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Maturitas 60 (2008) 131137

Androgen disruption and toxicity tests of Butea superba Roxb., a traditional herb used for treatment of erectile dysfunction, in male rats
Wichai Cherdshewasart a, , Papong Bhuntaku a,b , Rattana Panriansaen c,d , Winai Dahlan e , Suchinda Malaivijitnond a,b
c

Department of Biology, Faculty of Science, Chulalongkorn University, Phyathai Road, Patumwan, Bangkok 10330, Thailand Primate Research Unit, Faculty of Science, Chulalongkorn University, Phyathai Road, Patumwan, Bangkok 10330, Thailand Program of Biotechnology, Faculty of Science, Chulalongkorn University, Phyathai Road, Patumwan, Bangkok 10330, Thailand d Program of Applied Thai Traditional Medicine, Faculty of Science and Technology, Suan Sunandha Rajabhat University, Dusit, Bangkok 10300, Thailand e Lipid and Fat Sciences Research Center, Faculty of Allied Health Science, Chulalongkorn University, Phyathai Road, Patumwan, Bangkok 10330, Thailand
b

Received 13 March 2008; received in revised form 20 April 2008; accepted 24 April 2008

Abstract Objective: To evaluate the effects of the tuberous powder of Butea superba Roxb. (Leguminosae) on blood testosterone and luteinizing hormone (LH), and toxicity in male rats. Methods: Adult male Wistar rats were orally treated with 0, 10, 100, 150 or 200 mg/kg BW/day of B. superba powder suspension in 0.7 ml distilled water for 90 consecutive days. Blood samples were collected every 30 days and submitted to testosterone and LH analysis. On the 90th day of treatment, blood and the main organs were collected for haematological and histopathological analysis, respectively. Results: The adverse effects found included an increase in spleen relative weight, and increased serum level of alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in rats treated with 150 mg/kg BW/day B. superba powder. At 200 mg/kg BW/day treatment, rats showed signicant decreased and increased blood levels of neutrophil and eosinophil, respectively, and a decrease in serum creatinine levels. Serum hormonal analysis revealed a dose-dependent decrease in testosterone, but not LH, in rats treated with 150 and 200 mg/kg BW/day B. superba powder. Conclusion: Subchronic treatment of B. superba tuberous powder suspension at high doses in male rats exhibited adverse effects to blood chemistry, haematology, and blood testosterone level. The results of the study should initiate awareness of the possible adverse risk of over-dose consumption of B. superba products for treatment of erectile dysfunction (ED) in mature males. 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Androgen disruption; Butea superba; Flavonoids; Phytoestrogen; Testosterone

Corresponding author. Tel.: +66 2 2185379; fax: +66 2 2185386. E-mail address: cwichai@sc.chula.ac.th (W. Cherdshewasart).

0378-5122/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2008.04.011

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1. Introduction Hormonal disruption is currently being investigated because it may initiate a biphasic response in the human body, disturbance of endocrine function and/or hormonal replacement action. Estrogen disruption is the most investigated case, especially that resulting from phytoestrogens [1], which can cause an adverse animal physiology such as sex hormone uctuation [2]. Phytoestrogens initiate hormonal replacement actions in impaired estrogen synthesis animals for example, ovariectomized rats [35] and aging monkeys [6,7], but also initiate hormonal disruption in normal female including monkeys [810] and rats [11]. Androgen disruption was rst recorded in sh, caused by either androgenic or anti-estrogenic substance in the environment [12] but currently no signicant effect in higher vertebrates has been reported. Butea superba Roxb. (Leguminosae) is commonly found in Thai deciduous forests and has the domestic name of Red Kwao Krua. The plant tubers have long been consumed as a traditional medicine for the promotion of male sexual vigor. The efcacy of the plant powder has been demonstrated in a human clinical trial with effective treatment of erectile dysfunction (ED) in Thai males [13] comparable to that seen in a worldwide clinical trial-based evaluation of sildenal [14]. Recently, the plant products are becoming popular as traditional and alternative medicines, dietary supplements and topical products including for the promotion of male vigor. The Thai FDA has approved many traditional recipes that are comprised of B. superba as a major ingredient with limited claims for tonic purposes. We therefore setup a rat subchronic study to evaluate the possible toxicity and hormonal, especially testosterone and luteinizing hormone (LH) disruption. The results might open the possibility of not only to x a safe dose for human consumption but also to establish the rst record of androgen disruption in higher vertebrate studies.

was done in comparison with the voucher specimen no. BCU 1046 [15]. The tubers were washed, sliced, ovendried at 70 C, and powdered to a size of 100 mesh. The freshly prepared suspension, by dissolving plant powder with distilled water in a nal volume of 0.7 ml, was used for oral administration. 2.2. Animals and treatment Adult male Wistar rats, 130 10 days of age, were obtained from the National Laboratory Animal Center, Mahidol University, Thailand. The animals were housed in stainless cages with sawdust bedding at ve animals/cage, in a light- and temperature-controlled room (light on 06.0020.00 h) temperature 25 1 C at the Primate Research Unit, Chulalongkorn University, Bangkok, Thailand. Rat chow diet (Pokaphan Animal Feed Co., Ltd., Bangkok, Thailand) and water were provided ad libitum. The experimental protocol was approved in accordance with guideline for the care and use of laboratory animals by Chulalongkorn University. Seventy-ve adult male rats of body weight 280300 g were divided into ve groups. Fifteen rats per group were orally treated with 0 mg/kg BW (BS-0), 10 mg/kg BW (BS-10), 100 mg/kg BW (BS-100), 150 mg/kg BW (BS-150) or 200 mg/kg BW (BS-200) of B. superba powder suspension for 90 consecutive days. The rat body weights were recorded weekly. 2.3. Haematology and blood chemistry At the end of the experiment, blood samples were collected by cardiac puncture under ether anesthesia from 9.00 to 10.00 a.m. and were partly used for haematology. From the remainder blood serum was prepared by centrifugation at 1000 g for 30 min and kept at 20 C for blood chemistry analysis, including blood urea nitrogen (BUN), creatinine, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), cholesterol, and triglyceride. The assays were performed with automated analytical systems at the service laboratory of the Lipid and Fat Sciences Research Center, Faculty of Allied Health Science, Chulalongkorn University. Fifteen replicates of serum from each of the treated groups and control group were run at the same analysis.

2. Materials and methods 2.1. Plant materials Fresh tubers of B. superba were collected in Lampang Province, Thailand. The identication of the plant

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2.4. Necropsy After the blood sampling, the rats were kept under anesthesia and were subjected to necropsy. The brain, liver, kidney, spleen, testis and epididymis were removed and weighed. Body weight measured on the day of necropsy was used to calculate the relative organ weight. The organs were preserved in 10% (w/v) neutral phosphate buffer formalin. Liver, kidney, spleen, and testis xed-tissue were embedded in parafn and submitted for microtome sectioning at 5 m and staining with haematoxylin and eosin. The pathology of the tissue slides was examined under a light microscope. 2.5. Hormonal assays Serum LH levels were measured using NIDDK kits for rat LH as previously described [4] using rat LH-RP2 for iodination and anti-rat LH-S11 antiserum. The results obtained are expressed in terms of the rat LHRP-2 reference standards. Serum testosterone levels were measured by the established radioimmunoassay method of the World Health Organization after samples were extracted by diethyl ether [16,17]. To minimize the inter-assay variation, all samples were assayed in a single run for each hormone. The intra-assay coefcients of variation were 5.7% for LH and 7.0% for testosterone. 2.6. Statistical analysis Data are presented as the mean S.E.M., and a test of homogeneity of variance was performed followed by the least signicant difference (LSD) test and Duncans multiple range tests by SPSS program. A value of P < 0.05 was considered statistically signicant.

Fig. 1. Body weight of male rats treated with B. superba for 90 consecutive days in comparison with the negative control.

with 150 mg/kg BW (Table 1). However, the apparent increase in spleen weight showed no apparent dose-dependency and remains to be conrmed. The histology of the spleen, liver, kidney and testes all exhibited a normal appearance in comparison with the control. 3.2. Blood chemistry and haematology The blood chemistry marker assayed showed no clear dose-dependent or signicant changes with increasing B. superba powder suspension doses except for a biphasic increase in ALP and AST levels which were signicant only at 150 mg/kg BW (Table 2) and creatinine which was decreased in comparison with the control at the treated dose of 200 mg/kg BW, with no evidence of a dose-dependent at least within this range of treatment time scale and administered doses. 3.3. Testosterone and LH There was a potentially dose-dependent decrease in testosterone levels which was signicantly different from the control (P < 0.05) at the two highest tested doses (150 and 200 mg/kg BW) (Table 3). In contrast, a slight but dose-dependent observed decrease in the LH levels was however, insignicant (P > 0.05).

3. Results 3.1. Animal growth and histology analysis There was no signicant difference in either the growth rate measured by living body weight (Fig. 1) or the relative weight of the main body organs of male rats treated with all doses of B. superba, when compared with the control except for the increase in relative weight of the spleen (13.67%) in rats treated

4. Discussion Treatment with B. superba powder as an oral administered suspension showed no discernable adverse

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Table 1 Effects of orally administered B. superba powder suspension for 90 days on relative weight of the main organs in male rats Organ Brain Liver Kidney Spleen Testes Epididymis BS-0 5.85 25.20 6.06 1.83 9.76 2.77 0.14 0.56 0.17 0.17 0.26 0.12 BS-10 5.63 25.34 6.07 1.98 9.41 2.74 0.09 0.49 0.09 0.07 0.18 0.10 BS-100 5.78 24.30 5.94 2.00 9.46 2.94 0.12 0.49 0.13 0.11 0.16 0.15 BS-150 55.53 25.03 6.10 2.08 9.01 2.82 0.08 0.11 0.01 0.02* 0.12 0.08 BS-200 5.61 25.70 6.51 1.67 9.77 2.83 0.18 0.52 0.18 0.08 0.26 0.14

The data in each group are derived from 15 rats and are expressed in g/1000 g unit as mean S.E.M. *Signicant difference at P < 0.05. Table 2 Effects of orally administered B. superba powder suspension for 90 days on blood chemistry and haematology in male rats Parameter ALP (U/L) ALT (U/L) AST (U/L) BUN (mg/dl) Creatinine (mg/dl) Uric acid (mg/dl) Triglyceride (mg/dl) Cholesterol (mg/dl) Hematocrit (%) Lymphocyte (%) Monocyte (%) Neutrophil (%) Eosinophil (%) Basophil (%) BS-0 133.47 58.78 167.82 25.81 0.74 1.24 65.09 67.09 45.60 71.29 2.60 25.11 1.00 12.64 3.80 7.34 1.50 0.02 0.13 6.39 2.98 1.18 2.72 0.60 2.52 0.00 BS-10 148.94 60.77 165.00 26.32 0.73 1.46 56.80 65.62 44.13 69.60 4.00 25.40 1.00 14.10 2.33 8.88 1.92 0.02 0.17 3.31 2.55 1.90 3.80 1.00 3.55 0.00 BS-100 161.62 57.67 142.29 25.22 0.74 1.49 57.76 68.02 44.13 71.00 3.20 24.80 1.00 20.28 2.49 7.23 1.71 0.03 0.19 3.08 3.23 1.34 2.60 0.44 2.50 0.00 BS-150 194.67 69.79 218.05 27.59 0.76 1.65 62.56 71.08 43.15 78.27 3.86 19.00 1.00 15.65* 8.15 31.96* 2.43 0.029 0.19 5.94 4.48 0.92 3.11 1.06 3.53 0.00 BS-200 168.50 62.38 120.63 21.46 0.58 1.23 65.75 62.63 47.38 78.70 4.67 15.13 1.50 10.40 2.90 15.56 0.90 0.01* 0.22 5.54 1.99 0.65 2.97 1.36 2.42* 0.05*

The data in each group are derived from 15 rats and are expressed as (w/v) or % in mean S.E.M. *Signicant difference from negative control at P < 0.05.

effect upon male rat growth rates at the doses (0 200 mg/kg BW) and duration (090 days) administered in this study. The results from the haematology showed a signicant adverse effect on blood neutrophil and eosinophil at high (200 mg/kg BW) doses. It is possible that the high doses of plant chemicals could either inuence the respective blood stem cell lineage division and differentiation within the haematopoietic tissues or alternatively, inuence chemokine and cytokine levels or molecular responses to them. Potentially haematological toxicity was also demonstrated with the increase in the relative weight of the spleen because

two of the main functions of the spleen are principally, the destruction of aging erythrocytes and secondarily, humoral immunity. However, the haematocrit value of this treated group retained normal and no dosedependent response was seen with higher and lower doses which in contrast to 150 mg/kg BW showed no signicant difference in spleen weights to the control. The results from the blood chemistry showed the inuence of the high doses of plant chemicals with elevated serum ALP and AST levels, a trait normally associated with liver malfunction. Another potential sign of altered liver malfunction is the decreased level of

Table 3 Effects of orally administered B. superba powder suspension for 90 days on blood testosterone and LH in male rats Hormone (g/ml) Testosterone LH BS-0 129.54 15.78d 0.20 0.02ab BS-10 79.04 5.84bc 0.26 0.04b BS-100 97.22 16.50cd 0.14 0.02ab BS-150 18.47 1.80a 0.11 0.01a BS-200 46.07 3.37ab 0.16 0.05ab

The data in each group are derived from 15 rats and are expressed as g/ml unit in mean S.E.M. Means not sharing a common superscript letter in the same row are signicantly different (P < 0.05) as determined by Duncans multiple range test.

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creatinine after the highest dose of treatment with B. superba. The cytotoxicity of B. superba crude extract has previously been investigated in the antiproliferation effect upon the estrogen-dependent MCF-7 cells, in which the plant extract derived from the same source of plant materials used in this experiment exhibited an IC50 of 370.91 g/ml [15]. Five chemicals, namely medicarpin, formononetin, 7,4 -dimethoxyisoavone, prunetin, and 7-hydroxy6,4 -dimethoxyisoavone were isolated from the plant tubers with formononetin and prunetin exhibiting IC50 values of 37.30 and 71.10 g/ml in tests with the human epidermoid carcinoma of cavity cell line (KB) (ATCC CRL-5804), respectively, and 32.70 and 47.30 g/ml with the breast cancer cell line (BC) (NCL-H 187), respectively [18]. Medicarpin exhibited cytotoxicity in the sea urchin egg assay and tumor cell lines in cultures [19]. 7,4 -Dimethoxyisoavone showed cancer chemopreventive potential in a mouse mammary organ culture assay [20]. Formononetin exhibits estrogenic activity [21] through ER and ER and afnity to progesterone receptor (PR) and androgen receptor (AR) [22]. Testosterone and LH were analyzed in B. superbatreated male rats to screen for potential androgen disruption. The results indicated a dose-dependent decreased of only blood testosterone but not LH and this was signicantly different from the control in the rats treated with high doses of plant powder. The implication is that testosterone disruption is signicant, at least after 90 days of consumption of high doses of B. superba powder. The testosterone disruption clearly seen in this study is an antagonist of the reported androgenic effect initiated by consumption of other plants. Lepidium meyenii (Maca) consumption showed positive androgenic effects on the testes, and epididymis but not the seminal vesicles in humans [23], and to seminal vesicles, sperm count, and sperm motility in male rats [24]. Hibiscus macranthus and Basella alba extracts also exhibited clear androgenic trophic-activity in rat testes by promoting testosterone synthesis [25]. In this study the blood LH level was not signicantly changed after B. superba consumption for 90 days and thus any androgenic disruption had occurred in the treated male rats, it may not have exhibited systemic results, but rather restricted only to certain androgenic tissues. Certainly, histological and weight abnormalities of the testes and epididymis were not found and thus any

androgen disruption was presumably mainly affected altered testosterone biosynthesis or metabolism. Even though testosterone was strongly disrupted in a dosedependent manner, with the reduction of 39.0, 24.9, 85.7 and 64.4% in 10, 100, 150 and 200 mg/kg BW doses, respectively, in comparison with the control, only the later two highest doses were signicantly different from the control. However the male sexual organs were found to be normal in these groups with no signs of necrosis or degradation, suggesting the possibility that these organs could perform their functions normally. If so, this may derive from a supplemental androgenic action of phytochemicals in B. superba similar to the supplemental estrogenic action of phytoestrogens in P. mirica in ovariectomized rats [35]. This would be evidence of the presence of a phytoandrogen in B. superba. However, this experiment was performed in adults, not in aged male rats. Testosterone disruption may not occur in aged male rats at the same high dose of B. superba because of a signicantly lower level of endogenous testosterone. When using any herbal treatment for the purpose of promoting of male sexual potency, there is a need to be wary of not only androgen disruption but also of general adverse effects. In another animal study with a higher dose (1250 mg/kg BW), a different source of plant material (Chiang Mai Province) and a shorter period of treatment (8 weeks), there were no abnormalities found in the prostate gland, seminal vesicle and liver or kidney functions rather only a larger testis size with an increased sperm count was found for the highest dose [26,27]. Herbs contains varied amount of mixed active compounds that are under the inuence of metabolic activation by liver enzymes [28,29]. This may be one reason why the responses of rats treated with B. superba powder were not exactly a dose-dependent. We assigned the dose of 200 mg/kg BW/day as the maximum dose because the tuberous powder of our plant materials could not be completely dissolved at higher concentration due to its high starch content. Nevertheless, B. superba may be an alternative for herbal treatment of ED because Yohimbe, used for the same purpose, also created toxicity at high dose consumption [30]. Even Eurycoma longifolia Jack (Tonkat Ali), an herbal plant used for the promotion of male sexual potency in Malaysia, yielded increasing sexual qualities in middle-aged male rats [31], but as a dose which was higher than B. superba in this

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study. Isoavonolignans and both butesuperins A and B isolated from B. superba tubers showed antioxidant activity but no report on toxicity or hormonal disruption [32]. The study on acetylcholinesterase inhibitor activity of B. superba did not mention any toxicity either [33]. Flavonoid and avonoid glycoside isolated from B. superba tubers show a strong inhibition of the cAMP phosphodiesterase, PDE4 without test results on toxicity and androgen disruption [34]. Taking these data into account, one could postulate that the cytotoxicity, including altered blood chemistry and haematology markers of B. superba were possible partly derived from some of their known chemical ingredients at high doses. There are very few reports on the subtypes of androgen receptors, let alone the interaction with plant androgen mimics [35]. However, some studies on phytochemical interaction with the androgen receptor have been published, for example daidzein is found to be a phytoandrogen [36]. B. superba has not yet been analyzed for androgenic/anti-androgenic activities in vitro. Thus we cannot reliably explain the possible mechanism(s) of the interaction of the phytochemicals in B. superba with the rat androgen receptor(s) in this study. Certainly the human clinical trials of B. superba revealed a signicantly increased in the number of successive male sexual performances with an increased penis erection and successful penis penetration [13] with no signicant change in blood chemistry, haematological analysis and blood testosterone levels. The human trial dose was approximately 20 mg/kg BW/day which is 10 times lower than the dose used in the adult male rats. We conclude that the effective dose for ED treatment in human consumption is far too low to initiate adverse effect to the human body. Human physiology and population genetics are both much more varied than in male inbred rats with additionally greater variance of diets and lifestyles in humans. In this experiment, the male rats were fed with a single-type diet, lived in a controlled environment throughout the 3month period, and thus were subject to far fewer biotic and abiotic variances than the human clinical trials. Thus, the toxicity found in rats after B. superba treatment may not happened in human at the same degree. However, this is to our knowledge, the rst report on the potential androgen disruption in a higher vertebrate supporting that there should be additional studies

in lower vertebrates and other higher vertebrates, especially at the hormonal level including the conrmation of the existence of an androgen-disruption phenomena. No matter which phytochemicals initiate this effect or how it is achieved; the benets of the study will be enormous and benecial to mature males. If the adverse toxicity is limited when consume at a low dose and promotion of male sexual libido without androgen disruption, it is possible that B. superba powder will become an herbal alternative aid to promote male sexual potency without a signicant risk for human health. Besides, the crude extract prepared from B. superba tuberous powder may possible to be manufactured into a topical product to apply directly to the penis to promote in situ erection. This topical and temporally restricted application should help to avoid the main human organs and blood cells from the possible adverse toxicity derived from the continuous long-term oral consumption of high dose of B. superba powder for the purpose of treatment of ED.

Acknowledgements The authors wish to thank the Graduate School and Ratchadaphiseksomphot Endowment Fund of the Research affairs of Chulalongkorn University, and the National Center for Genetic Engineering, National Science and Technology Development Agency, Ministry of Science and Technology, for grant supports.

References
[1] Cornwell T, Cohick W, Raskin I. Dietary phytoestrogen and health. Phytochemistry 2004;65:9951016. [2] Jaroenporn S, Malaivijitnond S, Wattanasirmkit K, et al. Effects of Pueraria mirica, an herb containing phytoestrogens, on reproductive organs and fertility of adult male mice. Endocrine 2006;30:93101. [3] Cherdshewasart W, Kitsamai Y, Malaivijitnond S. Evaluation of the estrogenic activity of the wild Pueraria mirica by vaginal cornication assay. J Reprod Dev 2007;53:38593. [4] Malaivijitnond S, Kiatthaipipat K, Cherdshewasart W, et al. Different effects of Pueraria mirica, a herb containing phytoestrogens, on LH and FSH secretion in gonadectomized female and male rats. J Pharmacol Sci 2004;96:428 35. [5] Malaivijitnond S, Chansri K, Kijkuokul P, et al. Using vaginal cytology to assess the estrogenic activity of phytoestrogen-rich herb. J Ethnopharmacol 2006;107:35460.

W. Cherdshewasart et al. / Maturitas 60 (2008) 131137 [6] Trisomboon H, Malaivijitnond S, Watanabe G, et al. The estrogenic effect of Pueraria mirica on gonadotrophin levels in aged monkeys. Endocrine 2006;29:12934. [7] Trisomboon H, Malaivijitnond S, Cherdshewasart W, et al. The inuence of Pueraria mirica herb containing phytoestrogens on the urinary gonadotropin and estradiol levels in aged menopausal monkeys. Anim Sci J 2007;78:37886. [8] Trisomboon H, Malaivijitnond S, Watanabe G, et al. Estrogenic effect of Pueraria mirica on the menstrual cycle and hormones related ovarian function in cyclic female cynomolgus monkeys. J Pharmacol Sci 2004;94:519. [9] Trisomboon H, Malaivijitnond S, Watanabe G, et al. Ovulation block by Pueraria mirica: a study of its endocrinological effect in female monkeys. Endocrine 2005;26:339. [10] Trisomboon H, Malaivijitnond S, Cherdshewasart W, et al. Assessment of urinary gonadotropin and steroid hormone proles of female cynomolgus monkeys after treatment with Pueraria mirica. J Reprod Dev 2007;53:395403. [11] Jaroenporn S, Malaivijitnond S, Wattanasirmkit K, et al. Assessment of fertility and reproductive toxicity in adult female mice after long-term exposure to Pueraria mirica herb. J Reprod Dev 2007;53:9951005. [12] Subramantan P, Amutha C. Sewage-induced alterations in the sex of Oreochronis mossambicus; a plausible cue of endocrine disruption. Toxicol Environ Chem 2006;8:51526. [13] Cherdshewasart W, Nimsakul N. Clinical trial of Butea superba, an alternative herbal treatment of erectile dysfunction. Asian J Androl 2003;5:2436. [14] Cappelleri JC, Rosen RC. The sexual health inventory for men (SHIM): a 5 year review of research and clinical experience. J Impot Res 2005;17:30719. [15] Cherdshewasart W, Cheewasopit W, Picha P. The differential anti-proliferation effect of white (Pueraria mirica), red (Butea superb), and black (Mucuna collettii) Kwao Krua plants on the growth of MCF-7 cells. J Ethnopharmacol 2004;93:255 60. [16] Su SB, Donaldson A, Jeffcoate SL, editors. Method manual: World Health Organization collaborating center for research and reference services in the immunoassay of hormone in human reproduction. 8th ed. Geneva, Switzerland, 1986. p. 1120. [17] Malaivijitnond S, Varavudhi P. Evidence for morphine-induced galactorrhea in male cynomolgus monkeys. J Med Primatol 1998;27:19. [18] Ngamrojanavanich N, Loontaisong A, Pengpreecha S, et al. Cytotoxic constituents from Butea superba Roxb. J Ethnopharmacol 2007;109:3548. [19] Milit ao GC, Pinheiro SM, Dantas IN, et al. Bioassay-guided fractionation of pterocarpans from roots of Harpalyce brasiliana Benth. Bioorg Med Chem 2007;15:668791. [20] Jang DS, Park EJ, Hawthorne ME, et al. Potential cancer chemopreventive constituents of the seeds of Dipteryx odorata (tonka bean). J Nat Prod 2003;66:5837.

137

[21] Booth NL, Overk CR, Yao P, et al. Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoavones and estrogenic activity. J Agric Food Chem 2006;54:127782. [22] Beck V, Unterrieder E, Krenn L, et al. Comparison of hormonal activity (estrogen, androgen and progestin) of standardized plant extracts for large scale use in hormone replacement therapy. J Steroid Biochem Mol Biol 2003;84:25968. [23] Gonzales GF, Cordova A, Gonzales C, et al. Lepidium meyenii (Maca) improved semen parameters in adult men. Asian J Androl 2001;3:3013. [24] Gonzales GF, Ruiz A, Gonzales C, et al. Effect of Lepidium meyenii (Maca) roots on spermatogenesis of male rats. Asian J Androl 2001;3:2313. [25] Moundipa PF, Ngouela S, Kamchouing P, et al. Effects of extracts from Hibicus macranthus and Basella alba mixture on testosterone production in vitro in adult rat testes. Asian J Androl 2006;8:1124. [26] Manosroi A, Sanphet K, Saowakon S, et al. Effects of Butea superba on reproductive systems in rats. Fitoterapia 2006;77:4358. [27] Pongpanparadon A, Aritajat S, Saenphet K. The toxicity of Butea superba, Roxb. Southeast Asian J Trop Med Public Health 2002;33:1558. [28] Ku WW, Bigger A, Brambilla G, et al. Strategy for genotoxicity testing-metabolic considerations. Mutation Res/Genet Toxicol Environ Mutagen 2007;627:5977. [29] Cherdshewasart W, Sriwatcharakul S. Metabolic activation promotes estrogenic activity of the phytoestrogen-rich plant. Maturitas 2008;59:12836. [30] Al-Majed AA, Al-Yahya AA, Al-Bekari AM, et al. Reproductive, cytological and biochemical toxicity of Yohimbe in male Swiss albino mice. Asian J Androl 2006;8:46976. [31] Ang HH, Ngai TH, Tan TH. Effects of Eurycoma longifolia Jack on sexual qualities in middle aged male rats. Phytomedicine 2003;10:5903. [32] Ma K, Ishikawa T, Seki H, et al. Isolation of new isoavonolignans, butesuperins A and B, from a Thai miracle herb, Butea superba. Heterocycles 2005;65:893900. [33] Ingkaninan K, Temkitthawon P, Chuenchom C, et al. Screening for acetylcholinesterase inhibitory activity in plants used in Thai traditional rejuvenating and neurotonic remedies. J Ethnopharmacol 2003;89:2614. [34] Roengsumran S, Petsom A, Ngamrojanavanich N, et al. Flavonoid and avonoid glycoside from Butea superba Roxb. and their cAMP phosphodiesterase inhibitory activity. J Sci Res Chulalongkorn Univ 2000;25:16976. [35] Dugger BN, Morris JA, Jordan CL, et al. Androgen receptors are required for full masculinization of the ventromedial hypothalamus (VMH) in rats. Horm Behav 2007;51: 195201. [36] Chen J-J, Chang H-C. By modulating androgen receptor coactivators, daidzein may act as a phytoandrogen. Prostate 2007;67:45762.