Anti-Androgen Effects of Extracts and Compounds from Ganoderma lucidum

Anti-Androgen Effects of Extracts and Compounds from Ganodermalucidum

by Jie Liua), Sadaaki Tamuraa), Kenji Kurashikia), Kuniyoshi Shimizua), Kiyoshi Nodab), FumikoKonishib), Shoichiro Kumamotob), and Ryuichiro Kondo*a)

a) Department of Forest and Forest Products Science, Faculty of Agriculture, Kyushu University,Fukuoka, 812-8581, Japan (phone: þ81-92-6422811; fax: þ81-92-6422811;

e-mail: [email protected])b) Research Laboratories, Chlorella Industry Co., Ltd., Fukuoka 833-0056, Japan

The 30% EtOH extracts of Ganoderma lucidum Fr. Karst (Ganodermateceae) showed weak 5a-reductase inhibitory activity and binding ability to androgen receptor. When LNCaP (lymph-nodecarcinoma of the prostate) cells were treated with the EtOH extracts, cell proliferation was inhibited.Treatment with the extracts significantly inhibited the testosterone-induced growth of the ventralprostate in castrated rats. These results showed that G. lucidum might be a useful ingredient in thetreatment of androgen-induced diseases, such as benign prostatic hyperplasia or prostate cancer. Fromthe 30% EtOH extracts, we isolated ganoderiol F, which showed binding activity to androgen receptorand inhibited LNCaP cell proliferation, as one of the active compounds in the 30% EtOH extracts.

Introduction. – Androgen-mediated diseases such as prostate cancer, hirsutism,acne, androgenic alopecia, and benign prostatic hyperplasia (BPH) have becomeserious problems among the modern population [1]. Prostate cancer is one of the mostfrequently diagnosed malignancies and is the second leading cause of cancer death inAmerican men. BPH, in particular, is one of the most common ailments seen in oldermen; 40% of men 50 to 60 years of age, and 90% of men 80 to 90 years of age have beendiagnosed with BPH. The principal prostatic androgen is dihydrotestosterone (DHT),which is formed by the steroid enzyme 5a-reductase from its substrate testosterone [2].In several androgen target tissues, like the prostate, testosterone is converted to DHT,which is the most potent natural androgen. This process amplifies the androgenicresponse, perhaps because of the higher affinity of the androgen receptor (AR) forDHT than for testosterone [3]. Both 5a-reductase and DHT perform critical roles,physiologically and pathologically, in man. The plasma level of DHT has been reportedto be elevated in patients with either BPH or prostatic cancer.

The AR is a transcriptional activator that, in a multistep process, transducesextracellular signals to target tissues. The AR regulates transcription in response toandrogens, and has a key role in the regulation of prostate growth and the maintenanceof prostatic function [4]. It is classically understood that, after ligand binding (mainlyDHT), the ligand– AR complex with associated proteins translocates into the nucleus,binds to the consensus sequence of androgen response elements [5], and regulates theexpression of androgen-responsive genes (ARGs) [6]. Prostate-specific antigen (PSA)

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� 2009 Verlag Helvetica Chimica Acta AG, Z�rich

is an ARG known to be under the control of the AR, and is a well-accepted marker forthe diagnosis and prognosis of prostate cancer. Amplification of the AR can lead to thedevelopment of prostatic diseases or androgen-refractory prostate cancer. Therefore,the first step in the process of transcriptional regulation is the binding of DHT to theAR. The androgen antagonist can suppress DHT-induced prostate regrowth.

The inhibition of 5a-reductase and AR has become a pharmacological strategy forthe treatment of BPH and prostate cancer as well as other DHT-related disorders suchas acne and male pattern baldness [7].

For thousands of years, mushrooms have been known as a source of medicine. InEast Asia, the fruiting body of the fungus Ganoderma lucidum has been usedmedicinally for centuries. It has long been used as a folk medicine to treat varioushuman diseases such as cancer, hypertension, hepatitis, nephritis, and others [8].Although the inhibitory effects on the proliferation and migration of prostate cancercells by hot H2O extracts of G. lucidum [9] have been reported, AR binding activity andsuppression of androgen-induced prostate cell growth by 30% EtOH extracts of G.lucidum have never been reported. We reported that 99.5% EtOH extracts of G.lucidum showed the anti-androgen effects, which came from 5a-reductase inhibitoryactivity [10]. The EtOH extracts of G. lucidum also suppressed the testosterone-induced prostate regrowth in animal experiment. Until now, the safety of 99.5% EtOHextracts of G. lucidum had not been guaranteed. G. lucidum is a popular medicinalmushroom, and its dried powder and liquor are currently used worldwide in the form ofdietary supplements. As the alcohol content in the liquor is ca. 30%, we changed thesolvent for extraction from the 99.5% EtOH to 30% EtOH to insure the safety and theeffectiveness of the EtOH extracts of G. lucidum. In this study, we isolated fivecompounds, 1 – 5. They are identified as ganoderic acid A (1) [11], ganoderic acid F (2)[12], ganodermanontriol (3) [13], ganoderiol A (4) [13], and ganoderiol F (5) [14](Fig. 1). We also demonstrated the in vitro and in vivo anti-androgenic activity of 30%EtOH extracts of G. lucidum.

Results and Discussion. – 5a-Reductase Inhibitory Activity of 30% EtOH Extractsof G. lucidum. In our previous screening of 19 edible and medicinal mushrooms, wediscovered that the fruiting body of G. lucidum showed higher suppressing effect ontestosterone-induced prostate regrowth [10]. The 99.5% EtOH extracts of G. lucidumalso showed higher 5a-reductase inhibitory activity [15]. The fruiting body of G.lucidum showed a very strong possibility to be used as health supplement. To use the G.lucidum as a health supplement, its safety must be insured. As there are no records onedible 99.5% EtOH extracts of fruiting body of G. lucidum, the safety of 99.5% EtOHextracts cannot be insured. On the other hand, the liquor of the fruiting body of G.lucidum has been used in China for a long time. As the EtOH in the liquor is ca. 30%,we changed the solvent for extraction from the 99.5% EtOH to 30% EtOH to insurethe safety and the effectiveness of the EtOH extracts of G. lucidum. With changing thesolvent, the components of the extracts were also changed. In this situation, thecomponents of 30% EtOH extracts include more hydrophilic compounds than those of99.5% EtOH extracts. The changing of compounds in extracts will also affect thebioactivity. These results led us to further investigate the 30% EtOH extracts of G.lucidum. The inhibitory concentration leading to 50% activity loss (IC50) was estimated

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to be 3000 mg/ml (Table 1). It should be noted that finasteride [16], which is known as apotent steroidal inhibitor, showed an IC50 value of 0.73 mm in our assay system.

Androgen-Receptor-Binding Activity of 30% EtOH Extracts of G. lucidum. We alsoexamined the androgen-receptor-binding activity. The binding-active concentrationleading to 50% fluorescence polarization loss (IC50) was estimated to be 49 mg/ml for30% EtOH extracts of G. lucidum (Table 2). It should be noted that DHT used as apositive control showed an IC50 value of 20 nm in our assay system.

Table 1. The Inhibitory Effect of 30% EtOH Extracts, CHCl3, BuOH, H2O Fraction, and Compounds 1–5 on 5a-Reductase

5a-Reductase inhibitory activity [%]

Compound/Fraction 667 mm 333 mm 200 mg/ml IC50

30% EtOH Extract 3000 mg/mlCHCl3 Fraction 45BuOH Fraction 22H2O Fraction 19Ganoderic acid A (1) 22Ganoderic acid F (2) 18Ganodermanontriol (3) 32 21Ganoderiol A (4) 6Ganoderiol F (5) 34 23Finasteride (positive control) 0.73 mm


Fig. 1. The chemical structures of the isolated compounds 1–5

The Inhibitory Effect of 30% EtOH Extracts of G. lucidum on the Prostate CancerCell. The LNCaP (lymph-node carcinoma of the prostate) human prostate cancer cellline is a well-established and androgen-dependent cell line [17]. LNCaP Cells retainmost of the characteristics of human prostatic carcinoma, like the dependence onandrogens, the presence of ARs, and the production of acid phosphatase and PSA [18].For these reasons, the LNCaP cell line becomes an attractive model for in vitro studieson the biology of human prostate cancer [19].

The effect of the 30% EtOH extracts on a prostate cancer cell is shown in Fig. 2.LNCaP Cells were incubated with varying concentrations of the 30% EtOH extracts(from 10 to 200 mg/ml) with or without testosterone or DHT for 3 d. The NR (¼ 3-amino-7-(dimethylamino)-2-methylphenazine) assay was performed to measure cellviability. In the absence of 30% EtOH extracts, testosterone alone apparentlystimulates the LNCaP cells to ca. 150% proliferation on average above the numberof cells in an untreated control, and DHT alone apparently stimulates the LNCaP cellnumber to increase by ca. 200% on average above the number of cells in an untreatedcontrol. The cell cytotoxic effect was caused by 30% EtOH extracts from 50 to 200 mg/ml when cultured without testosterone or DHT. Treating LNCaP cells with the 30%EtOH extracts in the presence of testosterone or DHT resulted in a dose-dependentinhibition of cell growth, which suggested that the inhibition of cell growth in thepresence of testosterone or DHT may come from the anti-androgen effect such as 5a-reductase inhibition and the binding to AR, but we cannot deny the cell cytotoxic effectover 50 mg/ml.

Growth Suppression of Rat Prostate by Administration of 30% EtOH Extracts of G.lucidum. Based on the results of 5a-reductase inhibitory activity, the AR-bindingexperiment, and the cell-proliferation inhibition experiment, we concluded that the30% EtOH extracts may inhibit the androgen-induced growth of prostate cancer cellsin vitro by inhibiting the 5a-reductase activity and binding to the AR. It still cannot bestated with certainty that the 30% EtOH extracts suppresses the growth of the ventralprostate, because some problems remain such as the absorption to the blood,metabolism in the blood, penetration through the cell membrane, etc. To determinewhether the 30% EtOH extracts suppresses ventral prostate growth, we designed an

Table 2. The IC50 Values of the Binding Activity of 30% EtOH Extracts, CHCl3, BuOH, H2O Fraction,and Compounds 1–5 on Androgen Receptor. n¼3.

IC50 [mg/ml] IC50 [mm]

30% EtOH Extract 49CHCl3 Fraction 73BuOH Fraction 40H2O Fraction 174Ganoderic acid A (1) 350Ganoderic acid F (2) 322Ganodermanontriol (3) >400Ganoderiol A (4) 470Ganoderiol F (5) 25DHT 0.02


experiment as follows. In the rats that received testosterone, administration of the 30%EtOH extracts reduced the increased weight of the ventral prostate (Fig. 3). Four daysafter castration, the weights of the rat prostates were markedly reduced, and the


Fig. 2. The inhibition effect of the 30% EtOH extracts of G. lucidum on the LNCaP cell growth. n¼3. T¼Testosterone, DHT¼dihydrotestosterone, *: P<0.05 against control, T, or DHT, **: P<0.01 against

control, T, or DHT.

Fig. 3. Effects of the fractions prepared from 30% EtOH extracts of G. lucidum on testosterone-inducedregrowth of the castrated rat prostrate. Each column represents the mean�S.D., n¼6. C¼Castrated rat,T¼ testosterone, F¼ flutamid (1 mg/kg of body weight), G¼30% EtOH extracts of G. lucidum, *: P<

0.05 against CþT, **: P<0.01 against CþT.

prostate size was recovered by sc injections of testosterone. Administration of the 30%EtOH extracts at the concentration of 1 and 0.1 mg/kg/d showed 26 and 36% prostategrowth inhibition, respectively. This result suggested that the growth suppression of theventral prostate was at least partially the result of the anti-androgen effects, i.e., 5a-reductase inhibition and the binding to the AR.

In our long-term effect assay, the 30% EtOH extracts of G. lucidum also suppressedthe prostate growth. The rats were not castrated and fed for 25 weeks. Administrationof the 30% EtOH extracts at the concentration of 0.002% in diet food showed 15%prostate growth inhibition. The weight of the testis, fat, spleen, adrenal, kidney, thymus,heart, lung, liver, brain, and hypophysis were not affected by the administration of 30%EtOH extracts of G. lucidum (Table 3). We also tested the serum of the rats. Fromthese results, we determined that the 30% EtOH extracts of G. lucidum specificallysuppressed regrowth of the prostate, but not of other organs.

5a-Reductase Inhibitory Activity and AR-Binding Activity of Each Fraction of 30%EtOH Extracts of G. lucidum. To determine the active principles of the 30% EtOHextracts of G. lucidum, we carried out 5a-reductase inhibitory activity-guidedfractionation. The 30% EtOH extracts was suspended in H2O, and extracted withCHCl3 and H2O-saturated BuOH successively, to give a CHCl3 fraction, a BuOHfraction, and a H2O fraction. The CHCl3 fraction showed the strongest inhibitoryactivity on 5a-reductase of 45% at 200 mg/ml, while the BuOH fraction and the H2Ofraction showed 5a-reductase inhibitory activity of 23 and 19% at 200 mg/ml,respectively (Table 1). We also examined the AR-binding activity. The binding-activeconcentration leading to 50% fluorescence polarization loss (IC50) was estimated to be73, 40, and 174 mg/ml for the CHCl3, BuOH, and H2O fraction, respectively (Table 2).

Table 3. The Effect of the 30% EtOH Extracts on Growth of the Rat Prostrate. Each column includes themean�S.D. n¼10.

Control 30% EtOH Extracts Inhibitory effect [%]

Body weight g 685�57 680�52 1Prostate mg/100 g body weight 144�24 122�20 15a)Testis left g/kg body weight 2.8�0.3 2.7�0.3 7

right g/kg body weight 2.9�0.3 2.9�0.2 4Adipose g/kg body weight 14�4 14�3 2Spleen g/kg body weight 0.19�0.04 0.17�0.04 7Adrenal left g/kg body weight 5.4�0.8 5.0�1.0 8

right g/kg body weight 5.6�0.8 5.5�1.0 2Kidney left g/kg body weight 3.2�0.6 3.1�0.4 4

right g/kg body weight 3.3�0.5 3.1�0.3 5Thymus mg/100 g body weight 8�3 10�5 �22Heart g/kg body weight 2.3�0.2 2.4�0.3 �2Lung g/kg body weight 2.7�0.2 2.7�0.3 2Liver g/kg body weight 27.8�3.2 26.8�3.9 4Brain g/kg body weight 3.8�0.2 3.6�0.3 6Hypophysis mg/100 g body weight 29�15 34�18 �16

a) p< 0.05 against control.


Growth Suppression of Rat Prostate by Administration of Each Fraction of 30%EtOH Extracts of G. lucidum. We next conducted animal tests of each fraction. Fourdays after castration, the weights of the rat prostates were markedly reduced. Theprostate weights recovered by sc injection of testosterone, but not completely. In therats that received testosterone only, the prostate weight was 46.6�12.5 mg/100 g ofbody weight. Administration of CHCl3 fraction at the concentration of 0.01 mg/kgshowed higher suppression effects on the prostate. In the rats that received testosteroneand CHCl3 fraction, the prostate weights were 34.6�8.6 mg/100 g of body weight(Fig. 4). It should be noted that the body weights were almost the same in four groups.The BuOH and H2O fraction had no effect on the weight of the prostate in the castratedrats that received testosterone at the concentration of 0.01 mg/kg/d. These resultssuggested that the CHCl3 fraction inhibited prostatic regrowth through the inhibitionof 5a-reductase activity and having a direct effect on the AR.

The Inhibitory Effect of the CHCl3 Fraction on the Prostate Cancer Cells. Weperformed a cell proliferation experiment to test the results of CHCl3-fractiontreatment. Testosterone alone apparently stimulates LNCaP cell numbers by ca. 100%on average above the number of cells in the untreated control, and DHT alone has thesame effect. Treatment of LNCaP cells with the CHCl3 fraction resulted in a dose-dependent inhibition. LNCaP Cell growth was 50% lower than that of in thetestosterone-stimulated controls (Fig. 5) at higher concentrations of 80 mg/ml CHCl3

fraction in the presence of testosterone. The same inhibition result of LNCaP cellgrowth was also observed in the DHT assay. When the CHCl3 fraction was added to thecell without androgen, the cell number did not decrease. The inhibition effect onandrogen-induced cell proliferation at this concentration was not caused by the celltoxicity. We detected triterpenoids in the CHCl3 fraction by qualitative HPLC analysis.From the results of these experiments, it is reasonable to conclude that the triterpenoidsare the principle active compounds in the CHCl3 fraction. At concentrations of 60 to80 mg/ml, the light proliferation of the LNCaP cells can be observed. In our preliminaryexperiments, there were many triterpenoids in this fraction. Some of the isolated

Fig. 4. Effects of CHCl3, BuOH, and H2O fraction on testosterone-induced regrowth of the castrated ratprostrate. Each column represents the mean�S.D., n¼6. C¼Castrated rat, T¼ testosterone, CH¼

CHCl3 fraction, Bu¼BuOH fraction, H¼H2O fraction, *: P<0.05 against CþT.


triterpenoids showed the increased proliferation effects (data not shown). In the CHCl3

fraction, the inhibitory effect of triterpenoids on cell proliferation is the balance effectbetween those inhibiting proliferation and those increasing proliferation. In higherconcentration, those inhibitory-effect triterpenoids inhibit the cell growth, but in lowerconcentration, they do not affect the cell growth.

5a-Reductase Inhibitory Activity of Each Isolated Compound. Ganoderic acid A(1), ganoderic acid F (2), ganodermanontriol (3), ganoderiol A (4), and ganoderiol F(5) were isolated from the CHCl3 fraction (Fig. 1). The 5a-reductase inhibitory activityis shown in Table 1. It should be noted that finasteride, which is known as a potentsteroidal inhibitor, showed an IC50 value of 0.73 mm in our assay system. All of thesecompounds exhibited weak 5a-reductase inhibitory activities. Compared with otherganoderma alcohols and ganoderma acids [20], these compounds were not very activeinhibitors of 5a-reductase.

AR-Binding Activity of Each Isolated Compound. In the AR competitor assay,ganoderiol F (5) showed the strongest binding activity to androgen receptor (Table 2).In this experiment, we used DHT as the positive control. DHT showed an IC50 value of0.02 mm in our assay system. Ganoderiol F gave the IC50 value of 25 mm for AR.

The Inhibitory Effect of Ganoderiol F (5) on the Prostate Cancer Cell. Consideringthe results of 5a-reductase inhibitory activity and AR-binding experiments, ganoderiolF (5) did not show potent inhibitory activity for 5a-reductase activity and exhibitedbinding activity to the AR. The effect of ganoderiol F (5) on prostate cancer cells isshown in Fig. 6. LNCaP Cells were incubated with varying concentrations of 5 (5 to20 mm) with or without testosterone or DHT for 3 d. Ganoderiol F (5) is the only

Fig. 5. The inhibition effect of CHCl3 fraction on the LNCaP cell growth. n¼3. T¼Testosterone. DHT¼dihydrotestosterone, *: P<0.05 against control, T, or DHT, **: P<0.01 against control, T, or DHT.


compound among the five compounds that showed proliferation inhibition on LNCaPcells (data not shown). Compound 5 showed no inhibition of LNCaP cell growth whencultured without testosterone or DHT. Interestingly, treating LNCaP cells with 5 in thepresence of testosterone or DHTresulted in a dose-dependent inhibition of cell growth.These results suggested that the inhibition of cell growth in the presence of testosteroneor DHT was not the result of the cytotoxic effect, but came from an anti-androgeneffect such as the binding to AR.

The fungus G. lucidum (Reishi, Mannentake, or Lingzhi) has been used forcenturies in East Asia to treat various human diseases such as hepatitis, hepatopathy,hypertension, nephritis, bronchitis, and cancers [21] [22]. Its dried powder wasespecially popular as a cancer-chemotherapy agent in the Imperial Court of ancientChina [23]. Some of the triterpenoids such as ganoderic and lucidic acids, recentlyisolated from Ganoderma, have demonstrated cytotoxicity against mouse sarcoma andmouse lung-carcinoma cells in vitro [24]. Intraperitoneal administration of H2O-solublepolysaccharides isolated from Ganoderma has been found to inhibit the growth ofsarcoma-180 solid tumors in mice [25]. In addition, polysaccharides from Ganodermaalso potentiate the production of cytokines which subsequently suppress theproliferation of HL-60 and U937 leukemic cell lines [26].

The use of herbal therapies in alternative medicine has been increasing, and,although the number of cancer patients using herbal dietary supplements is not exactlyknown, there is evidence of the increasing use of dietary supplements in cancertreatment [27]. G. lucidum is one of the herbs in the herbal mixture PC-SPES, whichhas shown activity against hormone-refractory disease in two prostate cancer patients[28]. PC-SPES is labeled as a mixture of eight herbs used in Chinese and Westernmedicine: Chrysanthemum, Isatis, Licorice, Lucid ganoderma, Pseudoginseng, Rabdo-sia Rubescens, Saw Palmetto, and Scute (Scutellaria baicalensis (huangqin), or

Fig. 6. The effect of ganoderiol F (5) on the LNCaP cell growth. n¼3. T¼Testosterone; DHT¼dihydrotestosterone, *: P<0.05 against control, T, or DHT.


Skullcap). PC-SPES is sold in the USA as a nutritional supplement. The name PC-SPES comes from PC for prostate cancer and SPES, Latin for �hope�. Extracts of PC-SPES have demonstrated estrogenic effects [29] and decreased the growth of hormone-sensitive as well hormone-insensitive prostate cancer cells [30]. Our results suggest thatthese effects might be related to not only the anticancer effects of G. lucidum but alsoits anti-androgen effects. Since excessive 5a-reductase activity has been proposed to bea possible contributing factor in prostate-cancer development and progression, thedevelopment and progression of prostate cancer may also be affected by dietscontaining inhibitors of 5a-reductase.

In this experiment, the treatment with 30% EtOH extracts at 1 and 0.1 mg/kg/dsignificantly inhibited the growth of the ventral prostate induced by testosterone in rats.The yield of ganoderiol F (5) in the 30% EtOH extracts of G. lucidum was determinedas 0.1%, leading to the conclusion that 5 was one of the active compounds in the 30%EtOH extracts. However, it should be noted that G. lucidum has a variety oftriterpenoids with structures similar to that of 5, so not just one compound such as 5 butalso other structurally similar compounds might contribute to the anti-androgenicactivity. Since ganoderiol F (5) shows AR-binding activity and inhibits the androgen-induced growth of LNCaP cells, the growth inhibition might be related to thecompetitive binding to AR with DHT.

The authors are grateful to Mr. Shuhei Kaneko (Fukuoka Prefecture Forest Research and ExtensionCenter) and Mr. Yoshitaro Suimi (Bisoken and Co., Ltd.) for providing mushroom samples.

Experimental Part

General. G. lucidum (BMC9049) was obtained from Bisoken Inc. (Fukuoka, Japan). The mushroomwas identified by Mr. S. Kaneko, Fukuoka Prefecture Forest Research and Extension Center. Thevoucher specimen (BMC9049) was deposited with the herbarium of the Department of Forest and ForestProducts Sciences, Kyushu University in Japan. The fruiting body was dried and ground to powder beforeuse. Unless otherwise specified, chemicals were obtained from Sigma Aldrich Japan Co., Ltd. (Tokyo,Japan). Org. solvents were purchased from Wako Pure Chemical Industries Co. (Osaka, Japan). [4-14C]Testosterone was obtained from Perkin-Elmer Japan Co., Ltd. (Kanagawa, Japan).

30% EtOH Extracts of G. lucidum. Dried and chipped G. lucidum (200 g) was extracted with 30%EtOH at r.t. for 24 h by using blender. The extracts were filtered through ADVANTEC No. 2 filter paper,concentrated under vacuum, and then freeze-dried. The 30% extracts (10 g) were stored at �208 beforeassay.

The 30% EtOH extracts (10 g) were suspended in H2O (0.3 l) and extracted with CHCl3 (1 l�5) andH2O-sat. BuOH (2 l�5) successively. A part of CHCl3 extract (1.8 g) was treated with NaHCO3 soln. togive neutral CHCl3 fraction (0.2 g) and acidic CHCl3 fraction (1.6 g), resp. The extracts were stored at�208 before assay. Repeated column chromatography (CC) of CHCl3- and BuOH-soluble fractions ledto the isolation of compounds 1–5. They are identified as ganoderic acid A (1) [11], ganoderic acid F (2)[12], ganodermanontriol (3) [13], ganoderiol A (4) [13], and ganoderiol F (5) [14]. MS, NMR, andoptical-rotation data were in agreement with published data (Fig. 1).

Preparation of Rat Microsomes. Rat liver from female SD rats (7-weeks age) was prepared by amethod reported by Shimizu et al. with some modifications [31]. From two mature SD female rats, theliver was removed, and minced tissue was homogenized in 4 tissue volumes of medium A (0.32m sucrose,1 mm dithiothreitol, and 20 mm sodium phosphate, pH 6.5). The resulting supernatant from thecentrifugations was further centrifuged at 105,000g for 1 h twice. The washed microsomes weresuspended in 1 pellet volume of medium A, and the dispersion of microsomes was achieved using a


syringe with 18, 23, and 26 G needless in succession. The microsome suspension was stored at �808 justbefore use.

Measurement of 5a-Reductase Inhibitory Activity. A complete reaction mixture included 1 mm

dithiothreitol, 20 mm phosphate buffer (pH 6.5), 1.9 nCi [4-14C]testosterone, 150 mm testosterone,167 mm NADPH, and the microsomes (0.6 mg of protein) in a final volume of 0.3 ml. The concentrationof testosterone contributed by [4-14C]testosterone was negligible. The sample was added to the soln. ateach conc. The incubation was carried out for 10 min at 378. It was started by the addition of 10 mlmicrosomes to the pre-heated reaction soln. in a tube. After 10 min, the incubation was terminated byadding 10 ml of 3m NaOH. To extract metabolites, 1 ml of Et2O was added, and the tubes were capped andshaken. The org. phase was applied to a silica gel plate (Kieselgel 60 F254), and the plate was developed inAcOEt/hexane (7 : 3) at r.t. The radioactivity profile was determined with an imaging analyzer (FLA-5000 RF, Fuji Film Co., Ltd., Tokyo, Japan). The 5a-reductase activity was calculated from thepercentage of the extent of the conversion of [4-14C]testosterone to [4-14C]dihydrotestosterone.

Androgen Receptor (AR) Competitor Assay. The ability of each sample to interact with the AR wasevaluated by a fluorescence polarization (FP) method. This method enabled to determine the capacity ofthese competitor chemicals to displace a high-affinity fluorescent ligand (AL Green) from the purified,recombinant ligand-binding domain (LBD) of the human AR at r.t. Conceptually, the binding of afluorescent molecule to another molecule can be quantified by the change in its speed of rotation. Hence,the AR-fluorescent ligand bound complex (AR-AL Green) will rotate slowly and have a high FP value.Increasing concentration of competing ligand will displace the AL Green from the AR. Free AL Greenwill then rotate more rapidly and have a low FP value. Because the measured polarization is an averageof the free and bound AL Green molecules, it can be used to assess competitive displacement from theAR-LBD. Briefly, all samples were prepared as stock solns. in DMSO. Chemicals were serially diluted,over at least six log order concentrations, in triplicate in 20-ml volumes in assay buffer on a 384-well plate.No final DMSO concentrations exceeded the manufacturer�s recommendations, and, therefore, were notanticipated to alter fluorescence. A mixture of AR (25 nm final) and AL Green (1 nm final) was added in20-ml volumes to the serially diluted test chemicals. The plate was then incubated in the dark for ca. 4 h atr.t. The polarization was then measured on a Beacon 2000 FP instrument using 485-nm excitation and535-nm emission interference filters in polarization mode. The polarization values (mP) were plottedagainst increasing concentrations of the test extracts.

The Inhibitory Effect on the Prostate Cancer Cell. The AR-positive human prostate cancer LNCaPcells were obtained from American Type Culture Collection. The cells were maintained in RPMI 1640supplemented with 10% fetal bovine serum (FBS) at 378 in a 5% CO2, 95% air-humidified atmosphereincubator. The cells were used between passages 5–30 at a split ratio of 1 : 4 at each passage. The cellswere plated into a 24-well plate with 2�105/well density supplemented with 5% steroid-depleted (DCC-stripped) cFBS. After 24 h, the cells were treated with either vehicle control or androgens (testosteroneor DHT) for another 3 d. Cell proliferation was determined by the 3-amino-7-(dimethylamino)-2-methylphenazine (NR) method. The NR soln. was prepared at 5 mg/ml and diluted by culture medium to5 mg/ml. The NR extract soln. was made by using H2O and 50% EtOH (1% AcOH). The culture mediumwas changed to NR soln. and incubated for 3 h at 378, then the NR soln. was aspirated, and the cells werewashed with PBS twice. NR Extract soln. (500 ml) was added to each well to extract for 20 min at r.t. Theabsorbance of each well was measured at 540 nm.

Growth Suppression of the Rat Prostate. The assay for growth suppression of the rat prostate wasperformed as described by Fukuta et al. [32]. The testes of SD rats were removed at four weeks of ageunder light anesthesia with pentobarbital. After 4 d, testosterone (100 mg/body) was injected sc into therats once daily for 8 d. There were 6 rats for each group. Each sample suspended in 0.5% methylcellulosewas orally administered at different concentrations once daily for 8 d. The flutamide (10 mg/kg bodyweight) was used as the positive control and suspended in 0.5% methylcellulose, and orally administeredonce daily for 8 d. After 8 d, rats were anesthetized by pentobarbital. Then, their prostates were removedand their weights were determined.

Another assay was used to test the long-term effect of 30% EtOH extracts of G. lucidum. The 24-to-27-week-old SD rats were used. 0.002% EtOH extracts in CE-2 was used for 25 weeks. After 25 weeks,rats were anesthetized by pentobarbital. Then, their prostates were removed, and their weights were


determined. The weights of the testes, fat, spleen, adrenal, kidney, thymus, heart, lung, liver, brain, andhypophysis were measured.

This experiment was conducted according to the Guidelines for Animal Experiments in the Facultyof Agriculture and the Graduate Course, Kyushu University, and the Law (No. 105) and Notification(No. 6) of the Japanese Government.

Statistics. Results were expressed as means S.E.M. or S.D. Statistical significance of the animal testwas determined by Anova and Bonferroni-type multiple t-test, and the statistical significance of cellproliferation was determined by the t-test.

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Received January 10, 2008


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