.Sci (2016) 5(10): 891-909
obtained from the 15.6 μg/ml in the 1: 32 Micrococcus luteus was not subjected to
dilution at the O.D value of 0.34 for the any inhibition. Thus it shows negative
Methanolic extracts of the G.lucidum. action for those bacteria.

Cell viability (IC50) of 52.38% was From the Anticancer and Antibacterial
obtained from the 31.2 μg/ml in the 1:16 activity assays, it was proved that the
dilution at the O. D value of 0.33 for the Ethanol, Methanol and Aqueous extracts of
Ethanol extracts of G.lucidum. G.lucidum consists of some phytochemical
components, which controls the growth of
Similarly, cell viability (IC50) of 6.25 μg/ml micro organisms. The results for the
was obtained from the 1:8 at the O.D value Anticancer and Antibacterial activity were
of 0.32 dilutions for the Aqueous extracts of interpreted and described in the form of
the G.lucidum. Tables, Charts and Figures.

Antibacterial activity The liver cancer cell lines which are used in
the research purposes are HepG2, Huh 7,
After the incubation at 37°C for 24 hours, HeLA, SNU – 387, SNU -423, MC/9, Capan
the MHA plates were checked for the Zone -1, Hep 3 –B, WCH -17, H4TG, RTH – 149,
of Inhibition. The methanol extracts of G. BRL 3A, etc (www.atcc.org). The available
lucidum showed 20mm of zone formation cell lines used for the present study, such as
in Pseudomonas flourescens (MTCC 103) the Hep G2 and Vero cell lines supports the
and 10mm of zone was observed from the viral replication when transfected with HBV
methanol extracts of E.coli. Similarly, the DNA.
Bacillus thuringiensis (MCC 2095) and

Table.1 Cytotoxic effect of Methanol Extract on VERO Cell line

S.No Concentration (µg/ml) Dilutions Absorbance Cell Viability
(O.D) (%)
1 1000 Neat 0.36 80.00
2 500 1:1 0.38 84.44
3 250 1:2 0.40 88.88
4 125 1:4 0.41 91.11
5 62.5 1:8 0.41 91.11
6 31.2 1:16 0.42 93.33
7 15.6 1:32 0.42 93.33
8 7.8 1:64 0.43 95.55
9 Cell control - 0.45 100

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Table.2 Cytotoxic effect of Ethanol Extract on VERO Cell line

S.No Concentration (µg/ml) Dilutions Absorbance Cell Viability
(O.D) (%)
1 1000 Neat 0.35 77.77
2 500 1:1 0.36 80.00
3 250 1:2 0.37 82.22
4 125 1:4 0.40 88.88
5 62.5 1:8 0.42 93.33
6 31.2 1:16 0.43 95.55
7 15.6 1:32 0.43 95.55
8 7.8 1:64 0.44 97.77
9 Cell control - 0.45 100

Table.3 Cytotoxic effect of Aqueous Extract on VERO Cell line

S.No Concentration (µg/ml) Dilutions Absorbance Cell Viability
(O.D) (%)
1 1000 Neat 0.30 66.66
2 500 1:1 0.31 68.88
3 250 1:2 0.33 73.33
4 125 1:4 0.37 82.22
5 62.5 1:8 0.39 86.66
6 31.2 1:16 0.41 91.11
7 15.6 1:32 0.42 93.33
8 7.8 1:64 0.42 93.33
9 Cell control - 0.45 100

Table.4 Anticancer effect of Methanol Extract on HePG2 Cell line

S.No Concentration (µg/ml) Dilutions Absorbance Cell Viability
(O.D) (%)
1 1000 Neat 0.04 6.34
2 500 1:1 0.07 11.11
3 250 1:2 0.11 17.46
4 125 1:4 0.18 28.57
5 62.5 1:8 0.23 36.50
6 31.2 1:16 0.29 46.03
7 15.6 1:32 0.34 53.96
8 7.8 1:64 0.41 65.07
9 Cell control - 0.63 100

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Table.5 Anticancer effect of Ethanol Extract on HePG2 Cell line

S.No Concentration (µg/ml) Dilutions Absorbance Cell Viability
(O.D) (%)
1 1000 Neat 0.08 12.69
2 500 1:1 0.15 23.80
3 250 1:2 0.21 33.33
4 125 1:4 0.25 39.68
5 62.5 1:8 0.28 44.44
6 31.2 1:16 0.33 52.38
7 15.6 1:32 0.40 63.49
8 7.8 1:64 0.49 77.77
9 Cell control - 0.63 100

Table.6 Anticancer effect of Aqueous Extract on HePG2 Cell line

S.No Concentration (µg/ml) Dilutions Absorbance Cell Viability
(O.D) (%)
1 1000 Neat 0.10 15.87
2 500 1:1 0.16 25.39
3 250 1:2 0.22 34.92
4 125 1:4 0.27 42.85
5 62.5 1:8 0.32 50.79
6 31.2 1:16 0.39 61.90
7 15.6 1:32 0.44 69.84
8 7.8 1:64 0.51 80.95
9 Cell control - 0.63 100

Fig.1 Fruiting bodies of Ganoderma lucidum

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Fig.2 Dried mycelia & powder of Ganoderma lucidum

Fig.3 Soxhelet Extractor Fig.4 MTT Assay Microtitre Plate

Fig.5 HepG2 cell line Fig.6 VERO cell line

900