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ORIGINAL ARTICLE

Ganoderma lucidum is an inhibitor of testosterone-inducedprostatic hyperplasia in ratsA. Nahata & V. K. Dixit

Department of Pharmaceutical Sciences, Doctor Hari Singh Gour Vishwavidyalaya, Sagar (M.P.), India

Introduction

Nowadays, androgen-mediated diseases such as prostate

cancer, hirsutism, acne, androgenic alopecia and benign

prostatic hyperplasia (BPH) have become serious prob-

lems (Bartsch et al., 2002). Above all, BPH is one of the

most common ailments seen in older men; 40% of men

50–60 years of age and 90% of men 80–90 years of age

have been diagnosed with BPH. The principal prostatic

androgen is dihydrotestosterone, which is formed by the

steroid enzyme 5a-reductase from its substrate testoster-

one (Russell & Wilson, 1994). 5a-reductase is a mem-

brane-bound NADPH-dependent enzyme that catalyses

the reduction of testosterone to the more potent andro-

gen, dihydrotestosterone. The effect of dihydrotestosterone

is purely androgenic in that, unlike testosterone, it cannot

be transformed into oestrogen. As the weight of the semi-

nal vesicles depends on the 5a-reduced androgens, it is

important to regulate the level of dihydrotestosterone.

Two isoforms of 5a reductase have been cloned, expressed

and characterised (types 1 and 2) that display different tis-

sue expression patterns, enzyme kinetic parameters and

chromosomal localisation (Jenkins et al., 1991). Both iso-

zymes are overexpressed in BPH tissue (Iehle et al., 1999).

Because BPH therapy can reduce dihydrotestosterone

levels by blocking its conversion from testosterone, 5areductase inhibitors could be useful in the treatment

(Bartsch et al., 2000). In rats, the type 1 isozyme predomi-

nates in tissues such as liver, kidney, brain, lung and skin

but also exists in the prostate, whereas the type 2 isozyme

is more abundant in genital tissues such as the prostate.

A number of synthesised 5a-reductase inhibitors with

steroidal moiety have been reported. However, it should

be noted that these inhibitors have the potential to cause

adverse effects such as those reported for finasteride

(Uygur et al., 1998) —i.e., gynecomastia, impairment of

muscle growth and severe myopathy—due to their

structural similarity to steroidal hormones. Hence, the

Keywords

Benign prostatic hyperplasia—Ganoderma

lucidum—prostate-specific antigen—

testosterone—5a-reductase

Correspondence

Prof. Dr Vinod K. Dixit, Department of

Pharmaceutical Sciences, Doctor Hari Singh

Gour Vishwavidyalaya, Sagar (M.P.) 470003,

India.

Tel.: +919425647546;

Fax: +917582264236;

E-mail: [email protected];

[email protected]

Accepted: November 1, 2010

doi: 10.1111/j.1439-0272.2010.1155.x

Summary

The aim of the present study was to find out whether Ganoderma lucidum

(GL) can be used as a clinically effective medicine for the management of pros-

tatic hyperplasia. In vitro studies were conducted to assess the 5a-reductase

inhibitory potential of GL. A biochemical marker viz. b-sitosterol was identified

and characterised in the extracts utilising high-performance thin-layer chro-

matography. Testosterone (3 mg kg)1 s.c.) was administered to the rats along

with the test extracts (10, 20 and 50 mg kg)1 p.o.) and b-sitosterol (10 and

20 mg kg)1 p.o.) for a period of 28 days. Finasteride was used as a positive

control (1 mg kg)1 p.o.). GL extracts attenuated the increase in the prostate/

body weight ratio induced by testosterone. Petroleum ether extract exhibiting

the best activity. Ethanolic extract also exhibited significant activity. The urine

output also improved significantly, which emphasise the clinical implications of

the study. Testosterone levels measured weekly and prostate-specific antigen

(PSA) levels measured at the end of the study also support our claims. The

PSA levels decreased in the extract-treated groups, indicating their usefulness

in the treatment of benign prostatic hyperplasia. Histological studies have

shown a considerable improvement in the prostatic histoarchitecture in the

extract-treated groups when compared to the testosterone-treated group.

ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15 1

emergence of therapeutic materials having fewer side

effects- preferably, edible natural products- would be

highly desirable if their safety could be guaranteed.

Although there is no clear evidence that patients who

develop BPH will ultimately have prostate cancer, andro-

gens do influence the development of prostate cancer

(Ross et al., 1992; Giovannucci et al., 1997; Hsing et al.,

2002). The use of finasteride, the 5a-reductase inhibitor,

can lower the androgen levels in the prostate and reduce

the risk of prostate cancer (Thompson et al., 2003).

For thousands of years, mushrooms have been known

as a source of medicine. The fungi Ganoderma lucidum

(GL) has been used for centuries in East Asia. Its fruiting

body is called as ‘Reishi’ in Japan and ‘Lingzhi’ in China.

In these areas, GL has been a popular folk or oriental

medicine to cure various human diseases such as hepati-

tis, hypertension, hypercholesterolemia, gastric cancer

(Wasser & Weis, 1999; Yun, 1999) and lung cancer (Tang

et al., 2006). However, the precise mechanism and active

compounds of GL against these biological activities have

remained unclear.

Although the inhibitory effects on the proliferation and

migration of prostate cancer cells by GL (Jian et al.,

2004) and 5a-reductase inhibition and suppression of

androgen-induced prostate cell growth by GL (Fujita

et al., 2005; Liu et al., 2007) have been reported, the

clinical implications such as the urine output and the

testosterone levels and prostate-specific antigen levels

(PSA) have not been determined till date. Noguchi et al.

(2008) carried out a randomised, placebo-controlled

study in men with lower urinary tract symptoms (LUTS)

to evaluate safety and efficacy of an extract of GL and

also suggested a need for large-scale evaluation of phyto-

therapy in the treatment of these diseases. We have

demonstrated the basis for the future use of Ganoderma

in therapy by measuring the weekly urine output and tes-

tosterone levels. PSA levels were also measured as it is the

major determinant of diseased state of the prostate. Along

with these parameters, effect of GL extracts on hyperplas-

tic prostate was also tested using testosterone-induced

hyperplasia model. Petroleum ether extract that remained

untouched in the studies undertaken previously by above-

mentioned workers was included in the present study,

and it came out to be the best inhibitor of prostatic

hyperplasia induced by testosterone.

Materials and methods

Materials

Powdered fruiting body of GL was supplied by kind cour-

tesy of Dr. Prabhat Chouhan, Gano Excel Industries,

Kedah Darul Aman, Malaysia.

Preparation of extracts

GL powder was packed in soxhlet extractors and extracted

with petroleum ether (60–80 �C) till complete extraction.

The solvent from the petroleum ether extract (GLP) was

eliminated under reduced pressure (yield- 0.51% w/w).

The defatted marc was extracted with ethanol (95% v/v)

to obtain the ethanolic extract (GLE) (yield- 3.05% w/w).

The marc left after the ethanolic extraction was macerated

with distilled water for 24 h, and the aqueous extract

(GLA) was finally obtained by vacuum-drying (yield-

0.62% w/w).

Drugs and chemicals

Testosterone was obtained as a gift from Sun Pharma

Advanced Research Center (SPARC), Vadodara, Gujarat,

India. Finasteride and b-sitosterol were purchased from

Sigma Aldrich, St Louis, MO, USA. Ethylenediamine

tetraacetic acid (EDTA), sodium phosphate and sucrose

were purchased from HIMEDIA Pvt. Ltd., Mumbai,

India. Methanol, ethyl acetate and petroleum ether (60–

80 �C) were purchased from Qualigens Fine Chemicals

Pvt. Ltd., Mumbai, India. Testosterone ELISA kit was

purchased from United Biotech Inc., Mountain View, CA,

USA, and PSA ELISA kit was purchased from Cusabio

Biotech Co. Ltd., Newark, DE, USA. All other chemicals

used in the study were of analytical grade.

In vitro studies

With a view to explore the possibility that the extracts

may have some action on prostatic hyperplasia, the

extracts were screened for 5a-reductase activity, the key

enzyme involved in hyperplasia of the prostate. The

in vitro studies measured the 5a-reductase inhibitory

potential of the extracts and finasteride by determining

the concentration of testosterone in the reaction mixture

using HPLC (Pandit et al., 2008; Nandecha et al., 2010).

The detailed methodology is described in following

sections.

Preparation of enzyme solution

Human prostate (about 200 mg) procured from the

local hospital of Sagar was minced into small pieces

and homogenised in 10 ml of medium A (20 mm

sodium phosphate, pH 6.5, containing 0.32 m sucrose

and 1 mm EDTA). The homogenate was centrifuged at

716 g for 15 min. The supernatant was used as a

source of the enzyme viz., 5a-reductase. The concentra-

tion of enzyme in the supernatant was determined

by Bradford Method of Protein estimation (Bradford,

1976).

Ganoderma lucidum attenuates prostatic hyperplasia A. Nahata and V. K. Dixit

2 ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15

Preparation of test materials

Testosterone (1 mm solution in ethanol) and extracts

(1 mg ml)1) were prepared in ethanol (95%) with gentle

heating wherever necessary. EDTA solution (10 mg ml)1)

was made in distilled water. Finasteride (10 lg ml)1) was

prepared in ethanol.

Determination of optimum concentration of enzyme

It was determined by keeping the concentration of sub-

strate constant and varying the concentration of enzyme.

Testosterone solution (1 mm) was prepared in ethanol.

Reaction mixture (1 ml) was prepared by adding testos-

terone solution (0.1 ml), enzyme solution (0.1–0.9 ml)

and sodium phosphate buffer (20 mm). The reaction

mixture was incubated at 37 �C for 1 h. The reaction was

terminated by addition of 2 ml of ethyl acetate. The reac-

tion mixture was then shaken vigorously for 1 min, and

the ethyl acetate layer was separated. It was evaporated to

dryness, and the residue dissolved in 2 ml of methanol.

Testosterone content in methanolic solution was esti-

mated by HPLC (AT10; Shimadzu Corp., Kyoto, Japan).

The column was eluted isocratically with a mobile phase

of methanol : water (80 : 20) at a flow rate of

1.0 ml min)1 (Purdon & Lehman-McKeeman, 1997). The

volume of sample injected in the column was 20 ll, and

the wavelength was 245 nm. The optimum amount of

enzyme solution which is required for the conversion of

testosterone to dihydrotestosterone was found to be

0.8 ml.

Determination of inhibitory concentrations of extract

The reaction mixture (1.5 ml) was made by adding

0.1 ml of testosterone solution, 0.1 ml of EDTA solution,

0.1–0.5 ml of extract solutions for separate groups, opti-

mum amount of enzyme solution (i.e., 0.8 ml) and

sodium phosphate (20 mm) to a final volume of 1.5 ml.

Reaction mixture was incubated at 37 �C for 60 min, and

reaction was terminated by addition of 2 ml of ethyl ace-

tate. Rest of the procedure is same as described in the

section above.

Characterisation of extract and selection of marker

The in vitro studies led us to conclude that petroleum

ether and ethanolic extract of GL is having appreciable

inhibitory potential against 5a-reductase. Hence, thin-

layer chromatographic profiling was carried out to find

out the presence of various phytoconstituents. Aqueous

extract being less active was not included in further stud-

ies. GLP and GLE were co-chromatographed with a stan-

dard marker compound viz. b-sitosterol. When visualised

under UV at 254 nm, both the extracts revealed the pres-

ence of b-sitosterol. The solvent system used for GLP was

toluene : ethyl acetate (8 : 2) and that for GLE was chlo-

roform : methanol : toluene (8 : 2 : 1).

High-performance thin-layer chromatographic (HPTLC)

analysis

High-performance thin-layer chromatographic analysis

was performed to develop the characteristic fingerprint

profile for petroleum ether and ethanolic extracts of GL.

GLP and b-sitosterol, a biochemical marker, were dis-

solved in petroleum ether. Ten microlitres of the sample

solutions was applied, and the plate was developed in tol-

uene : ethyl acetate (8 : 2). Developed plates were scanned

densitometrically using a Camag TLC scanner 3 (CAMAG,

Muttenz, Switzerland) at 254 nm and documented. Simi-

larly, GLE was also analysed for its HPTLC profile and the

presence of marker compound. The procedure was the

same as followed in the case of GLP. The solvent system

for GLE was chloroform : methanol : toluene (8 : 2 : 1).

The percentage of b-sitosterol in GLP and GLE was calcu-

lated to be 28.30% and 18.82% respectively.

Moreover, the overlain UV spectra of standard b-sitos-

terol and the b-sitosterol present in the extract were

found to be superimposable on each other confirming the

presence of b-sitosterol in GL extracts.

Further HPTLC studies reported in American Herbal

Pharmacopoeia for Reishi mushroom GL were performed

with our samples to validate their identity. The HPTLC

profile of ethanolic extract in dichloromethane : methanol

(9 : 1) was found to be identical to that reported by the

pharmacopoeial text (American Herbal Pharmacopoeia,

2006).

In vivo studies

The results of the in vitro studies were encouraging as

appreciable 5a-reductase inhibitory activity was found in

the test extracts. Henceforth, to assess their in vivo effects

and to validate the findings of in vitro studies, in vivo

studies were performed.

Animals

Male Sprague-Dawley rats weighing 100–250 g (2–3

months old) were housed in polypropylene cages at room

temperature (25 ± 2 �C) and were fed on standard pellet

diet (Brooke Bond, Lipton, India) and water ad libitum.

The protocol for animal experimentation was approved by

the Institutional Animal Ethics Committee of B. R. Nahata

College of Pharmacy, Contract Research Center, Mandsaur,

Madhya Pradesh, India (Reg. No. 918/ac/05/CPCSEA).

Acute toxicity studies

Acute toxicity studies were performed following OECD

guidelines (OECD, 2001) (OECD 423 Acute Toxic Class

A. Nahata and V. K. Dixit Ganoderma lucidum attenuates prostatic hyperplasia


Method) (Roll et al., 1986). In all cases, 2000 mg kg)1

oral dose of the test extract was found to be safe as no

mortality was observed during the study. On the basis of

these studies, the doses of 10, 20 and 50 mg kg)1 p.o.

were selected for GLP and GLE.

Preparation of extracts

GLP and GLE were suspended in tween-80 solution

(0.2% v/v) for oral administration. Rats were given an

oral dose of 10, 20 and 50 mg kg)1 p.o. once daily for

28 days (Carbajal et al., 2004). Testosterone was dissolved

in arachis oil for s.c. injection (3 mg kg)1 s.c.). Finaste-

ride was suspended in tween-80 (0.2% v/v) and adminis-

tered per orally (1 mg kg)1 p.o.). b-Sitosterol was also

included in the study in doses of 10 and 20 mg kg)1 p.o.

and was suspended in tween-80 (0.2% v/v) for p.o.

administration.

Experimental design

Eleven groups containing six rats per group were created

for this study. Hyperplasia was induced by subcutaneous

administration of testosterone (3 mg kg)1) for 28 days in

all the groups except the vehicle-treated group. Rats were

treated with vehicle [tween-80 (0.2% v/v p.o.)] or finaste-

ride (1 mg kg)1, p.o.), GLP (10, 20 or 50 mg kg)1, p.o.),

GLE (10, 20 or 50 mg kg)1, p.o.) or b-sitosterol (10 or

20 mg kg)1, p.o.) before administration of arachis oil

(s.c.) or testosterone (3 mg kg)1 s.c).

Body and prostatic weights

Body weights were taken a day before the starting of the

treatment (baseline) and on the completion of the study

i.e. on 28th day of treatment. On day 29, animals were

anesthetised under light ether anesthesia and sacrificed.

The prostates were immediately dissected out and

weighed. Mean body weights and prostatic/body weight

ratios were calculated for each group.

Measurement of urine output

The urine output of individual animals were monitored

at the beginning of the study i.e. on day 0 and thereafter

weekly till the completion of the study i.e. 28th day. Met-

abolic cages were used for the purpose of urine collection.

Animals were kept for 24 h in the cages, and the urine

volume was recorded for each individual animal of each

group. During this period, animals had free access to

food and water.

Measurement of serum testosterone concentration

Testosterone levels of individual animals of each group

were measured weekly using testosterone ELISA kit. After

every 7 days, the effect of the test samples on the serum

testosterone levels was measured using ELISA reader

(BIOLINE BPR08). Blood was collected from the retro-

orbital plexus of the rats and was centrifuged at 2000 g

for 20 min to separate the serum. This serum was tested

for its testosterone content using the procedure supplied

with the kit (UBI MAGIWEL Total Testosterone kit

purchases from United Biotech Inc., Mountain View, CA,

USA). The UBI MAGIWEL testosterone quantitative test

is based on the principle of competitive solid-phase

enzyme immunoassay. The test sample competes with

enzyme-labelled testosterone for a fixed and limited num-

ber of antibody sites on the microtitre wells. In the assay

procedure, the testosterone standard or test serum is

incubated with the testosterone antibody and the testos-

terone–horseradish peroxidase conjugate in the anti-rabbit

IgG-coated well. In this solid-phase system, the antibody-

bound testosterone will remain on the well while

unbound testosterone will be removed by washing. A col-

our is developed when TMB substrate is mixed with the

antibody-bound testosterone–horseradish peroxidase

enzyme conjugate. After a short incubation, the enzyme

reaction is stopped, and the intensity of the colour is

measured with microreader at 450 nm.

Measurement of PSA

Prostate-specific antigen levels were measured for individ-

ual rats of each group to find the extent of hyperplasia

induced in the prostate by testosterone treatment. For

this purpose, PSA ELISA kit was utilised. The PSA ELISA

kit is intended for the quantitative determination of total

PSA. This kit was obtained from Cusabio Biotech Co.

Ltd. PSA was quantified by the method of Nilsson et al.

(1997). The PSA ELISA is a solid-phase, noncompetitive

immunoassay based upon the direct sandwich technique.

Calibrators, controls and samples were incubated together

with biotinylated anti-PSA monoclonal antibody and

horseradish peroxidase (HRP)-labelled anti-PSA monoclo-

nal antibody in streptavidin-coated microtitre stripes.

After washing, buffered substrate (TMB-HRP substrate)

that contains hydrogen peroxide and chromogen reagent

(3, 3¢, 5, 5¢ tetra methyl benzidine) was added to each

well, and the enzyme reaction was allowed to proceed.

The colour intensity was determined in the microtitre

plate spectrophotometer at 620 nm. Calibration curves

were constructed for each assay by plotting absorbance

versus the concentration of each calibrator. The concen-

tration of PSA in samples was then read from the calibra-

tion curve.

Histological studies

After prostatic weight measurements, the tissues were fixed

in 10% formalin (in normal saline). After 24 h, the tissues

were subjected to histological studies using microtome fol-

lowed by haematoxylin and eosin (H&E) staining. The



slides were observed under a microscope (Labovision trin-

ocular microscope) and the images recorded. One of the

authors, Prof. V.K. Dixit, who read the histology speci-

mens, was kept blinded to the treatment groups. The

observations are discussed in the section of histological

examinations later in the manuscript.

Statistical analysis

All results are expressed as mean ± SEM (n = 6). Com-

parisons between groups were performed using the Dun-

nett’s test using Graph pad Prism statistical software

(Graphpad Software Inc., La Jolla, CA, USA). P- and

F-values and degrees of freedom were calculated. P < 0.05

was considered to be statistically significant.

Results

HPTLC and characterisation of marker

Co-chromatography of GL extracts along with b-sitosterol

as a biochemical marker revealed the presence of b-sitosterol in

the extracts, with an Rf value of 0.95 for GLP (toluene : ethyl

acetate/8 : 2) and 0.93 for GLE (chloroform : methanol :

toluene/8 : 2 : 1) when analysed at 254 nm (Figs 1 and 2).

Overlain UV spectral interpretation further confirmed the

presence of b-sitosterol in the extracts (Fig. 3).

In vitro studies

The optimum concentration of the enzyme was found to

be 0.8 ml (270.0 lg protein calculated by Bradford

Fig. 1 High-performance thin-layer

chromatographic profile of petroleum ether

extract (GLP) and standard b-sitosterol in

toluene : ethyl acetate (8 : 2) at 254 nm.



method of protein estimation) (Bradford, 1976). Varying

concentrations of test samples were incubated with a

constant amount of testosterone and enzyme in reaction

mixture, and the residual testosterone content was deter-

mined after termination of reaction with ethyl acetate.

The residual testosterone content in reaction mixture

increased with increasing concentrations of GLP, GLE,

GLA and finasteride. The IC50 value i.e., the concentra-

tion of test compound required for 50% inhibition of the

control conversion of 1 mm testosterone, was calculated

by regression analysis. The IC50 values calculated for

GLP, GLE, GLA and standard 5a-reductase inhibitor i.e.

finasteride were 0.164 mg, 0.01 mg, 0.29 mg and 1.06 lg

respectively. Although GLE and GLP extracts showed

good activity, the activity of finasteride is about 10 times

greater. Relative inhibitory potency of the test material is

thus Finasteride > GLE > GLP > GLA (Table 1).

In vivo studies

The results of the in vitro studies paved the way for the

pharmacological screening of the extracts to evaluate their

potential against testosterone-induced hyperplasia in rats.

Results obtained are discussed in the following sections

Determination of body weight, prostatic weight and

prostate/body weight (P/BW) ratio of test groups

In testosterone-treated group, mean body weight and mean

prostatic weight showed a considerable increase after

28 days of treatment. In case of vehicle-treated group, no

Fig. 2 High-performance thin-layer

chromatographic profile of ethanolic extract

(GLE) and standard b-sitosterol in chloro-

form : methanol : toluene (8 : 2 : 1) at

254 nm.



appreciable increase in body weight was observed as noted

in the Table 2. While in case of finasteride-treated group, a

decrease was observed. Table 2 summarises the effects of

GLP (10, 20 and 50 mg kg)1 p.o.), GLE (10, 20 and

50 mg kg)1 p.o.) and finasteride (1 mg kg)1 p.o.) on pros-

tatic hyperplasia induced with testosterone. The P/BW

ratio calculated in case of vehicle-treated group was

1.47 ± 0.15. It was 7.27 ± 0.38 for testosterone-treated

negative control group and 2.62 ± 0.27 for finasteride-

treated positive control group. In case of GLP, P/BW ratios

were 4.59 ± 0.34, 3.30 ± 0.31 and 3.12 ± 0.30 for 10, 20

and 50 mg kg)1 doses respectively. Similarly in case of

GLE-treated groups, the ratios were 5.12 ± 0.44,

3.79 ± 0.16 and 3.27 ± 0.21 for 10, 20 and 50 mg kg)1

doses respectively. Similarly, b-sitosterol-treated groups

showed a P/BW ratio of 4.31 ± 0.15 and 4.18 ± 0.16 for 10

and 20 mg kg)1 dose respectively. Most of the values were

significant when compared to testosterone-treated group

and finasteride-treated groups. On the basis of mean

prostatic weights and P/BW ratios, we also calculated the %

recovery in P/BW ratio by test groups when compared

to testosterone-treated group. The increase induced by

testosterone was considered 100%, and all other test groups

were compared with this reading taken as control. The

reduction in weight induced by test extracts was compared

with testosterone-treated group. The formula used for

calculation of % recovery was as follows:

% Recovery by the test sample = A ) B

Where A = % increase in prostatic weight induced by

testosterone (considered 100%)

B = % increase in prostatic weight induced by test

sample

The % recoveries thus calculated for GLP-treated group

at doses of 10, 20 and 50 mg kg)1 were 46.10%, 68.35% and

71.57% respectively. In case of GLE, these recoveries were

36.99%, 59.94% and 68.94% for 10, 20 and 50 mg kg)1

respectively. b-Sitosterol-treated groups showed a recovery

of 51.07 (10 mg kg)1) and 53.31% (20 mg kg)1). Recovery

with standard finasteride (1 mg kg)1) was 80.10% (Table 2).

Thus, GLP proved to be better than GLE in this aspect.

Measurement of urine output

The urethra is pressed by the overgrowth of the prostate,

which results in the obstruction in urine flow. The mean

urine output was measured to denote the clinical implica-

tions of the study as urine flow is seriously obstructed in

case of hyperplasia of the prostate. In case of vehicle-

treated control group, there was practically no change in

urine output. As the hyperplasia progressed with testos-

terone treatment, the urine output was reduced, and

output was drastically reduced after 28 days of treatment

in testosterone-treated control group. When GL extracts

were administered along with testosterone, significant

improvement in urine output over testosterone-treated

control group were observed. The % obstruction in urine

Fig. 3 Overlain UV spectra of standard

b-sitosterol with b-sitosterol present in the

extract using high-performance thin-layer

chromatographic.

Table 1 5a-reductase inhibitory concentrations (IC-50) of treated

groups

S. No Group IC 50

1 Petroleum ether extract 0.164 mg

2 Ethanolic Extract 0.01 mg

3 Aqueous Extract 0.29 mg

4 Finasteride 1.06 lg



output for GLP-treated groups in doses of 10, 20 and

50 mg kg)1 were 3.94, 9.60 and 1.47% respectively as

against 77.15% in testosterone-treated group. Similarly,

when GLE was administered in graded doses of 10, 20

and 50 mg kg)1 along with testosterone, significant

improvement in urine output was recorded when com-

pared to testosterone-treated group. The % obstruction in

urine output for GLE-treated groups was 3.23, 3.46 and

0.48% respectively for doses of 10, 20 and 50 mg kg)1. In

b-sitosterol-treated group, the % obstruction recorded

was 7.53% (10 mg kg)1 p.o.) and 11.42% (20 mg kg)1

p.o.). In finasteride-treated positive control group, practi-

cally no obstruction in urinary output was recorded. The

relative efficacy of the extracts in reducing the obstruction

caused by testosterone can be stated in the following

order:

GLE50 > Finasteride > GLP50 > GLE10 > GLE20 >

GLP10 > GLP20 > b-sitosterol (10) > b-sitosterol (20).

The results are depicted in Table 3.

Measurement of serum testosterone concentration

The 5a-reductase inhibitory activity found in the extracts

during in vitro studies was validated during in vivo studies

by measuring serum testosterone concentration of various

groups. The extracts as well as finasteride treatment

increased the testosterone levels in the serum of the test

animals as seen by the levels determined by using testoster-

one ELISA kit. In normal vehicle-treated group, the levels

were unchanged during the study as measured on 0, 7, 14,

21 and 28th day of the study. Testosterone-treated group

showed a decrease in these levels as the study progressed,

and this decrease continued till the end of the study, which

implicates the activity of the enzyme (5a-reductase) in

the prostate of testosterone-treated animals. Treatment of

animals with exogenous testosterone caused its elevation in

serum. Simultaneous administration of extracts and

b-sitosterol along with testosterone led to further increase

in serum testosterone levels suggesting inhibition of

5a-reductase activity of the extracts and b-sitosterol. The

results are depicted in Fig. 4.

Measurement of prostate-specific antigen

PSA serum levels are abnormally elevated in patients with

prostate cancer, BPH and patients with prostate inflamma-

tory conditions (Catalona et al., 1995). The effect of the

administration of test extracts and finasteride along with

Table 2 Effect of test extracts of Ganoderma lucidum and b-sitosterol on prostatic weight

Treatment

(mg kg)1)

Body weight (g)

Prostatic

weight (mg)

Prostatic/body

weight ratio

(P/BW Ratio)

Treatment

effect on

P/BW (P2–P1)

% Increase

in prostate

weight

%

RecoveryaDay 0 Day 28

Blank control

(vehicle only)

110.75 ± 9.21 117.50 ± 14.93 170.15 ± 22.08 1.47 ± 0.15 0 0 0

Testosterone (T)

(3 mg kg)1 s.c.)

113.75 ± 36.36 138.75 ± 38.31 1028.41 ± 21.83 7.27 ± 0.38b 5.80 100 0

T + finasteride

(1 mg kg)1 p.o.)

127.50 ± 12.50 126.25 ± 7.46 337.67 ± 56.19 2.62 ± 0.27c 1.15 19.90 80.10

GLP 10 + T 165.00 ± 36.17 170.00 ± 33.41 560.30 ± 155.62 4.59 ± 0.34b,c 3.12 53.90 46.10

GLP 20 + T 95.00 ± 9.12 120.00 ± 8.165 545.45 ± 19.47 3.30 ± 0.31c 1.83 31.65 68.35

GLP 50 + T 106.50 ± 7.50 110.00 ± 4.08 366.32 ± 43.76 3.12 ± 0.30c 1.65 28.43 71.57

GLE 10 + T 87.50 ± 7.50 113.75 ± 7.46 599.98 ± 96.11 5.12 ± 0.44b,c 3.65 63.01 36.99

GLE 20 + T 99.00 ± 15.81 115.00 ± 9.57 429.42 ± 16.25 3.79 ± 0.16c,d 2.32 40.06 59.94

GLE 50 + T 102.50 ± 25.94 120.00 ± 23.45 408.30 ± 112.08 3.27 ± 0.21c 1.80 31.06 68.94

BS 10 + T 95.00 ± 5.00 105.00 ± 5.00 475.95 ± 20.45 4.31 ± 0.15b,c 2.84 48.93 51.07

BS 20 + T 105.00 ± 5.00 110.00 ± 0.00 424.50 ± 15.82 4.18 ± 0.16b,c 2.71 46.69 53.31

P < 0.0001

F = 20.387

Values are mean ± SEM (n = 6) anova followed by Dunnett’s test. Df = 8, 103.

GLP 10, GLP 20, GLP 50 – Petroleum ether extract of G. lucidum (10, 20 and 50 mg kg)1 p.o. respectively).

GLE 10, GLE 20, GLE 50 – Ethanolic extract of G. lucidum (10, 20 and 50 mg kg)1 p.o. respectively).

BS 10, BS 20 – b-Sitosterol (10 and 20 mg kg)1 p.o. respectively).a% Recovery in P/BW ratio by test groups when compared to testosterone-treated group.bP < 0.01 versus finasteride-treated group.cP < 0.01 versus testosterone-treated group (Dunnett’s test).dP < 0.05 versus finasteride-treated group.

P1 = P/BW ratio of blank group, P2 = P/BW ratio of test group.



testosterone on the PSA level in rats is an indication of the

hypertrophy of the prostate induced by testosterone. This

parameter was measured in the serum of the test animals of

various groups using PSA ELISA kit following the proce-

dure supplied with the kit. The normal PSA level in vehi-

cle-treated group was found to be 0.140 ± 0.087 lg l)1.

This level increased to 1.533 ± 0.731 lg l)1 in testosterone-

treated group. Finasteride-treated group showed a decrease

in PSA level to 0.900 ± 0.378 lg l)1 (P < 0.05 compared to

testosterone-treated group). GLP in doses of 10, 20 and

50 mg kg)1 p.o. showed levels of 0.896 ± 0.558 (P < 0.05),

0.507 ± 0.338 (P < 0.05) and 0.200 ± 0.002 (P < 0.01) lg l)1

respectively, which indicate the protective effects of GLP on

testosterone-induced hyperplasia. Decrease in PSA levels

was also observed with GLE treatment, which exhibited

levels of 1.395 ± 1.305 (P < 0.05), 0.782 ± 0.452 (P <

0.05) and 0.210 ± 0.021 (P < 0.01) lg l)1 for 10, 20 and

50 mg kg)1 respectively. The decreases observed were sig-

nificant compared to testosterone-treated group. These

observations indicate that GLP was more effective than

Table 3 Mean urine output of various groups measured weekly using metabolic cage

Treatment (mg kg)1)

Urine output (ml)Obstruction in

urine output (%)

(U0–U28)/U0 · 100Day 0 Day 7 Day 14 Day 21 Day 28

Blank (vehicle only) 1.02 ± 0.05 1.05 ± 0.06 1.10 ± 0.04 1.05 ± 0.09a 1.02 ± 0.02a –

Testosterone (3 mg kg)1 s.c.) 0.98 ± 0.09 0.85 ± 0.06 0.75 ± 0.08 0.37 ± 0.08b 0.22 ± 0.02b 77.15

Finasteride (1 mg kg)1 p.o.) + T 1.01 ± 0.07 0.97 ± 0.01 0.950 ± 0.02 0.970 ± 0.07a 1.000 ± 0.04a 0.99

GLP 10 + T 1.01 ± 0.07 1.00 ± 0.05 0.77 ± 0.06 0.82 ± 0.02b 0.97 ± 0.04a 3.94

GLP 20 + T 0.88 ± 0.25 0.85 ± 0.02 0.60 ± 0.09 0.67 ± 0.04b 0.80 ± 0.05a 9.60

GLP 50 + T 1.01 ± 0.05 1.00 ± 0.19 0.82 ± 0.10 0.95 ± 0.06b 1.00 ± 0.14a 1.47

GLE 10 + T 1.02 ± 0.08 1.01 ± 0.40 1.07 ± 0.11 0.97 ± 0.11c 0.98 ± 0.04a 3.23

GLE 20 + T 1.01 ± 0.02 1.00 ± 0.08 0.98 ± 0.04 0.92 ± 0.06b 0.97 ± 0.04a 3.46

GLE 50 + T 1.03 ± 0.08 1.02 ± 0.02 0.90 ± 0.08 0.95 ± 0.05b 1.02 ± 0.04a 0.97

BS 10 + T 0.99 ± 0.84 0.97 ± 0.12 0.90 ± 0.07 0.60 ± 0.13 0.92 ± 0.09c 7.53

BS 20 + T 0.875 ± 0.10 0.725 ± 0.18 0.62 ± 0.17 0.22 ± 0.07d 0.77 ± 0.11c 11.42

F = 3.318

P < 0.0001

F = 2.989

P = 0.001

F = 7.676

P < 0.0001

F = 7.979

P < 0.0001

U0 – urine output on day 0, U28 – urine output on day 28.

Values are mean ± SEM (n = 6) anova followed by Dunnett’s test. Df = (8, 78) 103.

GLP 10, GLP 20, GLP 50 – petroleum ether extract of Ganoderma lucidum (10, 20 and 50 mg kg)1 p.o. respectively).

GLE 10, GLE 20, GLE 50 – ethanolic extract of G. lucidum (10, 20 and 50 mg kg)1 p.o. respectively).aP < 0.01 compared to testosterone.bP < 0.01 compared to finasteride.cP < 0.05 compared to finasteride.dP < 0.01 compared to testosterone.

Fig. 4 Mean serum testosterone levels of

various groups measured weekly using

testosterone ELISA kit.



GLE in counteracting testosterone-induced hyperplasia.

b-Sitosterol-treated group also showed a decrease in PSA

levels to 0.880 ± 0.325 (P < 0.05) (10 mg kg)1 p.o.) and

0.445 ± 0.126 (P < 0.05) (20 mg kg)1 p.o.). The observa-

tions are depicted in Fig. 5.

Histological examinations

Control group (arachis oil)

Normal histological features of prostate gland are visible

showing the tubules of variable diameter and irregular

lumen. Lumens are filled with prostatic secretions. In

connective tissue, blood vessels and lymph vessels, matrix

is normal. At some places, aggregation of columnar cells

has been observed (Figs 6a and 7a).

Testosterone-treated group (3 mg kg)1 s.c.)

Tubules have become wider when compared to control.

The walls of tubules have thickened, and every tubule

almost has developed large involutions projecting into the

lumen, reducing the volume of the lumen compared to

control. The amount of the secretion in some tubules has

increased. The connective tissue has been compressed,

and blood vessels have dilated compared to control. The

distinct nucleus and normal sarcoplasmic texture is not

visible. Shape of the tubules has become obliterated

(Figs 6b and 7b).

Testosterone + GLP (10, 20 and 50 mg kg)1 p.o.)

Vacuolisation in the cells is clear. Nucleus is picnotic

(vacuole formation in outer layer of nucleus). Chromatin

material in nucleus is normally distributed. Lumen of the

tubules is normal and at some places slightly obliterated.

Involutions are few in number and even less than what

are observed in control. Connective tissue between the

tubules is reduced. Stroma is composed of smooth mus-

cles and connective tissue. A significant improvement

compared to testosterone-treated group can be easily

identified (Figs 6d–f and 7d).

Testosterone + GLE (10, 20 and 50 mg kg)1 p.o.)

With the better intraluminal secretions, tubules have

shown morphological improvement in the texture. Still

the epithelium is wide and thicker. When compared to

testosterone-treated group, the stroma (composed of con-

nective and smooth muscle cells) is normal. The appear-

ance of the transitional epithelium resembles that of

control. Connective tissue has increased appreciably as

the dose is increased, and at some places it resembles the

normal control group. Minor curvature in the epithelium

is also observed. Involutions in the epithelium are fewer

and thick (Figs 6g–i and 7e).

Testosterone + finasteride (1 mg kg)1 p.o.)

Normal distribution of stroma is seen. The projections

are not prominent as seen in the testosterone-treated

group. Although finasteride is antagonising the effects of

testosterone up to a good degree, several cells with their

increased volume are present throughout the transitional

epithelium. Cells with swollen nuclei are prominent at

many places. Reduced involutions in lesser number are

observed (Figs 6c and 7c).

Testosterone + b-sitosterol (10 and 20 mg kg)1 p.o.)

Lumen of the tubules is normal, and at some places

epithelium is slightly thicker than control. Stroma is

normal. The appearance of the transitional epithelium

resembles that of control. Overall, the effects induced by

testosterone are effectively antagonised in these groups

(Figs 6j, k and 7f).

Fig. 5 Mean prostate-specific antigen (PSA)

level of various groups measured using

PSA ELISA kit.



Discussion

A densitometric high-performance TLC analysis was per-

formed to develop the characteristic fingerprint profile for

the petroleum ether and alcoholic extract of GL. This can

be used as a tool for evaluation and standardisation of

the drug.

GL has been used as a remedy for a wide variety of ail-

ments, and its hidden therapeutic potentials are being

explored for various diseased conditions. The present

study is an approach in this direction where we have

identified the possible clinical implications of GL in BPH

and its major clinical symptoms viz. urinary problem,

which is a major cause of discomfort for most of the

patients suffering from this disease.

Testosterone is converted to more potent dihydrotes-

tosterone by the enzyme 5a-reductase present in prostate

homogenates (Steers, 2001; Dhanotiya et al., 2009;

Nandecha et al., in press). During in vitro studies, addi-

tion of GLP, GLE and GLA in reaction mixture showed

increased levels of unchanged testosterone in the reaction

mixture, suggesting inhibition of enzyme action by these

test materials. Furthermore, the inhibition of conversion

by these materials clearly reflects that enzyme activity is

blocked, and therefore more testosterone remains

unchanged in the reaction mixture. It was noted that

finasteride is about 10 times more potent in inhibiting

5a-reductase activity in in vitro studies. As least activity

was shown by GLA, it was not included in the studies

thereafter, and only GLP and GLE were taken up for

in vivo studies.

The results of the present investigations suggest that

GLP and GLE at different dose levels inhibit prostatic

hyperplasia induced with an exogenous supply of testos-

terone in a rat model.

The aetiology of BPH in humans is heterogeneous, and

no other species shows the same complexity of this disor-

der. Animal models of BPH studied to date do not appear

(a)

(e)

(h) (i) (j) (k)

(f) (g)

(b) (c) (d)

Fig. 6 Histopathological observations of the effect of test extracts of Ganoderma lucidum and standard b-sitosterol on testosterone-induced

hyperplasia (·100). L indicates Lumen, S indicates the stromal compartment, and arrows indicate the luminal involutions. (a) Vehicle-treated

control group. (b) Testosterone-treated group (3 mg kg)1 s.c.). (c) Finasteride-treated group 1 mg kg)1 p.o. (d) Petroleum ether extract-treated

group 10 mg kg)1. (e) Petroleum ether extract-treated group 20 mg kg)1. (f) Petroleum ether extract-treated group 50 mg kg)1. (g) Ethanolic

extract-treated group 10 mg kg)1. (h) Ethanolic extract-treated group 20 mg kg)1. (i) Ethanolic extract-treated group 50 mg kg)1. (j) b-Sitosterol-

treated group 10 mg kg)1. (k) b-Sitosterol-treated group 20 mg kg)1.



to fully mimic the stromal and epithelial changes with BPH

in humans (Mahapokai et al., 2000). Therefore, in vitro

and animal models are of limited value for the study of

BPH events. Spontaneous animal models are limited to

nonhuman primates and canines (hormone-induced BPH

in canines appears to be an especially replicate model of

human BPH), but ethical and economic problems have

reduced the applicability of these models. In particular,

BPH induced with testosterone or dihydrotestosterone do

not reproduce all findings of BPH in humans because the

pathogenesis of BPH is dependent on a functional andro-

genic signal involving several components (e.g. testosterone

synthesis in the testes, conversion of testosterone to dihyd-

rotestosterone, transportation of dihydrotestosterone to

target prostate tissues, binding of dihydrotestosterone to

androgenic receptor and the subsequent gene modulation)

(Deslypere et al., 1992; Zhou et al., 1995; Carson & Ritt-

master, 2003). BPH in humans also involves prostatic oes-

trogens and a-adrenergic receptors not fully reproducible

in other models. Thus, the model used in the present study

has limitations in predicting the effects of any treatment on

the management of BPH in humans.

Nevertheless, the effects of testosterone and dihydrotes-

tosterone on prostatic growth in rodents have previously

been documented and used to assess the effects of drugs

used for prostatic hyperplasia treatment, including saw

palmetto fruit lipid extract. (Paubert-Braquet et al., 1996;

Bombardelli et al., 1997). In the present study, in rats

administered with GLP and GLE along with testosterone,

the increase in prostatic weight and P/BW ratio were

attenuated after 28 days of oral treatment at different

doses when compared to testosterone-treated negative

control group. The weekly measurement of testosterone

concentration in serum also supports our findings. The

levels of testosterone are increased significantly after

14 days, and this increase is a result of the inhibition of

the enzyme 5a-reductase by the test extracts, the enzyme

being responsible for the conversion of testosterone to

dihydrotestosterone which is more potent than testoster-

one in causing inflammation of the prostate. Our extracts

proved to be inhibitors of 5a-reductase and hence

retained less harmful testosterone in the body.

Obstruction of urinary discharge is the major patho-

logical problem of clinical significance in BPH patients. It

was found that urine output was decreased drastically in

testosterone-treated group. Significant increase is observed

in urine output in extract- and finasteride-treated groups.

In most of the cases, urine output returned to the normal

values as on day 0 of the study. Obstruction in urine out-

put shown by testosterone-treated group was significantly

greater than extract and finasteride-treated groups. The

extracts GLP as well as GLE, finasteride and b-sitosterol

exhibited a significant improvement in urine output com-

pared to the testosterone-treated group. This finding is

significant as prostatic hyperplasia directly causes urinary

problems like painful micturition, reduced urine flow,

urinary urgency, etc. The results of the study indicate

possible use of the drug in stated conditions.

Further, PSA levels were measured at the end of the

study i.e. on 28th day. PSA is a protein produced by the

cells of the prostate gland. The PSA test measures the

level of PSA in the blood. PSA serum levels are abnor-

(a) (b) (c)

(d) (e) (f)

Fig. 7 Histopathological observations of the effect of test extracts of Ganoderma lucidum and standard b-sitosterol on testosterone-induced

hyperplasia (·400). L indicates Lumen, S indicates the stromal compartment, and arrows indicate the luminal involutions. (a) Vehicle-treated con-

trol group. (b) Testosterone-treated group (3 mg kg)1 s.c.). (c) Finasteride-treated group 1 mg kg)1 p.o. (d) Petroleum ether extract-treated group

50 mg kg)1. (e) Ethanolic extract-treated group 50 mg kg)1. (f) b-Sitosterol-treated group 20 mg kg)1.



mally elevated in patients with prostate cancer, BPH and

patients with prostate inflammatory conditions. If a

decrease in PSA levels is observed, it can be assessed that

the test sample in question is having protective effects on

the inflammatory conditions and hypertrophy of the

prostate induced by testosterone. Testosterone treatment

increased the PSA levels, which is an indication of hyper-

plasia, whereas finasteride reduced the PSA levels signifi-

cantly suggesting its protective effects.

Both the extracts and b-sitosterol significantly reduced

the PSA levels which are an indication of their 5a reduc-

tase activity and efficacy in the treatment of prostatic

hyperplasia.

The results of the study suggest that GLP prevented

prostatic hyperplasia significantly in a dose-dependent

manner with 50 mg kg)1 showing the best activity. GLE

also showed significant activity in a dose-dependent man-

ner. The in vitro studies cleared the mechanism of pre-

vention of prostatic hyperplasia induced by testosterone.

It is evident that GL extracts have 5a reductase inhibitory

activity. The weekly serum testosterone levels are sugges-

tive of the mechanism of action of the extracts and finas-

teride. The recoveries in urine output also suggest that

the extracts have a positive effect on hypertrophy of the

prostate. As b-sitosterol is a well-known molecule estab-

lished for the treatment of BPH (Martindale, 1989; The

Merck Index, 2006), the presence of b-sitosterol as a

major constituent in the extracts further supports our

observations. A number of clinical studies undertaken by

different scientific groups have supported clinical efficacy

of b-sitosterol in prostate disorders (Braeckman, 1994;

Berges et al., 1995; Klippel et al., 1997; Wilt et al., 1998;

Wilt et al., 1999). Our studies with b-sitosterol are indica-

tive of its protective effects on testosterone-induced

hyperplasia. The histological findings have shown the

recovery in the prostatic histoarchitecture particularly in

the cuboidal epithelial cells, intracellular luman, tubular

latency and shape which further support to make GL a

strong candidate for the management of prostatic hyper-

plasia. Further studies are necessitated to confirm the

effect of the drug on BPH in humans. The study is the

first attempt to analyse the effects of petroleum ether

extract of GL, and the attempt proved successful as GLP

showed better results than GLE in our experiments. The

study shows that GL holds sufficient promise to be used

as a drug for the prevention of BPH and is a firm

candidate for further clinical research in this area. It is

suggested, on the basis of these studies, to further under-

take clinical trails on GL to develop an effective nutraceu-

tical for the treatment of BPH.

The overall results clearly reflect the utility of extract in

BPH. Because of conversion of testosterone to dihydrotes-

tosterone, the prostate size is increased, thereby causing

obstruction in urinary output. The observed effect that

extracts do not allow the increase as reflected by urinary

output, P/BW ratios and histoarchitecture showed that

activity of administered testosterone was blocked by the

extract and resulted in recovery.

Acknowledgements

One of the authors, Alok Nahata, is grateful to All India

Council of Technical Education (AICTE), New Delhi for

providing National Doctoral Fellowship. Thanks are due

to Dr Prabhat Chouhan, Gano Excel Industries, Malaysia,

for generously providing powdered fruiting body of GL.

Sincere thanks go to the Director, B.R. Nahata Smriti

Sansthan, Contract Research Center, Mandsaur (M.P.),

for granting permission to carry out the in vivo studies.

Thanks are due to Sun Pharma Advanced Research Cen-

ter, Vadodara, India, for providing a gift of testosterone.

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