New Botanical Materials with Anti-Androgenic Activity

Enlargement of prostate, which affects 50% of men aged 60 and 90% of men by age 80, is commonly referred to as benign prostate hyperplasia (BPH) (Russell & Wilson, 1994). BPH is a slow, progressive enlargement of the fibromuscular and epithelial structures of the prostate gland (Cristoni et al., 2000). Substantial evidence indicates that the androgens testosterone (T) and dihydrotestosterone (DHT) contribute to the production of BPH (Lowe et al., 2003). The principal serum androgen T is converted by 5alpha-reductase (5aR) to DHT. DHT binds to androgen receptor (AR) in the prostate, where it initiates DNA synthesis (Marks, 2004). This action, in turn induces protein synthesis and abnormal growth of prostate. Current clinical evidence indicates that either the inhibition of 5aR or the inhibition of the binding of DHT to AR reverses the symptoms of BPH in human males. The effective drugs, finasteride as a 5aR inhibitor and flutamide as a binding inhibitor to AR are utiliezed clinically for treatment of BPH. Alternatively functional foods are eclectic selection as dietary approaches to prevent BPH in middle-aged men. Natural products are frequently used to care BPH in preference to therapeutic agents that can cause severe sideeffects. Plant extracts such as the lipid extracts of saw palmetto berry extract (SPE) have also been found to reduce the conversion of testosterone (T) to dihydrotestosterone (DHT) by inhibiting 5alpha-reductase (5aR) both in vitro and in vivo (Elliot, 2001). However, few products of natural origin besides SPE are believed to be effective against enlargement of the prostate. The scientifically-proven food materials are expected to be of benefit for prevention of BPH. In our preliminary study, the suppressive effects of natural food materials have been found in BHT model mice. We introduce here the suppressive effects against BHT of two materials, i.e., “banana peel” and “leaf of Houttuynia cordata”. The effects of these materials in the androgen-responsive LNCaP human prostate cancer cell line and in effects of BHT model mice were examined. These data presented here demonstrate that these materials inhibit the growth of the prostate and HCE has anti-androgenic activity.


Introduction
Enlargement of prostate, which affects 50% of men aged 60 and 90% of men by age 80, is commonly referred to as benign prostate hyperplasia (BPH) (Russell & Wilson, 1994).BPH is a slow, progressive enlargement of the fibromuscular and epithelial structures of the prostate gland (Cristoni et al., 2000).Substantial evidence indicates that the androgens testosterone (T) and dihydrotestosterone (DHT) contribute to the production of BPH (Lowe et al., 2003).The principal serum androgen T is converted by 5alpha-reductase (5aR) to DHT.DHT binds to androgen receptor (AR) in the prostate, where it initiates DNA synthesis (Marks, 2004).This action, in turn induces protein synthesis and abnormal growth of prostate.Current clinical evidence indicates that either the inhibition of 5aR or the inhibition of the binding of DHT to AR reverses the symptoms of BPH in human males.The effective drugs, finasteride as a 5aR inhibitor and flutamide as a binding inhibitor to AR are utiliezed clinically for treatment of BPH.Alternatively functional foods are eclectic selection as dietary approaches to prevent BPH in middle-aged men.Natural products are frequently used to care BPH in preference to therapeutic agents that can cause severe sideeffects.Plant extracts such as the lipid extracts of saw palmetto berry extract (SPE) have also been found to reduce the conversion of testosterone (T) to dihydrotestosterone (DHT) by inhibiting 5alpha-reductase (5aR) both in vitro and in vivo (Elliot, 2001).However, few products of natural origin besides SPE are believed to be effective against enlargement of the prostate.The scientifically-proven food materials are expected to be of benefit for prevention of BPH.In our preliminary study, the suppressive effects of natural food materials have been found in BHT model mice.We introduce here the suppressive effects against BHT of two materials, i.e., "banana peel" and "leaf of Houttuynia cordata".The effects of these materials in the androgen-responsive LNCaP human prostate cancer cell line and in effects of BHT model mice were examined.These data presented here demonstrate that these materials inhibit the growth of the prostate and HCE has anti-androgenic activity.

Drugs and chemicals
The chemicals used and their sources were as follows: testosterone propionate and dihydrotestosterone from Wako Pure Chemical Industries (Osaka, Japan); flutamide from Sigma-Aldrich Japan (Tokyo); finasteride from LKT Labs.(St.Paul, MN); and pentobarbital from Dainippon Sumitomo Pharma (Osaka, Japan).Other chemicals were of analytical grade.

Animal experiments
Male ddY mice (6-8 weeks of age) were purchased from SLC Inc., Shizuoka, Japan.The room was maintained at 24±1°C and 50±10% humidity under a 12 h light/dark cycle (lights on from 8:00 AM to 8:00 PM), and the animals had free access to water and food.Animal studies were conducted according to the 2006 guidelines entitled Notification No. 88 of the Ministry of the Environment in Japan and Guidelines for Animal Experimentation of Tokyo University of Marine Science and Technology with the approval of the Animal Care and Use Committee of Tokyo University of Marine Science and Technology.

Growth suppression of mouse prostates and seminal vesicles in testosteroneinduced BPH model mice
Assay of growth suppression in castrated mouse prostates and seminal vesicles was performed based on the OECD protocol.(Yamasaki et al., 2003) The testes of ddY mice were removed at 7 weeks of age under anesthesia by intraperitoneal injection of pentobarbital (50 mg/ml/kg).After 1 d, testosterone propionate (TP) was injected intraperitoneally into the mice once daily for 10 d.Enlargement of the reproductive organs in the mice was demonstrated dose-dependently due to TP in our preliminary studies as shown in Fig. 1A.The growth of these organs is androgen-dependent (Franck-Lissbrant et al., 1998).To evaluate the effects on reproductive organs, each of various doses of sample extract was suspended in 1% ethanol and orally administered to the BPH model mice once daily.After 10 d, the mice were weighed, and sacrificed by cervical dislocation.The lengths of the short www.intechopen.comNew Botanical Materials with Anti-Androgenic Activity 195 and long axes of the prostates were then measured with vernier calipers, and the seminal vesicles were removed and weighed.In particular, the weights of the seminal vesicles were sensitive to androgenic effects in our mice model.Finasteride (FI, 2 mg/kg) and flutamide (FL, 10 mg /kg) were used as positive control for the assay system.As shown in Fig. 1B, orally administration of flutamide suppressed weight of the seminal vesicles in BPH model mice with dose dependent manner (1-25 mg/kg/day) for 10 d.

Cell culture and growth studies of human prostate cancer cells
The LNCaP human prostate cancer cell line is a well-established androgen-dependent cell line.(Goldman et al., 2004) AR-positive human prostate cancer LNCaP cells were obtained from the Riken BRC Cell Bank (Tsukuba, Japan).The cells were plated onto a 96-well plate at a density of 2 × 10 5 /well and supplemented with 10% charcoal stripped fetal bovine serum (CSFBS) obtained from Invitrogen Japan (Tokyo).Twenty-four h later, the cells were treated with either vehicle control or androgens (T or DHT) in the presence and the absence of each concentration of assessed samples for another 3 d.Sample was dissolved in ethanol and added to the cells after further dilution so that the final volume of ethanol was 1% or less.After culture, cell proliferation was determined to measure cell viability by 3-[4,5dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay.(Hamid et al., 2004) The cells were treated with 1 mg/ml of MTT for 2 h, and precipitated dye was dissolved into dimethylsulfoxide.The absorbance of each well was measured at 570 nm.

Statistical analysis
Data were analyzed statistically by Student's t-test to determine significant differences in the data among the groups.The p values less than 0.05 were considered significant.The values were expressed as mean ± S.E.

Banana peel
Fig. 2. Banana peel extract (BPEx) prepared from fresh peel part from organically-cultivated bananas showed suppressive effects for enlargement of prostate in BPH model mice

Source material
The common banana (Musa spp.) is a tropical fruit that grows in the western hemisphere.Primarily viewed as a food source, the banana has a fleshy inside portion surrounded by an outer typically yellow peel (Fig. 2).The fleshy inside portion, or pulp, is edible when raw, and the peel is usually discarded.When ripe, bananas have a deep yellow rind with brown spots, and a creamy pulp, which is easily digested.Bananas are rich in carbohydrates and contain relatively large amounts of vitamins A, B and C and the minerals potassium and phosphorous (Proteggente et al., 2002;Blades et al., 2003).However, banana peel has not been studied nutritionally and pharmaceutically as a source of bioactive compounds.In this report, we examined the effects of banana peel extract (BPEx) on androgen-induced enlargement of accessory reproductive organs in castrated mice.To elucidate the mechanisms of action, the effects of BPEx on the androgen-responsive LNCaP human prostate cancer cell line were investigated.The data presented here indicate that BPEx inhibits growth of the prostate and that BPEx has anti-androgenic activity through inhibition of 5aR.Fresh banana peel (2.4 kg) of organically-cultivated bananas were cut and extracted with methanol at room temperature for 2 days.The extracts were filtered, concentrated under a vacuum, and freeze-dried.BPEx (56 g) was stored in a refrigerator before assay.

Suppressive effect of benign prostate hyperplasia in mouse
Our research group has been shown that BPEx treatment (10 d of administration at 0, 50, 100, and 200 mg/kg per orally) dose-dependently reduced the prostate size and the weights of seminal vesicles in the BPH model mice.Then, the effects of BPEx on the growth of mouse prostates and seminal vesicles were studied as compared with those of flutamide and finasteride in the BPH model mice for 10 d.The short and long axes of the ventral prostates and the weights of the seminal vesicles were severely reduced, and when TP (2 mg/kg i.p.) was injected, significant growth of prostates and seminal vesicles was induced.BPEx (200 mg/kg) produced a reduction in prostate weight.Finasteride (2 mg/kg) and flutamide (10 mg/kg), well-known anti-androgens, showed larger reductions in prostate weight.Similar results were observed with regard to seminal vesicle weights.When DHT (6 mg/kg, i.p.) was injected in place of T, significant growth of the prostate and seminal vesicles was induced.BPEx did not inhibit these effects of DHT (Akamine, 2009).These results indicate that orally treatment of BPEx suppress the action of testosterone against enlargement of the reproductive organs in vivo.

Effects of parts of banana fruit on mouse prostate and seminal vesicles
Testosterone propionate (TP, 2 mg/kg, i.p.) and sample solution (p.o.) were treated to castrated mice once daily for 10 d (Fig. 3).The lengths of the long axes (A) of the prostates of mice were measured vernier calipers, and seminal vesicles (B) were removed and weighed.Cast, castration only; Cont, control; Pe, banana peel (200 mg/kg); Ed, edible part (200 mg/kg).Each value represents the mean ± S.E., n=5.*: p<0.05; **: p<0.01 vs. control (TPtreated without samples).Fresh banana fruits separated into two parts, peel and edible part to evaluate suppressive effects of the extracts ("Pe" and "Ed", respectively) on BPH model mice.The inhibitory effects of BPEx on the growth of mouse prostates and seminal vesicles were estimated after 10 d of administration.BPEx dose-dependently reduced the prostate size and the weights of seminal vesicles, and showed almost maximal effect at 200 mg/kg.These results indicate that some components in banana peel will be required to suppress prostate enlargement in this model.

Inhibitory effects of BPEx on prostate cancer cells
The effects of BPEx on the proliferation of prostate cancer cells (LNCaP cells) were investigated.LNCaP cells show most of the characteristics of human prostatic carcinoma, such as dependence on androgens, the presence of ARs, and the production of acid phosphatase and prostate-specific antigen.The LNCaP cell line is used as an attractive model for in vitro studies of the biology of human prostate cancer.LNCaP cells were incubated with different concentrations of BPEx (3.13-100 μg/ml) with and without T or DHT for 3 d.In the absence of BPEx, T alone stimulated LNCaP cell number about 20.5 ± 0.5 x 10 5 /well, and DHT alone stimulated LNCaP cell numbers to about 30.4 ± 2.1 x 10 5 /well.

Leaf of Houttuynia cordata
Fig. 5. Houttuynia cordata extract (HCE) prepared from fresh leaf part showed suppressive effects for enlargement of prostate in BPH model mice
The fresh Houttuynia cordata were cut and extracted with 99.5% methanol at room temperature for 2 days.The extract were filtered, and concentrated under a vacuum.The extract was stored in the refrigerator before assay.
We examined the effects of H. cordata extract (HCE) in the androgen-responsive effects in BPH model mice and LNCaP human prostate cancer cell line.The data presented here demonstrate that HCE inhibits the growth of the prostate with anti-androgenic activity.In the further experiments, the suppressive effects of HCE were compared with flutamide (FL, 10 mg/kg/day) and finasteride (FI, 2 mg/kg/day) in BPH model mice.As shown in Fig. 7, the size of long-axis length of prostates (A) and the weight of seminal vesicles (B) were severely reduced by HCE ingestion, and when T was injected, a significant growth of prostates and seminal vesicles was induced.Flutamide and finasteride are well known anti-androgen drugs, showed a larger reduction in prostate and seminal vesicle.HCE showed suppressive effect on reproductive organs as same as these medicine at these experimental conditions.

Inhibitory effects of HCE on LNCaP cells
The effects of HCE on the proliferation of prostate cancer cells (LNCaP cells) were investigated.After incubation, the cell numbers in T-treated and DHT-treated groups were defined as 100%.As shown in Fig. 8 finasteride, 5aR inhibitor, showed dose-dependent inhibition in T-treated and DHT-treated LNCaP cells.However, its inhibitory effects on DHT-treated group are relatively weak.In contrast, flutamide, androgen receptor antagonist, showed inhibition both in T alone and DHT alone groups at the similar concentrations.On the other hand, the treatment of HCE to LNCaP cells in the presence of T and DHT resulted in the concentration-dependent inhibition of cell proliferation at the same concentrations as same as case of flutamide treatment.These results indicate that HCE suppress enlargement of prostate with a different mechanism with finasteride, 5aR inhibitor.Proliferation of prostate cancer cells (LNCaP) were evaluated cell viability (% of control) by MTT assay after 3 days incubation with or without samples at 6.25, 25, 100 µg/ml in the presence of testosterone (T: 10 mg/ml) or dihydrotestosterone (DHT: 10 mg/ml).HCE: Houttuynia cordata extract; FI: finasteride; FI: flutamide.Each data presents as mean±S.E.(n=4).

Discussion
We studied the in vivo potency of BPEx and HCE, as new botanical materials in immature castrated mice.We evaluated these biological effects in the androgen-responsive LNCaP human prostate cancer cell line and in effects of BHT model mice.
BPEx inhibited TP-induced growth of prostates and seminal vesicles in castrated mice, although it was less potent than finasteride at the same doses.While BPEx inhibited the action of TP, it did not inhibit DHT-induced organ growth.In addition, BPEx inhibited cell proliferation in the presence of T, but not in the presence of DHT.These results suggest that BPEx suppressed the growth of prostates and seminal vesicles by inhibiting the conversion of T to DHT, rather than by blocking the binding of androgen and its receptor.BPEx may have reduced prostate size and seminal vesicle weight by inhibiting 5aR.In further study, we found that treatment of LNCaP cells with BPEx inhibited Tinduced cell proliferation (Akamine et al., 2009).The inhibition of this effect of T might have be due, at least in part, to inhibition of 5aR or to antagonism of androgen binding to the AR (Lazier et al., 2004).Since 25 μg/ml of BPEx inhibited 5aR activity by about 30%, we expected to find that this dose would inhibit T action.Similar results have been found with finasteride, a well-known inhibitor of 5aR (McConnell et al., 1992;Sudduth & Koronkowski, 1993), at doses above 25 μg/ml.Similar results were also found with flutamide, an antagonist of androgen binding to the AR (Bergman, & Eriksson, 1996;Sundblad et al., 2005).On the other hand, treatment of LNCaP cells with BPEx in the www.intechopen.com Prostate Cancer -Original Scientific Reports and Case Studies 202 presence of DHT did not result in a dose-dependent inhibition of cell growth.These results suggest that inhibition of cell growth in the presence of BPEx was not the result of a cell cytotoxic effect, but rather was due to an anti-androgen effect, such as inhibition of 5aR.According to a previous report describing discrimination between cytotoxic and cytostatic effects of integrants on LNCaP cells (Romijn et al., 1988), cytotoxic effects are detectable as a decrease in MTT conversion to a level below that of the starting cells.In our experiment, growth of androgen-induced LNCaP cells was suppressed without decreasing from the starting level in the presence of BPEx during the experimental period.Therefore, a cytostatical effect was estimated for suppression of BPEx on LNCaP cell growth.However, since no index markers related to apoptotic signaling were measured in our experiments, the possibility of a non-cytostatical effect should be considered and confirmed in further detailed investigation.There are generally two ways to suppress prostate regrowth in animal experiments: by inhibiting 5aR activity, and through the use of an androgen receptor antagonist (Imperato-McGinley et al., 1992).An androgen antagonist can suppress DHT-induced prostate and seminal vesicle regrowth (Geller et al., 1981) Therefore, blocking DHT from binding to androgen receptors in the prostate and seminal vesicle is considered to be a possible mechanism of action (other than 5aR inhibition) (Nakayama et al., 1997).To examine this possibility, the effects of BPEx on prostate growth induced by DHT were investigated.If the suppression of prostate growth is caused only by inhibition of 5aR, DHT-induced prostatic regrowth should not be suppressed.Ten d after castration, the weights of mouse prostates were markedly reduced, and prostate size was restored by i.p. injections of TP or DHT.BPEx had no effect on the sizes of the prostate and seminal vesicle of castrated mice that received DHT, whereas flutamide, an androgen receptor antagonist, significantly reduced prostate weight (Fig. 3).In our study, blood levels of testosterone and samples were not measured, but in vivo and in vitro experiments gave mutually correlated results with regard with their mechanism of action.These results suggest that BPEx inhibited prostate growth by inhibiting 5aR activity rather than by exerting a direct effect on the androgen receptor, although the detailed mechanism including bioavailability remains unclear.Recently, there is research reports on components of the banana peel: dietary fiber and pectin (Happi Emaga et al., 2008), and phytosterol from unripe pulp and peel (Oliveira et al., 2008) have been reported.In particular, phytosterols are able to be the active principle in BPEx, due to their anti-androgen activity based on structural similarity.Investigation to elucidate the active components in banana peel is in progress.In this study, we found that BPEx can have anti-androgenic activities through in vitro 5aR inhibitory activity and in vivo growth suppression of prostates and seminal vesicles from castrated mice.In this study, we have shown that HCE inhibited the T-induced growth of prostates and seminal vesicles in castrated mice and that HCE inhibited T-and DHT-induced cell proliferation in NLCaP cells at the same concentration.The inhibition of this effect of T may be due, at least in part, to the inhibition of 5aR or to antagonism of androgen binding to the AR.And treatment of LNCaP cells with HCE in the presence of DHT result in the dosedependent inhibition of cell growth.These results were seen with flutamide, which is an antagonist of androgen binding to the AR.These results suggested that the inhibition of cell proliferation in the presence HCE was not the result of a cell cytotoxic effect, but rather was www.intechopen.com New Botanical Materials with Anti-Androgenic Activity 203 due to an anti-androgen effect, such as inhibition of AR.These results suggest that HCE suppressed the growth of prostates and seminal vesicles by blocking the binding to AR, rather then by conversion of T to DHT.HCE may have reduced prostate size and seminal vesicle weight by blocking the binding of androgen and its receptor.
There are generally two ways to suppress prostate regrowth in animal experiments: by inhibiting 5aR activity and by blocking the binding of androgen and its receptor.An androgen antagonist can suppress DHT-induced prostate and seminal vesicle regrowth.Therefore, blocking DHT from binding to androgen receptors in the prostate and seminal vesicle was considered to be possible mechanism of action (other than 5aR inhibition) of the extract of HCE.To examine this possibility, the effects of HCE on LNCaP cells growth induced by DHT were investigated.These results suggest that HCE inhibited prostate growth by inhibiting androgen receptor rather than by having a direct effect on the 5aR, although the detailed mechanism unclear.In this study, we found that HCE may have antiandrogenic activities through in vitro androgen binding to the AR and in vivo growth suppression of prostate and seminal vesicles from castrated mice.Active compounds showing anti-androgen activity in HCE are of scientific interest.Since, the clinical implications of this activity are currently unknown, further research is needed before is needed before HCE can for the treatment of BPH.
For several years, SPEx has been used as a popular phytotherapeutic agent in the treatment of BPH, but its active component and mechanism of action have not been fully elucidated (Hill & Kyprianou, 2004).Banana peel is usually considered useless and is discarded, but the anti-androgenic activity of BPEx might be useful in the treatment of BPH patients.And leaf of H. cordata have been used as folk medicine, however, never used for treatment BPH with suppressive effect of androgenic functions.Since the clinical implications of this activity are currently unknown, further research is needed before BPEx can be used in the treatment of BPH.

Conclusion
In this chapter, the androgen-responsive effects of the two natural extracts from banana peel extract (BPEx) and leaf of H. cordata extract (HCE) were shown in BPH model mice and LNCaP human prostate cancer cell line.The data presented here revealed that BPEx was estimated to inhibit the growth of the prostate in BPH model mice by its inhibitory activity against 5-alpha reductase (5aR).And HCE was estimated to inhibit BPH in mice by its antiandrogenic activity different from 5aR inhibition.Therefore, these new botanical materials can be promising most likely candidates as potential material for preventing benign prostate hyperplasia, and it is able to continue to be one of the best preventive medicinal foods to keep our good health in the future.

Fig. 1 .
Fig.1.TP-induced regrowth of seminal vesicles (A) and its suppressive effects by flutamide (B) in castrated mice Testosterone propionate (TP; 2, 10, and 50 mg/kg) was injected i.p. into castrated mice (7weeks-old) once daily for 10 days.Flutamide (1, 5, and 25 mg/kg) suspended in 1% ethanol and orally administered once daily.The seminal vesicles were removed and weighed and compared with castrated mice as a control.Cast: castrated control, Cont: control (TPtreated), and FL: flutamide.Each value represents the mean ± S.E., n=5.**: p<0.01,***: p<0.005 vs control.To evaluate the effects on reproductive organs, each of various doses of sample extract was suspended in 1% ethanol and orally administered to the BPH model mice once daily.After 10 d, the mice were weighed, and sacrificed by cervical dislocation.The lengths of the short

Fig. 3 .
Fig. 3. Effects of extracts of peel (Pe) and edible part (Ed) of banana fruit on TP-induced regrowth of prostates and seminal vesicles in castrated mice

Fig. 4 .
Fig. 4. Inhibitory effects of BPEx on the proliferation of LNCaP cells induced by testosterone (T) or dihydrotestosterone (DHT)

Fig. 8 .
Fig. 8. Inhibitory effects of HCE, Flutamide (FL), and Finasteride (FI) on LNCaP cell proliferation in the presence of testosterone (T) or dihydrotestosterone (DHT).Each point shows the mean of the separated duplicate experiments.
The suppressive effects of HCE on reproductive organs were investigated in testosteroneinduced BPH model mice.The assay to evaluate the effects of HCE on castrated mice prostates and seminal vesicles was performed based on the Hershberger assay as a mean of rapidly developing in vivo.The testes of ddY mice were removed at 7 weeks of age under anesthesia with intraperitoneal injection of pentobarbital (50 mg/ml/kg).After one day of orchiectmy, testosterone propionate (TP) 2 mg/kg was injected intraperitoneally (i.p.) into the mice once daily for 10 days.HCE was administered once daily.After 10 days, mice were weighted and sacrificed by cervical dislocation.The lengths of the long axes of prostate were measured by vernier calipers, and seminal vesicles were removed and weighed.As shown in Fig.6, HCE suppress prostates and seminal in a dose-dependent manner at 20, 100, and 500 mg/kg.