A Comparative evaluation of in vitro anti‑inflammatory and antifungal activity of Ganoderma lucidum strains DARL‑4 and MS‑1

Abstract

Background: Ganoderma lucidum commonly known as Reishi is a lignicolous high value medicinal mushroom belonging to family Ganodermataceae. DARL-4 is an indigenous strain and MS-1 is an exotic Malaysian strain which is in vitro cultivated under sterile condition. The main aim of this study is a comparative evaluation of in vitro anti-inflammatory and antifungal activity of G. lucidum strains DARL-4 and MS-1. Materials and Methods: The hydroalcoholic extract of G. lucidum strains DARL-4 and MS-1 was screened for in vitro anti-inflammatory activity using inhibition of albumin denaturation technique at different concentration. Diclofenac (100 µg/ml) was used as standard reference drug. In vitro antifungal activity of hydroalcoholic extract of G. lucidum strains DARL-4 and MS-1 was evaluated by agar well diffusion method using Candida albicans as a fungal strain. Fluconazole was used as standard drug. Results and Discussion: The % inhibition of denaturation produced by hydroalcoholic extract of DARL-4 and MS-1 was comparable with that produced by diclofenac. MS-1 shows more significant anti-inflammatory activity than DARL-4. DARL-4 and MS-1 show moderate antifungal activity with a zone of inhibition of 19 ± 0.21and 21 ± 0.36 mm, respectively, as compared to the standard (fluconazole) having zone of inhibition of 30 ± 0.03 mm. Conclusion: MS-1 possesses more significant anti-inflammatory and antifungal activity as compared to DARL-4.

Full text

International Journal of Green Pharmacy • Jan-Mar 2018 (Suppl) • 12 (1) | S126
A Comparative evaluation of in vitro
anti‑inflammatory and antifungal activity of
Ganoderma lucidum strains DARL‑4 and
MS‑1
Swati1, A. Tiwari1, P. S. Negi2, H. S. Meena2
1Department of Science, Devsthali Vidyapeeth, College of Pharmacy, Rudrapur, Uttarakhand, India,
2Department of Pharmacy, Defence Institute of Bio-Energy Research, Field Station, Pithoragarh – 262 501,
Uttarakhand, India
Abstract
Background: Ganoderma lucidum commonly known as Reishi is a lignicolous high value medicinal mushroom
belonging to family Ganodermataceae. DARL-4 is an indigenous strain and MS-1 is an exotic Malaysian strain
which is in vitro cultivated under sterile condition. The main aim of this study is a comparative evaluation of in vitro
anti-inflammatory and antifungal activity of G. lucidum strains DARL-4 and MS-1. Materials and Methods: The
hydroalcoholic extract of G. lucidum strains DARL-4 and MS-1 was screened for in vitro anti-inflammatory activity
using inhibition of albumin denaturation technique at different concentration. Diclofenac (100 µg/ml) was used as
standard reference drug. In vitro antifungal activity of hydroalcoholic extract of G. lucidum strains DARL-4 and
MS-1 was evaluated by agar well diffusion method using Candida albicans as a fungal strain. Fluconazole was used
as standard drug. Results and Discussion: The % inhibition of denaturation produced by hydroalcoholic extract of
DARL-4 and MS-1 was comparable with that produced by diclofenac. MS-1 shows more significant anti-inflammatory
activity than DARL-4. DARL-4 and MS-1 show moderate antifungal activity with a zone of inhibition of 19 ± 0.21and
21 ± 0.36 mm, respectively, as compared to the standard (fluconazole) having zone of inhibition of 30 ± 0.03 mm.
Conclusion: MS-1 possesses more significant anti-inflammatory and antifungal activity as compared to DARL-4.
Key words: Ganoderma lucidum, Anti-inflammatory activity, antifungal activity, albumin denaturation, agar well
diffusion
Address for correspondence:
Dr. Abhishek Tiwari, Devsthali Vidyapeeth,
College of Pharmacy, Rudrapur, Uttarakhand, India.
E-mail: abhishekt1983@gmail.com
Received: 03-10-2017
Revised: 27-12-2017
Accepted: 10-03-2018
INTRODUCTION
Ganoderma lucidum commonly known
as Reishi is a lignicolous high value
medicinal mushroom belonging to family
Ganodermataceae. G. lucidum (W.Curst.:Fr.)
P. Karst. (Higher Basidiomycetes) is well known
for nutraceutical and pharmaceutical properties
for promoting human health. G. lucidum has
been reported to show antitumor, hypotensive,
cytoxicity, antioxidant, anti-allergic,
antimicrobial, hepatoprotective, hypolipidemic,
anti-diabetic, and anti-inflammatory effects.[1]
G. lucidum contains bioactive comp onents main ly
triterpenoids, steroids, glycoproteins, and
polysaccharides.[2-5] These bioactive components
play a role in maintaining a good health and
fulfill the nutritional requirements. Wild
G. lucidum is less abundant in nature, and thus,
it is not available sufficiently for nutraceutical
product development so its’ in vitro cultivation
is developed for its easy availability for nutraceutical and
pharmaceutical development. DARL-4 is an indigenous strain
and MS-1 is an exotic Malayasian strain which is in vitro
cultivated under sterile condition.
Inflammation is a normal protective response to tissue
injury which involves enzyme activation, mediator release,
fluid extravasations, cell migration, tissue breakdown, and
repair.[6] It is frequently associated with pain and involves
the increase in vascular permeability, increase of protein
denaturation, and membrane alterations.[7] Inflammation is a
ORIGINAL ARTICLE

Swati, et al.: In vitro anti‑inflammatory and antifungal evaluation of Ganoderma lucidum strains
International Journal of Green Pharmacy • Jan-Mar 2018 (Suppl) • 12 (1) | S127
physiologic defense mechanism that helps the body to protect
itself against infection, burn, toxic chemicals, allergens, or
other noxious stimuli. Inflammations are mainly as acute
and chronic inflammations.[8] The present non-steroidal anti-
inflammatory drugs (NSAID’s) are commonly used drugs
for treating inflammation, and the long-term use of NSAID’s
causes severe side effects. For this reason, in recent time, a
search for other alternatives seems necessary and beneficial.
Pathogenic fungi are fungi that cause disease in humans.
Most of the incidence of fungal infections increase due to
the weak immune system related to HIV, cancer, and other
diseases.
Candida albicans is responsible for a wide range of
superficial and systemic infections.[9] There has been an
increase in resistance by C. albicans to conventionally
produced antimicrobials recently, leading to the search of a
new antifungal agent.[10,11]
The main aim of this study is a comparative evaluation
of in vitro anti-inflammatory and antifungal activity of
G. lucidum strains DARL-4 and MS -1.
MATERIALS AND METHODS
Collection of Fruiting Bodies of G. lucidum Strains
DARL‑4 and MS‑1
In vitro cultivated samples of G. lucidum strains DARL-4
and MS-1 were collected from the polyhouse of DIBER,
Pithoragarh, between April and May. The strains were
authenticated by Mycology Department, DIBER, field
station, Pithoragarh. Samples were then air-dried and grinded
into powdered form.
Preparation of Crude Extract
50 g of DARL-4 and MS-1 were extracted with hydroalcohol
by the cold maceration process. The extracts were filtered
with the help of Whatman No. 1 filter paper and evaporated
to dryness. The extracts were finally lyophilized and stored
in a desiccator.
Inhibition of Albumin Denaturation
The anti-inflammatory activity of hydroalcohol extracts of
G. lucidum strains DARL-4 and MS-1 were performed using
inhibition of albumin denaturation method and shown in
Figure 1[12-14] followed with slight modifications. The reaction
mixture (2 ml) was containing test extracts of different
concentrations (100–500 µg/ml) or 100 µg/ml diclofenac
(SAID) and 1% aqueous solution of bovine albumin fraction.
The sample extracts were incubated at 37°C for 20 min and
then heated to 51°C for 20 min, and after cooling the samples,
the turbidity was measured at 660 nm. Percentage inhibition
of denaturation was calculated from control where no drug
was added. The experiment was performed in triplicate. The
percentage inhibition was calculated using the following
formula:
Percentage inhibition Abs control - Abs sample
1Abs
=
()
×00 /ccontrol
In vitro Antifungal Activity of G. lucidum strains
DARL‑4 and MS‑1
In vitro antifungal activity of hydroalcohol extracts of
DARL-4 and MS-1 was performed using agar well diffusion
method and shown in Figure 2.[14-17]
Preparation of Culture Medium
Sabouraud’s dextrose agar media (Hi Media) were used for
in vitro antifungal activity. For the preparation of media,
dextrose (40 g), peptone (10 g), and agar (20 g) were accurately
weighed, dissolved in distilled water, and autoclaved at
121°C for 15 min. pH of the media was maintained at 5.6.
Standard Preparation
Fluconazole was used as a standard antifungal agent and
prepared in sterile distilled water to give a final concentration
of 10 µg/ml.
Figure 1: Percentage inhibition of hydroalcohol extracts of
DARL-4 and MS-1 on albumin denaturation
Figure 2: Evaluation of antifungal activity of hydroalcohol
extract of Ganoderma lucidum strains DARL-4 and MS-1

Swati, et al.: In vitro anti‑inflammatory and antifungal evaluation of Ganoderma lucidum strains
International Journal of Green Pharmacy • Jan-Mar 2018 (Suppl) • 12 (1) | S128
Sample Preparation
The hydroalcohol extracts of DARL-4 and MS-1 were
dissolved in dimethyl sulfoxide (DMSO) to give the final
concentration of 20 mg/ml.
Preparation of Inoculum
The suspension of fungus was prepared by McFarland
Nephelometer standard method. The culture of C. albicans
was used for the preparation of fungal suspension. An
inoculum was prepared by suspending the isolated colony in
2 ml of 0.9% w/v of normal saline solution and then mixed to
form a smooth suspension.
Procedure for In vitro Antifungal Activity
The Sabouraud’s dextrose agar media (Hi Media) were
poured in sterile Petri plates and allowed to solidify. After
that, the prepared inoculum was poured onto the surface of
agar plates and spreaded by a glass spreader. The flamed
sterile borer (21 mm in diameter) was used and the medium
was bored, and then 0.1 ml of standard and test samples
were added in each bore. A control having DMSO was
also maintained. The above procedure was carried out in
aseptic condition under Laminar Air Flow. The plates
were then incubated at 28°C for 72 h. Finally, the zone of
inhibition in each plate was measured and the test samples
were compared with the standard. The experiment was run
in triplicate.
Table 1: Effect of hydroalcohol extract of DARL-4 on albumin denaturation
Treatment Concentration (µg/ml) Absorbance (660 nm) % Inhibition
Control - 0.19±0.24 -
DARL-4 100 0.098±1.23 48.42±0.79
DARL-4 200 0.082±0.29 56.84±0.98
DARL-4 300 0.066±0.59 65.26±0.74
DARL-4 400 0.054±1.20 71.57±0.78
DARL-4 500 0.042±0.035 77.89±1.02
Diclofenac 100 0.034±0.42 81.84±1.24
Table 2: Effect of hydroalcohol extract of MS-1 on albumin denaturation
Treatment Concentration (µg/ml) Absorbance (660 nm) % inhibition
Control - 0.19±0.24 -
MS-1 100 0.096±0.90 49.47±0.90
MS-1 200 0.079±0.28 58.42±0.28
MS-1 300 0.062±1.04 67.36±1.02
MS-1 400 0.047±0.059 75.15±0.59
MS-1 500 0.037±1.12 80.52±0.24
Diclofenac 100 0.034±0.42 81.84±1.24
RESULT
In vitro anti-inflammatory activity of hydroalcohol extracts of
DARL-4 and MS-1 was investigated by albumin denaturation
method. The results are indicated in Tables 1 and 2. The
hydroalcohol extracts of DARL-4 and MS-1 were used in
the concentration range of 100–500 µg/ml, and it showed a
concentration-dependent inhibition of albumin denaturation.
Diclofenac, a SAID, showed the maximum inhibition 81.84%
at the concentration of 100 µg/ml. MS-1 and DARL-4 show
80.52 ± 0.24% and 77.89 ± 1.02% maximum inhibition at
concentration of 500 µg/ml. MS-1 shows more significant
anti-inflammatory activity in concentration-dependent
inhibition of albumin denaturation than that of DARL-4.
The results of antifungal activity of hydroalcohol extracts
of DARL-4 and MS-1 against the fungal strains C. albicans
are shown in Table 3. The hydroalcohol extracts of
MS-1 (21 ± 0.36 mm) produced the highest zone of inhibition
against C. albicans as compared to DARL-4 (19 ± 0.21 mm).
The standard antifungal control used (fluconazole 10 µg/ml)
formed a desirable zone of inhibition of 30 ± 0.03 mm. There
were no zones of inhibitions formed by the negative control.
The hydroalcohol extract of MS-1 possesses more significant
antifungal activity.
DISCUSSION
Fungi are responsible for many infectious diseases.[18]
G. lucidum and other Ganoderma species have been used

Swati, et al.: In vitro anti‑inflammatory and antifungal evaluation of Ganoderma lucidum strains
International Journal of Green Pharmacy • Jan-Mar 2018 (Suppl) • 12 (1) | S129
to treat various bacterial and fungal diseases. This might be
due to the presence of rich phytochemical constituents such
as polysaccharides, phenol, triterpenoids, and flavonoids.
There has been an increase in resistance by fungal strains to
conventionally produced antifungal agents recently, leading
to the search of a new antifungal agent. The agar well
diffusion method had shown that the tested hydroalcohol
extracts of DARL-4 and MS-1 have moderate antifungal
activity against the tested C. albicans.
Denaturation of proteins is a well-known cause of
inflammation. When proteins are denatured, they lose their
biological functions. Production of autoantigen in certain
arthritic disease is due to denaturation of protein.[19,20]
In the present study, the hydroalcohol extracts of DARL-4
and MS-1 are capable of inhibiting albumin denaturation.
MS-1 shows more significant anti-inflammatory activity as
compared to that of DARl-4.
CONCLUSION
The results from the present study reported that in vitro
cultivated G. lucidum strains DARL-4 and MS-1 used as
an ideal bio-pharmaceutics. The hydroalcohol extracts
of DARL-4 and MS-1 possessed significant anti-
inflammatory and antifungal activity. This might be due
to the presence of rich phytochemical constituents such
as polysaccharides, phenols, flavonoids, and terpenoids.
MS-1 which is an exotic Malaysian strain was found to
have more significant anti-inflammatory and antifungal
activity when compared with DARL-4 which is an
indigenous strain. This study is suggested that G. lucidum
can be used as anti-inflammatory and antifungal agent in
the development of new drug.
ACKNOWLEDGMENT
The authors would like to thank the Director of Defence
Institute of Bio-Energy Research DRDO, Pithoragarh, for
providing laboratory facilities to carry out research work.
A special thanks to Dr. K.P Singh, Scientist C, for his
guidance and Ms. Seema Singh and Mr. Abhishek, Senior
Research fellow, DRDO, Pithoragarh, for their kind support
and motivation.
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G. lucidum strains DARL-4 and MS-1
Plant extract Zone of inhibition (mm)
DARL-4 (hydroalcohol
extract)
19±0.21
MS-1 (hydroalcohol
extract)
21±0.36
Fluconazole 30±0.03
Control -
G. lucidum: Ganoderma lucidum

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Source of Support: Nil. Conflict of Interest: None declared.