Journal of Ethnopharmacology 121 (2009) 123129

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Androgenic activity of the Thai traditional male potency herb, Butea superba Roxb., in female rats
Suchinda Malaivijitnond a, , A-ngun Ketsuwan a,b , Gen Watanabe c,d , Kazuyoshi Taya c,d , Wichai Cherdshewasart a
a

Primate Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University, Phyathai Road, Bangkok 10330, Thailand Interdepartment of Physiology Program, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan d Department of Basic Veterinary Science, the United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
b c

a r t i c l e

i n f o

a b s t r a c t
Aim of the study: Butea superba Roxb. (Leguminosae) is a well-known Thai male potency herb with androgenic and anti-estrogenic activities. We evaluated whether oral administration of Butea superba has an androgenic or anti-estrogenic activity in female rats. Materials and methods: Normal and ovariectomized adult female rats were each subdivided into ve groups, DW, BS-10, BS-50, BS-250 and TP, and gavaged with 0, 10, 50 and 250 mg/kg BW/day of the crude of Butea superba and subcutaneously injected with 6 mg/kg BW/day of testosterone propionate (TP), respectively, during the treatment period. Results: In intact rats, only BS-250 increased the uterine thickness and the number of uterine glands, and could induce a prolonged diestrous phase. In ovariectomized rats, treatment with BS-50 as well as BS-250 increased the uterine thickness and the number of uterine glands. However, serum luteinizing hormone (LH) levels were also increased. TP reduced serum follicle stimulating hormone and LH levels with the appearance of anestrous cycle, and could signicantly increase the relative uterine weight and thickness and the number of uterine glands in both intact and ovariectomized rats. Conclusions: Orally administered Butea superba tubers have an androgenic effect on the reproductive organs of intact and ovariectomized rats, and exhibit anti-estrogenic activity on LH secretion in ovariectomized rats. 2008 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 31 July 2008 Received in revised form 7 October 2008 Accepted 9 October 2008 Available online 1 November 2008 Keywords: Androgenic activity Butea superba Roxb. Female rats Testosterone propionate Uterus

1. Introduction Kwao Krua herbs are a group of leguminous plants which have been used for centuries as traditional herbal medicines in Thailand (Suntara, 1931). The herbs are believed to contain rejuvenating and aphrodisiac properties which can promote youth and longevity. The popular use of white Kwao Krua (Pueraria mirica Airy Shaw and Suvatabandhu), based on its estrogenic activity, has become a major focal point for modern research of Kwao Krua herbs (Malaivijitnond et al., 2004, 2006; Trisomboon et al., 2004a, 2004b, 2005, 2006; Jaroenporn et al., 2006, 2007; Urasopon et al., 2007, 2008). However, in comparison the red Kwao Krua, Butea superba Roxb. (Legumi-

Abbreviations: AR, androgen receptor; BS, Butea superba Roxb.; D, day; DW, distilled water; ER, estrogen receptor; FSH, follicle stimulating hormone; LH, luteinizing hormone; P, proestrous phase; TP, testosterone proprionate. Corresponding author. Tel.: +66 2 2185275; fax: +66 2 2185256. E-mail address: Suchinda.m@chula.ac.th (S. Malaivijitnond). 0378-8741/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.10.014

nosae), with its proposed aphrodisiac potency, remains relatively unknown. Recently, Butea superba initiated interest about its potential medicinal applications after it was claimed that it has potential for the treatment of erectile dysfunction in mature human males (Cherdshewasart and Nimsakul, 2003), whilst in rats Butea superba could promote penile blood ow (Tocharus et al., 2006), and therefore aids erection, via the inhibition of cAMP phosphodiesterase activity (Roengsumran et al., 2000; Ingkaninan et al., 2003). Accordingly, most of the research on Butea superba has focused on its potential androgenic effects on the reproductive function in male animals. It was reported that feeding of Butea superba to male rats at doses of 150 and 200 mg/kg BW/day for 90 days induced a decrease in testosterone levels and a slight decrease in luteninzing hormone (LH) levels (Cherdshewasart et al., 2008). In contrast, Butea superba at doses of 2, 25, 250 and 1250 mg/kg BW/day had no discernable effect on the weights of the seminal vesicles, prostate gland and testis in male rats after daily feeding for 8 weeks (Manosroi et al., 2006). Furthermore, the anti-estrogenic activity of Butea superba has also been tested in vitro where Butea

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superba had an anti-proliferation effect (or anti-estrogenic effect) on the growth of the estrogen responsive human breast epithelial adenocarcinoma MCF-7 cells at concentrations of 10, 100 and 1000 g/ml, although a dose of 1000 g/ml was required for the less estrogen responsive HeLa cells (Cherdshewasart et al., 2004a, 2004b). However, to date the in vivo and in vitro studies are still equivocal as to whether Butea superba has an androgenic or antiestrogenic activity. Indeed, although Butea superba has been used for both men and women (Suntara, 1931), to the best of our knowledge no research has tested its activity in female animals. The present experiment was, therefore, designed to evaluate the effect of orally administered Butea superba on serum gonadotropin levels and reproductive organs (weights and histology) in adult cyclic and ovariectomized female rats. The doses of the powder suspension of Butea superba used in this study (0, 10, 50 and 250 mg/kg BW/day) were calculated from the treatment dosages and toxicity results of the previous studies (Cherdshewasart and Nimsakul, 2003; Manosroi et al., 2006; Cherdshewasart et al., 2008). 2. Materials and methods 2.1. Animals Adult female Wistar rats aged 60 days, weighing 200250 g were obtained from the National Laboratory Animal Center, Mahidol University, Nakhon Pathom, Thailand. They were housed in stainless steel cages with sawdust bedding at 5 animals/cage in a room with controlled lighting (lights on 06002000 h) and temperature (25 1 C) at the Primate Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University. The animals were fed with rat chow diet (Pokaphan Animal Feed Co., Ltd., Thailand) and water ad libitum and were acclimatized to the surroundings for 2 weeks before the onset of the study. The experimental protocol was approved by the Animal Ethical Committee in accordance with the guide for the care and use of laboratory animals prepared by Chulalongkorn University. 2.2. Experimental procedure One hundred adult female rats with a regular estrous cycle (45 days) for at least three consecutive cycles were selected for this study. They were divided into two main groups: intact ovary and ovariectomy. Each of these two main groups were randomly subdivided into ve treatment groups (10 rats/group), DW, BS-10, BS-50, BS-250 and TP. In the DW, BS-10, BS-50, and BS-250 groups, rats were gavaged with a suspension of 0, 10, 50 and 250 mg/kg BW/day of Butea superba in 0.7 ml distilled water, respectively, during the treatment period. In the TP group, rats were subcutaneously injected with 6 mg/kg BW/day of testosterone propionate (TP) in 0.2 ml of sesame oil. The experimental schedule was separated into three periods: pre-treatment, treatment and post-treatment. 2.2.1. Intact group During the pre-treatment period, the rst day of study period (day 1) was started when rats showed a proestrous phase (P) on the 1st estrous cycle. One milliliter blood samples were collected from each rat, designed as P1 , and they were gavaged with distilled water. The duration of pre-treatment period was for three-consecutive estrous cycles. The treatment period commenced when the rats showed a proestrous phase on the 4th estrous cycle, when blood samples (P4 ) were collected as above and the rats were either gavaged with distilled water (DW), Butea superba tuber powder suspension (BS-10, -50 and -250) or injected with TP for six consecutive estrous cycles. Blood samples were further collected on the proestrous phase of the 7th and 10th estrous cycles (P7 and

P10 ), respectively. At the end of treatment period, half of rats (i.e. ve rats from each group) were randomly selected and euthanized under the ether. The ovary and uterus were dissected out, weighed and then xed in 10% (w/v) neutral buffered formalin solution and manipulated for histological examination as described previously (Jaroenporn et al., 2007). The remaining ve rats in each group were subjected to the post-treatment period. In the post-treatment period, rats from all groups were gavaged with distilled water only for three consecutive estrous cycles. On the proestrous phase of the last estrous cycle (the 13th estrous cycle, P13 ) rats were collected, a blood sample taken and the rats euthanized. The ovary and uterus were dissected out, weighed and xed as described. All the blood samples were immediately separated after harvesting by centrifugation at 1000 g at 4 C for 20 min and the sera used for determination of the LH and follicle stimulating hormone (FSH) levels. Throughout the experimental periods all rats were weighed once a week and the vaginal cytology was checked daily as described previously (Malaivijitnond et al., 2006). 2.2.2. Ovariectomy group During the pre-treatment period when the rats showed a diestrous phase on the rst estrous cycle they were collected, a blood sample was taken and then they were ovariectomized, and this day was designed as D14 . They were kept for 14 days recovery period before submitting to the study. The experimental protocol in this group was similar to the intact group; however, the durations for pre-treatment, treatment and post-treatment period were 15, 30 and 15 days, respectively, and so blood samples were collected from the rats every 15 days which was designed as D1 , D16 , D31 , D46 and D61 . 2.3. The preparation of Butea superba suspensions The tuberous roots of Butea superba Roxb. were collected from Lampang Province, Thailand during summer 2000 (voucher specimen no. BCU 11046, Cherdshewasart et al., 2004a). The Butea superba roots used throughout this study were the same lot. The root was sliced and dried at 7080 C, pulverized in a mortar, and ltered through a 100- m mesh. The ltered powder was kept in dark bottles as a stock at room temperature. During treatment, the dried powder of Butea superba was mixed with distilled water to a stock suspension from which the required dilutions were made to a nal volume of 0.7 ml of a nal dose of 0, 10, 50 and 250 mg/kg BW. The suspension was administered to the rats during 08000900 h using a gastric feeding needle. 2.4. The preparation of testosterone propionate Testosterone propionate powder (Sigma, Merck, USA) was weighed and dissolved in a small volume of absolute ethanol. After the powder was completely dissolved, the sesame oil was added and the solution was allowed to stand at room temperature to evaporate the ethanol. This stock solution was then diluted with sesame oil to give a nal dose of 6 mg/kg BW/day/200 l sesame oil. The stock testosterone propionate solution was kept in the dark bottles at room temperature until used. The solution was subcutaneously injected to rats between 0800 and 0900 h. 2.5. Histological analysis After overnight xation in formalin, the ovary and uterus were dehydrated in a series of ethanol gradients, cleared in xylene and embedded and blocked in parafn prior to slicing into 5 m sections and staining with hematoxylin and eosin as reported (Humason, 1972; Jaroenporn et al., 2007). The permanent slides of

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ovary and uterus sections were then examined under an Olympus light microscope and photographed. 2.6. Serum LH and FSH level determination The concentrations of rat serum FSH and LH were measured by radioimmunoassay techniques using reagents obtained from the National Hormone and Pituitary Program. Iodination preparations were rat NIDDK-rat FSH-I-5 and rat LH-I-5. The antisera were antirat FSH-S11 and anti-rat LH-S11. The results obtained are expressed in termed of the rat FSH-RP-2 and rat LH-RP-3 reference standards (Malaivijitnond et al., 2004). To minimize interassay variations, all samples were run in a single assay. The intraassay coefcients of variation of FSH and LH were 7.27 and 3.10%, respectively. 2.7. Statistical analysis The data are expressed as the mean 1 S.E.M. Comparison of the ovary and uterine weights collected at the end of treatment and post-treatment periods were analyzed by Students t-tests. Differences in serum hormone levels between the pre-treatment, treatment and post-treatment periods in each group, and between the DW, BS and TP groups in each experimental period, were analyzed by one-way analysis of variance (ANOVA) with post hoc test by LSD test. In all cases signicance was set at p < 0.05. 3. Results 3.1. Effects of Butea superba on serum gonadotropin levels and reproductive organs in intact (normal) female rats 3.1.1. Serum FSH and LH levels The average serum FSH levels in rats from the control (DW) and all three Butea superba dose (BS) groups revealed no signicant difference throughout the study period, both within each treatment group over time, and between treatment groups (all p > 0.05). Serum FSH levels were signicantly decreased (p < 0.01) in the TP group, from P7 of treatment period to P13 of post-treatment period (Fig. 1), and this decrease was signicantly lower than that seen in the control (DW) and all three doses of the BS groups (p < 0.01). The patterns of changes of serum LH levels in all ve groups of intact rats were broadly similar to those of FSH levels (Fig. 1), with no signicant changes within or between the control (DW) and all treatment (BS) groups throughout the 13 assayed estrous cycles, but with a signicant decrease in the sera LH levels being observed in the TP group from P7 to P13 . 3.1.2. Body weights Relative to day 1, the mean rat body weights increased slightly, but not statistically signicantly, over the 50 days period for the control (DW) and all three BS treatment groups, but showed no signicant difference between each other or the control (DW) group at each time point throughout the study period. In contrast, the mean body weights of rats in the TP group increased more than the control and BS treatment groups and, indeed, from day 29 and 36 onwards were signicantly heavier than at the start and than the control and BS groups, respectively (Fig. 2). 3.1.3. Relative weights of reproductive organs and estrous cycles There were no signicant differences in the average ovarian or uterine wet weights either between the control (DW) and all three BS groups, or between the treatment and post-treatment periods in the control and each BS group (data not shown). Again, in contrast, the average wet ovarian weights in the TP group at both treatment and post-treatment periods were signicantly lower than
Fig. 1. Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels in intact female rats treated with distilled water (DW), Butea superba tuber powder at 10, 50 and 250 mg/kg BW/day (BS-10, -50 and -250, respectively) and testosterone propionate at 6 mg/kg BW/day (TP). The blood samples were collected on the proestrous phase (PX ) of the estrous cycle. The data are expressed as the mean 1 S.E.M., except for those of all three BS groups that the S.E.M. values do not be shown because the lines were overlapped and difcult to understand. * and **p < 0.05 and 0.01, respectively compared to the DW group.

those of the DW and BS groups and, additionally, the weight in the treatment period was signicantly lower than the post-treatment period. However, uterine weights in the TP group differed between treatment periods, being signicantly higher than DW in the treatment period but declining to normal by the post-treatment period (data not shown).

Fig. 2. Body weights in intact female rats treated with distilled water (DW), Butea superba tuber powder at 10, 50 and 250 mg/kg BW/day (BS-10, -50 and -250, respectively) and testosterone propionate at 6 mg/kg BW/day (TP). The data are expressed only the mean because the S.E. values were overlapped and difcult to understand. *p < 0.05 compared to pre-treatment level (day 1). a p < 0.05 compared to the DW group.

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Fig. 3. Daily monitoring of vaginal cytology in intact female rats treated with distilled water (DW), Butea superba tuber powder at 10, 50 and 250 mg/kg BW/day (BS-10, -50 and -250, respectively) and testosterone propionate at 6 mg/kg BW/day (TP). P, E and D = proestrous, estrous and diestrous phases, respectively. Thick and thin lines represent the treatment and post-treatment period, respectively.

Rats treated with DW, BS-10 and BS-50 showed regular estrous cycles of 45 days throughout the study period (Fig. 3). However, all the rats in the BS-250 group showed irregular estrous cycles with a prolonged diestrous phase (leukocyte cells) during the treatment period, and this was particularly marked in 4/10 of the rats. However, all rats in the BS-250 group returned to regular estrous cycles during the post-treatment period. In contrast, the TP group rats showed anestrous cycles with the appearance of leukocyte cells after the rst estrous cycle of treatment period, and did not recover normal estrous cycles in the post-treatment period studied.

3.2. The effects of Butea superba on serum gonadotropin levels and reproductive organs in ovariectomized rats 3.2.1. Serum FSH and LH levels On the day that rats were in the diestrous phase of the estrous cycle and were ovariectomized (D14 ), serum FSH levels in all ve groups of rats were signicantly lower than in their normal intact counterparts which were collected the blood samples on the proestrous phase (D1 ) (1.77 0.19 ng/ml at D14 of ovariectomized rats compared to 8.42 0.41 ng/ml at D1 of intact rats). However, this increased dramatically to levels higher than that seen in normal intact rats by D1 , 14 days after the ovariectomy (1.77 0.19 ng/ml at D14 and 20.70 1.53 ng/ml at D1 ). Compared to the pre-treatment levels (D1 ), serum FSH levels in DW and all three BS groups continued to increase over the next 31 days and thereafter tended to asymptote from day 46 to 61 (Fig. 5), with no signicant differences in serum FSH levels between rats in the DW and all three BS groups. As observed with the normal (intact) rats, serum FSH levels in TP group also congruently rose over the rst 16 days of the pre-treatment period, they signicantly decreased after initiation of treatment (D31 , 15 days after is the rst time point assayed) until the last day of the study period (D61 ). Serum LH levels in all ve groups of rats also signicantly increased in the 14 days after the ovariectomy (0.41 0.11 ng/ml at D14 and 16.83 3.29 ng/ml at D1 , respectively) and continued to increase in the 15 days of pre-treatment period (Fig. 5). However, although compared to the pre-treatment levels (D1 ), serum LH lev-

3.1.4. Histology of ovary and uterus The histology of the ovaries in all ve groups studied (DW, BS-10, BS-50, BS-250 and TP) showed a normal structure with numerous primary, secondary and Graaan follicles and with no signicant differences between groups (data not shown). The histology of the uterus in the BS-10 and BS-50 groups showed a simple columnar epithelium during the treatment period, but a lower density of uterine glands was observed compared to the DW group (Fig. 4). In contrast, the simple columnar epithelium, number of uterine glands and uterine endometrial and myometrial thickness in BS-250 group as well as in TP group increased compared to the DW group and reverted back to normal morphology and levels during the post-treatment period.

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Fig. 4. Uterus morphology in intact and ovariectomized female rats treated with distilled water (DW), Butea superba tuber powder at 10, 50 and 250 mg/kg BW/day (BS-10, -50 and -250, respectively) and testosterone propionate at 6 mg/kg BW/day (TP) after treatment period. EP = epithelial cell, E = endometrium, L = uterine lumen, and U = uterine gland. Scale bars = 50 m.

els in the control and all three BS groups continued to signicantly increase (p < 0.01) up to D31 and reached a plateau thereafter. On the other hand, serum LH levels in TP group signicantly declined to the D14 level after 15 days of TP injection (D31 ) until the last day of study period (D61 ). There were no signicant differences in serum LH levels between DW and BS-10 groups. The levels attained in BS-50 (at D31 D61 ) and BS-250 (at D31 ) were signicantly higher than the control (DW) group.

3.2.2. Body weights, uterine weights and estrous cycle After ovariectomy, the body weights of rats in DW, BS and TP groups signicantly increased throughout the study period, and the body weights of the ovariectomized rats at the end of study period (Fig. 6) were higher than those of the intact rats (Fig. 2). However, there were no signicant differences between the body weights of rats in different treatment groups at all time points (Fig. 6). There was neither any signicant difference in the relative uterine weights between treatment and post-treatment periods in DW and all three BS groups, nor between the same four treatment groups at the same period of study (data not shown). As with normal (intact) rats, the uterine weights of the TP group rats were signicantly increased in comparison to those of the other four treatment groups, and declined signicantly in the post-treatment period. However, in the case of the ovariectomized rats despite the marked decline between treatment and post-treatment periods, the posttreatment period uterine weights of ovariectomized rats were still

Fig. 5. Serum follicle stimulating hormone and luteinizing hormone levels in ovariectomized rats treated with distilled water (DW), Butea superba tuber powder at 10, 50 and 250 mg/kg BW/day (BS-10, -50 and -250, respectively) and testosterone propionate at 6 mg/kg BW/day (TP). The data are expressed as the mean 1 S.E.M., except for those of all three BS groups that the S.E.M. values do not be shown because the lines were overlapped and difcult to understand. OVX = ovariectomy. * and **p < 0.05 and 0.01, respectively compared to the DW group.

Fig. 6. Body weights in ovariectomized rats treated with distilled water (DW), Butea superba tuber powder at 10, 50 and 250 mg/kg BW/day (BS-10, -50 and -250, respectively) and testosterone propionate at 6 mg/kg BW/day (TP). The data are expressed only the mean because the S.E. values were overlapped and difcult to understand. OVX = ovariectomy. *p < 0.05 compared to pre-treatment level (day 1).

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signicantly higher than those of the corresponding rats from the DW and BS groups. After the ovariectomy, which was conducted on the diestrous phase (D14 ), rats from all ve different treatments showed only leukocyte cells in vaginal smears throughout the treatment and post-treatment study periods (data not shown). 3.2.3. Histology of uterus Ovariectomized rats had a smaller size of uterus than those of the intact cyclic rats (Fig. 4). Rats from the DW and BS-10 groups in the treatment period showed thin endometrial mucosa layers, the epithelial lining of which was a layer of cuboidal cells of low height. The number and diameter of uterine glands also decreased. However, in the BS-50 and BS-250 groups, the endometrium and myometrium of the uterus were thicker than those of DW and BS-10 groups. The number and diameter of uterine glands in BS-50 group were not signicantly different from the DW group, but were significantly increased in the BS-250 group. The size of uterus in TP group was larger than those of the DW and all three BS groups, whilst the epithelial lining, uterine endometrial and myometrial thickness and number and diameter of uterine glands were all higher than those of the DW group. In general, the uterus histology in DW, BS and TP groups at the post-treatment period showed the same changes as in the treatment period with no signicant differences between treatment and post-treatment periods being observed in all ve groups of rats. The exceptions are that the number and diameter of uterine glands in the BS-50, BS-250 and TP groups in the post-treatment period were lower than those of the treatment period, and the TP group also showed a decrease in epithelial cell lining and uterine size in the post-treatment period. 4. Discussion The androgenic and/or anti-estrogenic effects of Butea superba on serum gonadotropin levels and reproductive organs in adult cyclic female and ovariectomized rats were examined. Distilled water (DW) and testosterone propionate (6 mg/kg BW/day) (TP) were used as a negative control and a positive control of androgenic or anti-estrogenic activity, respectively. The estrogenic activity of Butea superba in this study was compared with previous results where the estrogenic activity of synthetic estrogens and Pueraria mirica tuberous powders were tested on gonadotropin levels and reproductive organs (Malaivijitnond et al., 2004, 2006; Jaroenporn et al., 2006, 2007). Generally, the doses recommended in traditional medicine for Butea superba in men are 5001000 mg/50 kg BW/day or 1020 mg/kg BW/day (Cherdshewasart and Nimsakul, 2003). Additionally, Manosroi et al. (2006) reported that feeding of Butea superba at the doses of 2, 25, 250 and 1250 mg/kg BW for 8 weeks to rats had no effect on blood cell counts, and liver and kidney function. Thus the doses of Butea superba used in the present study were 10, 50 and 250 mg/kg BW. In adult cyclic (intact) rats, we found that Butea superba in the dose range of 10250 mg/kg BW/day had no effect on secretion of the FSH and LH from the pituitary gland. Certainly, serum levels of both LH and FSH were signicantly decreased in rats from the TP group when compared to those of the DW group. TP has been reported to show an androgenic effect to suppress the secretion of FSH and LH from the pituitary and GnRH from the hypothalamus (Porkka-Heiskanen et al., 1992). Body weights of the adult cyclic rats in all BS groups did not signicantly differ from those in the DW group, whereas the body weight of rats in the TP group were signicantly higher, in agreement with the fact that androgenic substances increase food intake and that TP is a potent anabolic

steroid (Earley and Leonard, 1979). An increase in circulating testosterone concentration results in an increase in fat-free mass and muscle size (Griggs et al., 1989). From these results (FSH and LH sera levels and body weight), it appears that Butea superba at the doses of 10250 does not have a signicant androgenic effect on intact female rats. Although no signicant change was observed in uterine weight following Butea superba treatment, at the highest dose tested (250 mg/kg BW/day) the number of uterine glands and the uterine endometrial and myometrial thickness were higher than those of the control group during the treatment period. In agreement with the increase in the number of uterine glands, the uterine weight of rats fed with 6 mg/kg BW/day of TP also increased during the treatment period, in accord with the report that subcutaneous injections of TP at 5 mg/kg BW/day for 4 days signicantly increased the uterine weight in adult ovariectomized rats (Lundeen et al., 1997). Although the uterus is a classical 17 -estradiol target tissue, which has been used intensively to study gonadal-hormone-regulated cell proliferation, the uterotropic effect caused by the TP is likely to be mediated via the androgen receptor (AR) (Weihua et al., 2002; Nantermet et al., 2005). The uterus is organized into the endometrium, which is comprised of secretory epithelial cells and surrounding mesenchymal cells, and the myometrium, an outer muscle layer. In females, AR is abundant in this organ, being found exclusively in the stroma, and mainly in the myometrium, where they colocalized with the ER (Weihua et al., 2002). In addition, AR interacts with ER /ER in controlling cell proliferation in the uterus (Weihua et al., 2002; Nantermet et al., 2005). Thus mibolerone, a nonaromatizable AR-selective agonist, induced dosedependent increases in the total uterine area, as increases in both endometrial and myometrial cross-sectional area, mirroring the observed changes in uterine weight. Moreover, the endometrial gland cells remained relatively cuboidal after mibolerone treatment (Nantermet et al., 2005), which is similar to that observed in this study in both the TP and BS-250 treatment groups. In addition, the BS-250 group showed a prolonged diestrous phase of the estrous cycle which is similar to the persistent diestrous phase, with only leukocyte cells being observed, in the TP group. The persistent diestrous phase in the TP group is likely to have been induced by the androgenic activity of TP on the suppression of gonadotropin secretion and the consequent dysfunction of ovary in intact rats. Accordingly, TP at 6 mg/kg BW/day decreased the relative ovarian weights in this study. Taken together it is plausible that Butea superba has an androgenic effect directly on the uterus, and indirectly on the vaginal epithelium cells via the suppression of gonadotropin secretion, although the inuence was not strong enough to see a reduction in the serum gonadotropin levels. That the ovariectomies were complete was supported by the increase in serum FSH and LH levels (Porkka-Heiskanen et al., 1992). No effect of Butea superba on serum FSH and LH levels in the ovariectomized rats was observed in the BS-10 group, but in the BS-50 and BS-250 groups the serum LH levels were increased after Butea superba treatment for 15 days. This differs from the observation that in male rats that LH levels were decreased after a 90-day treatment with 150 and 200 mg/kg BW/day of Butea superba (Cherdshewasart et al., 2008). This discrepancy might be due to the shorter duration of treatment in this study (30 days), or to the gender of the animals used. However, since neither males were used in this study, nor ovariectomized females in that of Cherdshewasart et al. (2008), this discrepancy remains unresolved and it remains plausible that Butea superba might express an anti-estrogenic effect on LH levels in ovariectomized rats. Estrogens exhibit some catabolic activities (Earley and Leonard, 1979). Thus, a body weight gain can be observed in the ovariec-

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tomized rats in this study. In contrast, the body weights of ovariectomized rats were decreased after feeding with Pueraria mirica, a phytoestrogens-rich herb, or treatment with synthetic estrogens (Earley and Leonard, 1979; Urasopon et al., 2008). In contrast to the increase in body weights in intact female rats, there were no changes in the body weights in ovariectomized rats after either TP or BS treatment. Therefore, Butea superba potentially exhibited a similar androgenic activity on the body weights of the ovariectomized rats as TP. In all three BS and TP groups, the rat vaginal smears were only leukocyte cells throughout the experimental treatment and posttreatment periods, as found in rats from the DW group. This cell type appears in the diestrous stage of estrous cycle in the rat when the levels of endogenous estrogen or estrogenic substances in serum are very low. This result thus supports the notion that Butea superba at doses of 10250 mg/kg BW/day did not have any estrogenic activity on vaginal epithelium cells. In contrast, the number of uterine glands and the uterine thickness were increased in the BS-50 and BS-250 groups as well as in the TP group. Taken the above results together, we conclude that Butea superba is likely to have an androgenic effect on the reproductive organs in intact and ovariectomized female rats, and that it can exhibit the anti-estrogenic activity on LH secretion in ovariectomized rats. That the androgenic activity of Butea superba is elicited in females as well as in males makes it a choice of potential androgenic plants. Acknowledgements The authors thank Dr. Robert Butcher, Faculty of Science, Chulalongkorn University for proofreading of the manuscript. This study was supported in part by Interdepartment of Physiology, the Graduate School, and the grant for Primate Research Unit, Chulalongkorn University, Thailand and a Grants-in-Aid for Scientic Research (The 21st Century Center of Excellence Program, E-1) from the Ministry of Education, Culture, Sport, Science and Technology of Japan. References
Cherdshewasart, W., Nimsakul, N., 2003. Clinical trial Butea superba, an alternative herbal treatment for erectile dysfunction. Asian Journal of Andrology 5, 243246. Cherdshewasart, W., Cheewasopit, W., Picha, P., 2004a. The differential antiproliferation effect of white (Pueraria mirica), red (Butea superba), and black (Mucuna collettii) Kwao Krua plants on the growth of MCF-7 cells. Journal of Ethnopharmacology 93, 255260. Cherdshewasart, W., Cheewasopit, W., Picha, P., 2004b. Anti-proliferation effects of the white (Pueraria mirica), red (Butea superba), and black (Mucuna collettii) Kwao Krua plants on the growth of HeLa cells. Journal of Scientic Research of Chulalongkorn University 29, 2732. Cherdshewasart, W., Bhuntaku, P., Panriansaen, R., Dahlan, W., Malaivijitnond, S., 2008. Androgen disruption and toxicity tests of Butea superba Roxb., a traditional herb used for treatment of erectile dysfunction, in male rats. Maturitas 60, 131137. Earley, C.J., Leonard, B.E., 1979. Androgens, estrogens and their anti-hormones: effects on body weight and food consumption. Pharmacology, Biochemistry and Behavior 11, 211214.

Griggs, R.C., Kingston, W., Jozefowicz, R.F., Herr, B.E., Forbes, G., Halliday, D., 1989. Effect of testosterone on muscle mass and muscle protein synthesis. Journal of Applied Physiology 66, 498503. Humason, G.L., 1972. Animal Tissue Techniques. W.H. Freeman and Company, USA. Ingkaninan, K., Temkitthawon, P., Chuenchon, K., Yuyaem, T., Thongnoi, W., 2003. Screening for acetylcholinesterase inhibitory activity in plants used in Thai traditional rejuvenating and neurotonic remedies. Journal of Ethnophamacology 89, 261264. Jaroenporn, S., Malaivijitnond, S., Wattanasirmkit, K., Cherdshewasart, W., Watanabe, G., Taya, K., 2007. Assessment of fertility and reproductive toxicity in adult female mice after long-term exposure to Pueraria mirica. Journal of Reproduction and Development 53, 9951005. Jaroenporn, S., Malaivijitnond, S., Wattanasirmkit, K., Trisomboon, H., Watanabe, G., Taya, K., Cherdshewasart, W., 2006. Effects of Pueraria mirica, an herb containing phytoestrogens, on reproductive organs and fertility of adult male mice. Endocrine 30, 93101. Lundeen, S.G., Carver, J.M., McKean, M.-L., Winneker, R.C., 1997. Characterization of the ovariectomized rat model for the evaluation of estrogen effects on plasma cholesterol levels. Endocrinology 138, 15521558. Malaivijitnond, S., Chansri, K., Kijkuokul, P., Urasopon, N., Cherdshewasart, W., 2006. Using vaginal cytology to assess the estrogenic activity of phytoestrogen-rich herb. Journal of Ethnopharmacology 107, 354360. Malaivijitnond, S., Kiatthaipipat, P., Cherdshewasart, W., Watanabe, G., Taya, K., 2004. Different effects of Pueraria mirica, an herb containing phytoestrogens, on LH and FSH secretion in gonadectomized female and male rats. Journal of Pharmacological Sciences 96, 428435. Manosroi, A., Sanphet, K., Saowakon, S., Aritajat, S., Manosroi, J., 2006. Effects of Butea superba on reproductive systems of rats. Fitoterapia 77, 435438. Nantermet, P.V., Masarachia, P., Gentile, M.A., Pennypacker, B., Xu, J., Holder, D., Gerhold, D., Towler, D., Schmidt, A., Kimmel, D.B., Freedman, L.P., Harada, S., Ray, W.J., 2005. Androgenic induction of growth and differentiation in the rodent uterus involves the modulation of estrogen-regulated genetic pathways. Endocrinology 146, 564578. Porkka-Heiskanen, T., Laakso, M.-L., Stenberg, D., 1992. The effect of testosterone on serum gonadotropins of castrated rats kept under different lighting conditions. Biology of Reproduction 46, 11271129. Roengsumran, S., Petsom, A., Ngamrojanavanich, N., Rugsilp, T., Sittiwichienwong, P., Khorphueng, P., Cherdshewasart, W., Chaichantipyuth, C., 2000. Flavonoid and avonoid glycoside from Butea superba Roxb and their cAMP phosphodiesterase inhibitory activity. Journal of Scientic Research of Chulalongkorn University 25, 169176. Suntara, A., 1931. The Remedy Pamphlet of Kwao Kure Tuber of Luang Anusarnsuntarakromkarnpisit. Chiang Mai Upatipongsa Press, Chiang Mai, Thailand. Tocharus, C., Smitasiri, Y., Jeenapongsa, R., 2006. Butea superba Roxb. enhances penile erection in rats. Phytotherapy Research 20, 484489. Trisomboon, H., Malaivijitnond, S., Watanabe, G., Taya, K., 2004a. Estrogenic effect of Pueraria mirica on the menstrual cycle and hormones related ovarian function in cyclic female cynomolgus monkeys. Journal of Pharmacological Sciences 94, 5159. Trisomboon, H., Malaivijitnond, S., Suzuki, J., Hamada, Y., Watanabe, G., Taya, K., 2004b. Long-term treatment effects of Pueraria mirica phytoestrogens on parathyroid hormone and calcium levels in aged menopausal cynomolgus monkeys. Journal of Reproduction and Development 50, 639645. Trisomboon, H., Malaivijitnond, S., Watanabe, G., Taya, K., 2005. Ovulation block by Pueraria mirica: a study of its endocrinological effect in female monkeys. Endocrine 26, 3340. Trisomboon, H., Malaivijitnond, S., Watanabe, G., Cherdshewasart, W., Taya, K., 2006. The Estrogenic effect of Pueraria mirica on gonadotrophin levels in aged monkeys. Endocrine 1, 129134. Urasopon, N., Hamada, Y., Asaoka, K., Cherdshewasart, W., Malaivijitnond, S., 2007. Pueraria mirica, a phytoestrogen-rich herb, prevents bone loss in orchidectomized rats. Maturitas 56, 322331. Urasopon, N., Hamada, Y., Cherdshewasart, W., Malaivijitnond, S., 2008. Pueraria mirica, a phytoestrogen-rich herb, prevents bone loss in ovariectomized rats. Maturitas 59, 137148. Weihua, Z., Ekman, J., Almkvist, A., Saji, S., Wang, L., Warner, M., Gustafsson, J.-A., 2002. Involvement of androgen receptor in 17 -estradiol-induced cell proliferation in rat uterus. Biology of Reproduction 67, 616623.