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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];
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
ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15 3
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
Ganoderma lucidum attenuates prostatic hyperplasia A. Nahata and V. K. Dixit
4 ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15
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.
A. Nahata and V. K. Dixit Ganoderma lucidum attenuates prostatic hyperplasia
ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15 5
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.
Ganoderma lucidum attenuates prostatic hyperplasia A. Nahata and V. K. Dixit
6 ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15
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
A. Nahata and V. K. Dixit Ganoderma lucidum attenuates prostatic hyperplasia
ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15 7
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.
Ganoderma lucidum attenuates prostatic hyperplasia A. Nahata and V. K. Dixit
8 ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15
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.
A. Nahata and V. K. Dixit Ganoderma lucidum attenuates prostatic hyperplasia
ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15 9
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.
Ganoderma lucidum attenuates prostatic hyperplasia A. Nahata and V. K. Dixit
10 ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15
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.
A. Nahata and V. K. Dixit Ganoderma lucidum attenuates prostatic hyperplasia
ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15 11
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.
Ganoderma lucidum attenuates prostatic hyperplasia A. Nahata and V. K. Dixit
12 ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15
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.
References
American Herbal Pharmacopoeia (2006) Reishi mushroom
Ganoderma lucidum standards of analysis, quality control,
and therapeutics. In: American Herbal Pharmacopoeia and
Therapeutic Compendium. Anonymous. American Herbal
Pharmacopoeia, Scotts Valley, CA, pp 10–12.
Bartsch G, Rittmaster RS, Klocker H (2000) Dihydrotestoster-
one and the concept of 5a-reductase inhibition in human
benign prostatic hyperplasia. Eur Urol 37:367–380.
Bartsch G, Rittmaster RS, Klocker H (2002) Dihydrotestoster-
one and the concept of 5a -reductase inhibition on human
benign prostatic hyperplasia. World J Urol 19:413–425.
Berges RR, Windeler J, Trampisch HJ, Senge T (1995)
Randomised, placebo-controlled, double-blind clinical trial
of beta-sitosterol in patients with benign prostatic hyperpla-
sia. Beta-sitosterol Study Group. Lancet 345:1529–1532.
Bombardelli E, Morazzoni P, Small JK (1997) Serenoa repens
(13artram). Fitoterapia 68:99–113.
Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem 72:248–254.
Braeckman J (1994) The extract of Serenoa repens in the
treatment of benign prostatic hyperplasia: a multicenter
open study. Curr Ther Res 55:776–785.
Carbajal D, Arruzazabala ML, Mas R, Molina V, Rodriguez E,
Gonzalez V (2004) Effects of D-004, a lipid extract from
Cuban royal Palm fruit, on inhibiting Prostatic hypertrophy
induced with testosterone or dihydrrotestosterone in a rat
model: a randomized, controlled study. Curr Ther Res
65:505–514.
Carson CI, Rittmaster R (2003) The role of dihydrotestoster-
one in benign prostatic hyperplasia. Urology 61:2–7.
Catalona WJ, Smith DS, Wolfert RL, Wang TJ, Rittenhouse
HG, Ratliff TL, Nadler RB (1995) Evaluation of percentage
A. Nahata and V. K. Dixit Ganoderma lucidum attenuates prostatic hyperplasia
ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15 13
of free serum prostate-specific antigen to improve specificity
of prostate cancer screening. JAMA 274:1214–1220.
Deslypere JP, Young M, Wilson JD, McPhaul MJ (1992)
Testosterone and 5 c�-dihydrotestosterone interact differ-
ently with the androgen receptor to enhance transcription of
the MMTV-CAT reporter gene. Mol Cell Endocrinol 88:15–
22.
Dhanotiya R, Chauhan NS, Saraf DK, Dixit VK (2009) Effect
of Citrullus colocynthis Schard. on testosterone-induced
Benign Prostatic hyperplasia. J Complement Integr Med 6:art
29.
Fujita R, Liu J, Shimizu K, Konishi F, Noda K, Kumamoto S,
Ueda C, Tajiri H, Kaneko S, Suimi Y, Kondo R (2005)
Anti-androgenic activities of Ganoderma lucidum.
J Ethnopharmacol 102:107–112.
Giovannucci E, Stampfer MJ, Krithivas K, Brown M, Brufsky
A, Talcott J, Hennekens Ch, Kantoff PW (1997) The
CAG repeat within the androgen receptor gene and its
relationship to prostate cancer. Proc Natl Acad Sci
94:3320–3323.
Hsing AW, Reichardt JK, Stanczyk FZ (2002) Hormones and
prostate cancer: current perspectives and future directions.
Prostate 52:213–235.
Iehle C, Radvanyi F, Medina S, Ouafik LH, Gerard H, Chopin
D, Raynaud JP, Martin PM (1999) Differences in steroid 5a-reductase iso-enzymes expression between normal and
pathological human prostate tissue. J Steroid Biochem Mol
Biol 68:189–195.
Jenkins EP, Hsieh CL, Milatovitch A, Normington K, Berman
DM, Francke U, Russell DW (1991) Characterization and
chromosomal mapping of a human steroid 5a-reductase
gene and pseudogene and mapping of the mouse homolog.
Genomics 11:1102–1112.
Jian JH, Slivova V, Valachovicova T, Harvey K, Sliva D (2004)
Ganoderma lucidum inhibits proliferation and induces
apoptosis in human prostatae cancer cells PC-3. Int J Oncol
24:1093–1099.
Klippel KF, Hiltl DM, Schipp B (1997) A multicentric,
placebo-controlled, double-blind clinical trial of
beta-sitosterol (phytosterol) for the treatment of benign
prostatic hyperplasia. German BPH-Phyto Study group.
Br J Urol 80:427–432.
Liu J, Shimizu K, Konishi F, Noda K, Kumamoto S, Kurashiki
K, Kondo R (2007) Anti-androgenic activities of the
triterpenoids fraction of Ganoderma lucidum. Food Chem
100:1691–1696.
Mahapokai W, Van Sluijs FJ, Schalken JA (2000) Models for
studying benign prostatic hyperplasia. Prostate Cancer
Prostatic Dis 3:28–33.
Martindale (1989) The Extra Pharmacopoeia, 29th edn. In:
Reynolds JEF (ed.). The Pharmaceutical Press, London,
pp 1203
Nandecha C, Nahata A, Dixit VK (2010) Effect of Benincasa
hispida fruits on testosterone induced prostatic hypertrophy
in albino rats. Curr Ther Res Clin Exp 71: 331–343.
Nilsson O, Peter A, Andersson I, Nilsson K, Grundstrom B,
Karlsson B (1997) Antigenic determinants of
prostate-specific antigen (PSA) and development of
assays specific for different forms of PSA. Br J Cancer 75:
789–797.
Noguchi M, Kakuma T, Tomiyasu K, Yamada A, Itoh K,
Konishi F, Kumamoto S, Shimizu K, Kondo R, Matsuoka K
(2008) Randomized clinical trial of an ethanol extract of
Ganoderma lucidum in men with lower urinary tract
symptoms. Asian J Androl 10:777–785.
OECD 423 (2001) OECD Guideline For Testing of Chemicals
Acute Oral Toxicity – Acute Toxic Class Method Adopted:
17th December 2001.
Pandit S, Chauhan NS, Dixit VK (2008) Effect of Cuscuta
reflexa Roxb. on androgen-induced alopecia. J Cosmetic
Dermatol 7:199–204.
Paubert-Braquet M, Richardson FO, Servent-Saez N (1996)
Effect of Serenoa repens extract (Permixon) on estradiol/
testosterone-induced experimental prostate enlargement in
the rat. Pharmacol Res 34:171–179.
Purdon MP, Lehman-McKeeman LD (1997) Improved
high-performance liquid chromatographic procedure for
the separation and quantification of hydroxytestosterone
metabolites. J Pharmacol Toxicol Methods 37:67–73.
Roll R, Hofer-Bosse T, Kayser D (1986) New perspectives
in acute toxicity testing of chemicals. Toxicol Lett Suppl
31:86.
Ross RK, Bernstein L, Pike MC, Henderson BE, Lobo RA,
Stanczyk FZ, Pike M, Henderson B (1992) 5a-reductase
activity and risk of prostate cancer among Japanese and
US white and black males. Lancet 339:887–889.
Russell DW, Wilson JD (1994) Steroid 5a-reductase: two
genes/two enzymes. Annu Rev Clin Biochem 63:25–61.
Steers WD (2001) 5a-Reductase activity in the prostate.
Urology 58:17–24.
Tang W, Liu J, Zhao W, Wei D, Zhong J (2006) Ganoderic
acid T from Ganoderma lucidum mycelia induces
mitochondria mediated apoptosis in lung cancer cells.
Life Sci 80:205–211.
The Merck Index (2006). Encyclopedia of chemicals, drugs,
and biologicals, 14th edn. O’Neil MJ, Heckelman PE, Koch
CB, Roman KJ (eds). Merck Research Laboratories, Merck
& Co., Whitehouse Station, NJ, USA, pp 1475.
Thompson IM, Phyllis J, Goodman PJ, Tangen CM, Scott
Lucia M, Miller GJ, Ford LG, Lieber MM, Cespedes RD,
Atkins JN, Lippman SM, Carlin SM, Ryan A, Szczepanek
CM, Crowley JJ, Coltman CA (2003) The influence of
finasteride on the development of prostate cancer. N Engl
J Med 349:215–224.
Uygur MC, Gur E, Ariki AI, Altug U, Erol D (1998) Erectile
dysfunction following treatments of benign prostatic
hyperplasia: a prospective study. Andrologia 30:5–10.
Wasser SP, Weis AL (1999) Therapeutic effects of substances
occurring in higher Basidiomycetes mushrooms: a modern
perspective. Crit Rev Immunol 19:65–96.
Ganoderma lucidum attenuates prostatic hyperplasia A. Nahata and V. K. Dixit
14 ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15
Wilt TJ, Ishani A, Stark G, MacDonald R, Lau J, Mulrow C
(1998) Saw palmetto extracts for treatment of benign pros-
tatic hyperplasia: a systematic review. JAMA 280:1604–1609.
Wilt TJ, Macdonald R, Ishani A (1999) b-sitosterol for the
treatment of benign prostatic hyperplasia: a systematic
review. BJU Int 83:976–983.
Yun TK (1999) Update from Asia. Asian studies on cancer
chemoprevention. Ann NY Acad Sci 889:157–192.
Zhou ZX, Lane MV, Kemppainen JA (1995) Specificity of
ligand-dependent androgen receptor stabilization: receptor
domain interactions influence ligand dissociation and
receptor stability. Mol Endocrinol 9:208–218.
A. Nahata and V. K. Dixit Ganoderma lucidum attenuates prostatic hyperplasia
ª 2011 Blackwell Verlag GmbH Æ Andrologia xx, 1–15 15