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To compare the effects of a 12-week course of 5%Pueraria mirifica gel and placebo gel on the prevalence of bacterial vaginosis, vaginal fungi, vaginal pH, vaginal health index (VHI), and genitourinary symptoms in postmenopausal women.In a randomized, double-blinded, placebo-controlled study (TCTR20160517002), 60 postmenopausal women were randomly assigned to a 12-week course of eitherP. mirifica gel or identical placebo gel.Vaginal Nugent score, fungal culture, pH, VHI, and genitourinary symptoms were evaluated at baseline and after 12 weeks of treatment.After 12 weeks of treatment, the proportion of participants with an abnormal Nugent score in the P. mirifica and the placebo groups were 6.7 % (2/30) and 23.3 % (7/30), respectively (p = 0.006). The mean changes in Nugent scores and VHI were significantly higher in the P. mirifica group (p 0.05). There were no significant decreases in the prevalence of symptoms between the two groups after treatment (p 0.05).A 12-week course of treatment with 5 % P. mirifica vaginal gel in postmenopausal women with GSM has been proved to be effective in reducing indicators of bacterial vaginosis compared with placebo gel. Nevertheless, the effect on alleviating genital symptoms was not demonstrated.

The proliferation of protein abundances under influence of seasonal changes are involved in cellular plant metabolic pathways. The protein profiles in relevant with seasonal variations in Butea superba leaves collected in winter, summer and rainy season were evaluated by two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupoletime- of-flight tandem mass spectrometry. The 84 proliferated protein spots were sum up of total proteins, which were found in 3 season-collected plant leaves and were classified into 11 functional groups. Eight proteins which exhibited the alteration of abundant levels in different seasons were found involved in carbohydrate and energy metabolism, photosynthesis, secondary metabolism, stress, RNA metabolism, ROS scavenging and detoxifying, and protein destination and storage. There were 2 proteins exhibited obviously isozyme polypeptide sequences. The variable physiological status within the plant leaf might be influenced by the quantitative and qualitative proliferated protein presences with response to seasonal changes and thus allowing plant to survive during severe abiotic stresses during seasonal changes.

Pueraria mirifica-derived tuberous powder has been long-term consumed in Thailand as female hormone-replacement traditional remedies. The protein profiles of tubers collected in different seasons were evaluated. Phenol extraction, 2D-PAGE, and mass spectrometry were employed for tuberous proteome analysis. Out of the 322 proteins detected, over 59% were functionally classified as being involved in metabolism. The rest proteins were involved in defense, protein synthesis, cell structure, transportation, stress, storage, and also unidentified function. The proteins were found to be differentially expressed with respect to harvest season. Importantly, chalcone isomerase, isoflavone synthase, cytochrome p450, UDP-glycosyltransferase, and isoflavone reductase, which are all involved in the biosynthesis pathway of bioactive isoflavonoids, were most abundantly expressed in the summer-collected tubers. This is the first report on the proteomic patterns in P. mirifica tubers in relevant with seasonal variation. The study enlights the understanding of variance isoflavonoid production in P. mirifica tubers.

Seasonal changes are major factors affecting environmental conditions which induce multiple stresses in plants, leading to changes in protein relative abundance in the complex cellular plant metabolic pathways. Proteomics was applied to study variations in proteome composition of Butea. superba tubers during winter, summer and rainy season throughout the year using two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight tandem mass spectrometry. A total of 191 protein spots were identified and also classified into 12 functional groups. The majority of these were mainly involved in carbohydrate and energy metabolism (30.37 %) and defense and stress (18.32 %). The results exhibited the highest numbers of identified proteins in winter-harvested samples. Forty-five differential proteins were found in different seasons, involving important metabolic pathways. Further analysis indicated that changes in the protein levels were due mainly to temperature stress during summer and to water stress during winter, which affected cellular structure, photosynthesis, signal transduction and homeostasis, amino-acid biosynthesis, protein destination and storage, protein biosynthesis and stimulated defense and stress mechanisms involving glycolytic enzymes and relative oxygen species catabolizing enzymes. The proteins with differential relative abundances might induce an altered physiological status within plant tubers for survival. The work provided new insights into the better understanding of the molecular basis of plant proteomes and stress tolerance mechanisms, especially during seasonal changes. The finding suggested proteins that might potentially be used as protein markers in differing seasons in other plants and aid in selecting B. superba tubers with the most suitable medicinal properties in the future.

This study evaluated the estrogenic and antiestrogenic activities of native and in vitro hepatic metabolized tuberous extracts of wild Butea superba collected from 23 out of the 76 provinces in Thailand by yeast estrogen screening (YES). The YES screen used consisted of the human estrogen receptors hERα and hERβ and the human transcriptional intermediary factor 2 or human steroid receptor coactivator 1, respectively, together with the β-galactosidase expression cassette as the reporter. The relative potency, effectiveness and relative inductive efficiency were evaluated by determining the β-galactosidase activity (EC(50)) of each tuberous extract in relation to that induced by 17β-estradiol. Six pure compounds isolated from B. superba were tested in parallel and exhibited a maximum relative potency compared to 17β-estradiol of 15.5% and 5.27% in the respective hERα and hERβ assays. Eighteen and seventeen plant extracts were respectively found to interact with the hERα and hERβ receptors in the YES assays with higher relative potency and relative inductive efficiency with hERβ than with hERα. The selected plant extracts tested exhibited antiestrogenic activity. Coincubation with the rat liver S9 mixture also elevated the estrogenic potency of these plant extracts.

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERalpha and hERbeta and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the beta-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the beta-galactosidase activity (EC(50)) of the tuber extracts in relation to 17beta-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERalpha and hERbeta, respectively, with a higher relative estrogenic potency with hERbeta than with hERalpha. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17beta-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.

Objective Effects of Pueraria mirifica on bone loss in fully mature ovariectomized rats are examined. Methods Two series of experiments were performed. In the first series, rats were kept with their ovaries intact and divided into two groups; initial control (IC) and sham control (SH). The IC rats were sacrificed on day 1 and their data were kept as baseline control. The SH rats were subjected to sham operation on day 0 and gavaged daily with distilled water for 90 days. In the second series, rats were subjected to ovariectomy, divided into five groups and gavaged daily with 0.1 mg/kg B.W./day of 17-alpha-ethinylestradiol (EE), 0, 10, 100 and 1000 mg/kg B.W./day of P. mirifica (P0, P10, P100 and P1000, respectively) for 90 days. Changes of bone mineral density and bone mineral content were measured using peripheral Quantitative Computerized Tomography. Results Bone loss was significantly induced by ovariectomy and it was dose-dependently prevented by P. mirifica treatment for 90 days. The preventive effects of P. mirifica on bone loss depended on bone types (axial or long bone), bone sites (metaphysis or diaphysis), and bone compartments (trabecular and cortical). At P100 and P1000, bone loss was completely prevented both in trabecular bone mineral density and content. The effects of P. mirifica were, as expected, comparable to that in the EE group. Conclusion These results suggest that P. mirifica may be applicable to treat the osteoporosis in menopausal women; however, an undesirable side effect on stimulating reproductive organs should be concerned.

Mucuna collettii Lace is a Thai herb with a long record of consumption among mature Thai males for the promotion of sexual potency. The mutagenic and antimutagenic potentials of Mucuna collettii extract were carried out by using the Ames test pre-incubation method in the presence and absence of S9 mixture. Salmonella typhimurium strains TA 98 and TA 100 were applied as the tester strains. Prior to mutagenic and antimutagenic tests, the survival of the tester strains was performed by treating with the plant extract. Results showed Mucuna collettii extract exhibited strong cytotoxic effects in a dose-dependent manner. Toxicity of the plant was confirmed in mice in which negative adverse effect was found in kidney, uterus, ovary, and testis. Mucuna collettii extract in the presence and absence of S9 mixture was negative for mutagenic Ames test. Mucuna collettii extract in the presence and absence of S9 mixture was positive for antimutagenic Ames test towards either one or both of the tested mutagens: 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) and benzo(a)pyrene. The antimutagenic activity of the plant extract was confirmed in rec-assays. Micronucleus test demonstrated that Mucuna collettii extract at high dose and a long incubation time could induce micronucleus formation in tested animals, but less than the response of the positive control. The overall mutagenic and antimutagenic assays are further evidences for the antimutagenic potential of Mucuna collettii.

The aim of this study was to evaluate the estrogenic activity of tuberous samples of wild, phytoestrogen-rich Pueraria mirifica collected from 28 out of 76 provinces of Thailand by MCF-7 proliferation assay. The plant extracts were administered to MCF-7, ER alpha positive human mammary adenocarcinoma cell cultures, for 3 days at dosages of 0.1, 1, 10, 100 and 1,000 microg/ml and were compared with 17 beta-estradiol at concentrations of 10(-12)-10(-6) M. The mean P. mirifica population at 1 mug/ml exhibited significant proliferation. Two plant samples exhibited levels of proliferation in MCF-7 that were similar to 17beta-estradiol. The mean P. mirifica populations at 100 and 1,000 microg/ml exhibited significant cytotoxicity in MCF-7. Analysis of the estrogenic activity of puerarin, representative of major isoflavonoids in P. mirifica tubers, revealed proliferation in MCF-7 only at the highest dose (10(-6) M) that was 10(2)-10(5) times less active than 17 beta-estradiol. Puerarin and 17 beta-estradiol at concentration of 10(-12)-10(-6) M exhibited no cytotoxicity in MCF-7.

Pueraria mirifica is a tuberous plant enriched with active phytoestrogens. There is no established information about the factors influencing isoflavonoid storage in the tubers. We investigated the tuberous storage of the major isoflavonoids of 1-year-old plants. Four cultivars of P. mirifica were cultivated in the same field trial during the same period to establish a unique plant age and differentiation under the same environment and soil conditions. The tubers collected from the 1-year-old plants in the summer, rainy season and winter were submitted to an HPLC analysis with a gradient system comprising 0.1% acetic acid and acetonitrile. Five major isoflavonoids, puerarin, daidzin, genistin, daidzein and genistein, were adopted as standards. P. mirifica tubers of different cultivars collected in the same season exhibited significant differences in individual and total isoflavonoid contents, showing chemovariety. P. mirifica tubers of the same cultivar collected from different seasons also exhibited significant differences in individual and total isoflavonoid contents, showing the influence of season. In conclusion, the tuberous storage of major isoflavonoids in 1-year-cultivated plants was greatly diverse and was strongly influenced by the season and plant genetics.

We investigated a non-invasive method of specimen collection for determining the changes of reproductive hormones in aged menopausal monkeys after a long-term feeding of the Thai herb Pueraria mirifica (PM) containing phytoestrogens. Three groups of aged menopausal monkeys (n = three in each group) were fed daily with 10, 100, or 1000 mg of PM for a 90 day treatment period, and fed with distilled water for 30 and 60 days of the pre- and post-treatment periods, respectively. Urine samples were collected for 14 h daily every 5 days and assayed for follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol levels. The result showed that monkeys fed with PM 10, PM 100, and PM 1000 had a decrease in urinary FSH levels during the treatment period, followed by a rebound increase during the post-treatment period. Urinary estradiol levels tended to decrease and fluctuated between 4.28 and 266.71, 2.85–42.27, and 6.24–203.50% of the pre-treatment levels in those three groups, respectively. Decreases in urinary LH levels could not be observed in all the three groups. These results suggest that FSH could be a candidate marker to detect the estrogenic effects of phytoestrogens in aged menopausal monkeys when changes of urinary hormones need to be used as an indicator.

Objective: Estrogens and estrogen-like substances have been reported to play an important role in male bone homeostasis and to prevent bone loss. Pueraria mirifica (Leguminosae), a Thai herbal plant, containing a high amount of phytoestrogens was a choice of interest for this study. We examined the effects of crude P. mirifica on bone loss and influences on reproductive organs in male rats. Methods: Using fully mature and orchidectomized (ORX) rats, the effects of 0, 10, 100 and 1000 mg/kg B.W./day of P. mirifica and 0.1 mg/kg B.W./day of 17 alpha-ethinylestradiol (a positive control) were evaluated on bone mineral density (BMD) and bone mineral content (BMC) measured with a peripheral Quantitative Computerized Tomography (pQCT) densitometry. Results: Bone loss in trabecular and cortical bones of the various sites of axial bone (fourth lumbar vertebral body) and long bones (tibia and femur) after ORX was dose-dependently prevented by P. mirifica. The effects were specific on bone types and sites. The weights of the accessory sex organs, seminal vesicle and ventral prostrate gland, which significantly decreased after 3-month of ORX, were not altered by P. mirifica. Conclusion: The results suggest that P. mirifica treatment may be useful to prevent an osteoporosis in elderly hypogonadism subjects without influences on reproductive organs. © 2006 Elsevier Ireland Ltd. All rights reserved.

This study investigated the changes in the urinary hormone levels of female monkeys (Macaca fascicularis) after single-dose and long-term treatments with Pueraria mirifica (PM). The monkeys were separated into 3 groups (n=3) and orally treated with 10, 100, or 1,000 mg of PM in each group. Two series of experiments were performed. In the first series of experiments, the monkeys were orally treated with a single dose of PM. The experimental schedule was divided into a one menstrual cycle pretreatment period and a two menstrual cycle post-treatment period. In the second series of experiments, the monkeys were orally treated daily with PM for 90 days. The experiment schedule was divided into a one menstrual cycle pretreatment period, a three menstrual cycle treatment period, and a two menstrual cycle post-treatment period. Urinary samples were collected daily and assayed for the FSH, LH, estradiol, and progesterone levels. The results showed that there were no changes in the FSH, LH, estradiol, and progesterone levels after treatment with a single dose of 10, 100, or 1,000 mg of PM or after daily treatment with 10 mg of PM for 90 days compared with the levels observed during the pretreatment period. Daily treatment with 100 mg and 1,000 mg of PM for 90 days only produced a clear reduction in the urinary FSH levels. This suggests that changes of urinary FSH levels can be considered an indicator for study of estrogenic effects on hormonal levels in female monkeys.

We investigated the effect of Pueraria mirifica (PM) on gonadotrophin and estradiol levels in aged animals; nine menopausal cynomolgus monkeys were divided into three groups. Each group (n = 3) was fed with 10, 100, and 1000 mg/d of PM for 90 d. PM-10 induced the decrease of follicle stimulating hormone (FSH) levels on d 15-90 in one out of three monkeys. PM-100 and PM-1000 decreased FSH levels of all monkeys throughout the treatment period. After the treatment period, FSH levels continued to decrease for 5 and 10-20 d in PM-100 and PM-1000, respectively, and the levels rebounded in all groups thereafter. PM-10 decreased luteinizing hormone (LH) levels throughout the treatment period in one out of three monkeys and returned to the pretreatment levels immediately after stopping treatment. PM-100 and PM-1000 prominently decreased LH levels between d 10 and 90 during treatment and persisted until d 15-25 and d 20-30 for PM-100 and PM-1000, respectively, during the post-treatment period. Serum LH levels rebounded after returning to pre-treatment levels in a dose-dependent manner. Estradiol levels tended to decrease during the treatment period in all groups. The daily feeding of PM suppressed gonadotrophin levels in aged menopausal monkeys based on dose. Moreover, they can be recovered, and there is a direct correlation between dosage and recovery time. PM may be effective as an alternative medicine in menopausal women because the effects are not permanent.

To investigate the estrogenic effect of Pueraria mirifica (PM), a Thai herbal plant that contains many phytoestrogens, sexual skin coloration was studied in cynomolgus monkeys. Aged menopausal monkeys were divided into three groups. Each group (n=3) was fed 10, 100, or 1,000 mg of PM daily. The treatment schedule was divided into three periods, a 30-day pre-treatment period, 90-day treatment period, and 60-day post-treatment period. The results show that the sexual skin exhibited reddish coloration within 24 h after PM-treatment and remained this way for the first half of the PM-feeding period. The changes in sexual skin coloration were not dose-dependent. The present results indicate that PM had estrogenic action by increasing reddish sexual skin coloration in aged menopausal monkeys.

The effects of Pueraria mirifica (PM) on reproductive organs and fertility of adult male mice were investigated. Male mice were divided into four groups (10 mice/group). Groups 1-3 were orally treated with PM at doses of 0 (PM-0), 10 (PM-10), and 100 (PM-100) mg/kg BW/d in 0.2 mL distilled water, and group 4 was subcutaneously injected with 200 microg/kg BW/d of synthetic estrogen diesthylstilbestol (DES). The treatment schedule was separated into two periods: treatment and posttreatment (8 wk for each period). The PM-10 and PM-100 treatments had no effect on testicular weight, sperm number, and serum LH, FSH, and testosterone levels. Only the PM-100 treatment reduced weights of epididymes and seminal vesicle and the sperm motility and viability. Histopathological examination demonstrated that testis, epididymis, and seminal vesicle were normal in all doses of PM treatment. PM-treated males showed no alterations in mating efficiency and on causing pregnancy of their female partners. DES injection impaired all those parameters. Offspring fathered by the PM- and DES-treated males exhibited neither malformations nor change of body weight gains, and the reproductive organ weights of 50-d old pups were in the normal range. The present data clearly demonstrate that a long-term treatment of PM at doses 10 and 100 mg/kg BW/d, via oral route, does not alter a male fertility and a hypothalamus- pituitary-testis axis. Although PM-100 can cause some moderate impairment, no persistent effects were observed. Most of PM-treated mice increased the mating efficiency after stop treatment.

To investigate the effect of Pueraria mirifica (P. mirifica) containing phytoestrogens on reproductive systems, both sexes of rats were gonadectomized and treated orally with 0, 10, 100, and 1,000 mg/kg BW per day of P. mirifica suspended in water (abbreviated as P-0, P-10, P-100, and P-1000), respectively. The treatment schedule was separated into 3 periods: pre-treatment, treatment, and post-treatment. The duration for each period was 14 days. Blood samples were taken once a week. Serum LH and FSH levels were significantly increased within 1 week after gonadectomy; and there were no changes after administration of P-0, P-10, and P-100. However, the increase of LH levels in both sexes and FSH levels in females were attenuated within 1 week after P-1,000 treatment. The attenuation of LH levels in males was smaller than that of females. The decrease of gonadotropin levels was recovered within 1 week in males and 2 weeks in females, respectively, during the post-treatment period. The increase of uterine weight and vaginal cornification were observed in female rats treated with P-100 and P-1,000, whereas only the increase of epididymis weight was found in male rats treated with P-1,000. From this study, it can be concluded that P. mirifica can influence the reproductive functions in both sexes of rats, but the response in females is greater than in males. Keywords:: Pueraria mirifica, phytoestrogen, LH, FSH, vaginal cytology

Nucleotide sequences of the parathyroid (PTH) gene of 12 species of primates belonging to suborder Anthropoidea were examined. The PTH gene contains one intron that separates two exons that code the sequence of prepro and PTH, respectively. The intron of the PTH gene in Cebus apella, Callithrix jacchus, and Saguinus oedipus was 102 bp long, whereas a 103-bp intron was observed in the remaining species. Phylogenetic analysis using the nucleotide sequences of PTH revealed that these 12 species of primates of suborder Anthropoidea could be divided into two groups of the infraorder Platyrrhini (C. apella, C. jacchus, and S. oedipus) and the infraorder Catarrhini (Macaca fascicularis, Macaca fuscata, Cercopithecus aethiops, Papio hamadryas, Presbytes obscura, Hylobates lar, Pongo pygmaeus, Pan troglodytes, and Pan paniscus). The latter infraorder could be further subdivided into two subgroups belonging to the superfamily Cercopithecoidea (M. fascicularis, M. fuscata, C. aethiops, P. hamadryas, and P. obscura) and the superfamily Hominoidea (H. lar, P. pygmaeus, P. troglodytes, and P. paniscus). The deduced amino acid sequences of PTH gene between 12 species of nonhuman primates and human revealed no amino acid substitution in mature PTH among orangutans, chimpanzees, and humans. The results indicated that the PTH gene is very conserved among primates, especially between great apes and humans. The apes are the most suitable animals to be used for studying the bone metabolism and applying the knowledge to clinical use in humans.

We investigated the major leaf isoflavonoid contents of Pueraria mirifica from three different cultivars (PM-III, PM-IV, and PM-V) using reverse RP-HPLC analysis. The proportions and net levels of puerarin, daidzin, genistin, and daidzein in P. mirifica leaves were found to depend on the plant cultivar and to correlate with cultivation temperature and rainfall amount. The crude leaf-extracts were tested using the Yeast Estrogen Screen (YES) assay with both human estrogen receptors (hERα and hERβ). Their estrogenic activity was higher when determined by the YES system containing hERβ than that with hERα and was also higher when the Δsnq2 than the wildtype yeast was employed. The results open the possibility of selecting and cultivating certain P. mirifica cultivars at a farm scale to produce a sufficient supply of leaf material to act as a starting source for the commercial scale extraction of these major isoflavonoids.

Direct gene transfer to protoplasts is a widespread experimental procedure. In this review, it is discussed in relation to the fate and expression of transforming DNA and its genetic transmission upon integration, and expression (in)stability during successive meiotic generations. The results suggest that the major differences between Nicotiana plumbaginifolia and N. tabacum were not in DNA uptake but rather in the specific fate of the transforming DNA. Differences between integrative transformation versus transient gene expression are discussed

Pueraria mirifica is a Thai phytoestrogen-rich herb traditionally used for the treatment of menopausal symptoms. Pueraria lobata is also a phytoestrogen-rich herb traditionally used in Japan, Korea and China for the treatment of hypertension and alcoholism. We evaluated the mutagenic and antimutagenic activity of the two plant extracts using the Ames test preincubation method plus or minus the rat liver mixture S9 for metabolic activation using Salmonella typhimurium strains TA98 and TA100 as indicator strains. The cytotoxicity of the two extracts to the two S. typhimurium indicators was evaluated before the mutagenic and antimutagenic tests. Both extracts at a final concentration of 2.5, 5, 10, or 20 mg/plate exhibited only mild cytotoxic effects. The plant extracts at the concentrations of 2.5, 5 and 10 mg/plate in the presence and absence of the S9 mixture were negative in the mutagenic Ames test. In contrast, both extracts were positive in the antimutagenic Ames test towards either one or both of the tested mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and benzo(a)pyrene. The absence of mutagenic and the presence of anti-mutagenic activities of the two plant extracts were confirmed in rec-assays and further supported by a micronucleus test where both plant extracts at doses up to 300 mg/kg body weight (equivalent to 16 g/kg body weight plant tuberous powder) failed to exhibit significant micronucleus formation in rats. The tests confirmed the non-mutagenic but reasonably antimutagenic activities of the two plant extracts, supporting their current use as safe dietary supplements and cosmetics.

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 five 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 significantly 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.

Objective To evaluate the influences of seasonal changes and plant cultivars on estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica . Methods Three cultivars of P. mirifica ; PM-I, PM-II and PM-V, were grown in the same field trial for 3 years and random tubers collected during the summer, rainy season and winter seasons. Female Wistar rats were ovariectomized, kept for 14 days, randomly segregated into groups and treated with one of DW, 200 μg/100 g BW 17β-estradiol (E2) or tuberous powder of PM-I, PM-II and PM-V at dosages of 100, or 1000 mg/kg BW for the next 14 days. For the last 7 days of post-treatment period, rats received only DW. The vaginal cornification was recorded during the treatment and post-treatment period. The uterine tissues of the treated rats at the treatment and post-treatment periods were analyzed for uterine gland number and for the surface area of the myometrium, endometrium and lumen. In addition, ethanol tuberous extracts of PM-I, PM-II and PM-V was submitted to DPPH analysis. Results Vaginal cornification exhibited a dose-dependent response with plant samples collected during the winter and summer being more active than those collected in the rainy season. All plant samples-induced uterotrophic effects in the analysis at the treatment and post-treatment periods in a dose-dependent manner. The P. mirifica treated rats exhibited increasing uterine gland numbers and thickness of the endometrium and myometrium but a decreasing size of lumen, in comparison to the negative control. The results were more prominent in PM-I than other plants and also in plant samples collected during the winter and summer seasons than in the rainy season. DPPH assay of the ethanol tuberous extracts revealed variance in antioxidant activity. Conclusion The results of uterotrophic and vaginal cornification assays reveal that P. mirifica exhibits a dose-dependent estrogenic activity under the influence of both seasonal changes and plant cultivars, which is confirmed by DPPH assay.

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 significant 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.

Objective There is evidence that metabolic activation can increase the estrogenic activity of the phytoestrogen-rich herb in tests with HepG2 cells. Variation in both plant genetics and harvest season may also influence estrogenic activity of the plant materials. We evaluated the influence of in vitro metabolic activation by S9 mixture on the estrogenic activity of tuberous samples of different cultivars of the phytoestrogen-rich herb, Pueraria mirifica , harvested in different seasons. Methods Plant extracts were derived from the tubers of five plant cultivars collected during summer, rainy season and winter and administered to MCF-7 cultures, an ERα-positive human mammary adenocarcinoma cell line for 3 days at dosages of 0.1, 1, 10, 100 and 1000 μg/ml. These data were compared with the major plant isoflavonoids puerarin, daidzin, genistin, daidzein and genistein and with 17β-estradiol, at concentrations of 10 −12 to 10 −6 M. The test system was done in the absence and presence of the S9 mixture. Results The major plant isoflavonoids and the plant extracts exhibited variable degrees of estrogenic activities as evaluated by altered proliferation of the MCF-7 cell line which were significantly enhanced in the presence of the S9 mixture. Conclusion Metabolic activation of plant isoflavonoids at least in vitro by S9 mixture plays a significant role in amplification of the estrogenic activity of the phytoestrogen-rich plant. In addition, the estrogenic activities of the plant samples were potentially influenced by both seasonal changes and plant genetics.

The present study investigated the effects of long-term administration of Pueraria mirifica (PM) at non-toxic doses on the ovarian function and fertility of adult female mice based on evaluation of hematological and biochemical parameters. Female mice were divided into 4 groups (36 mice/group). Groups 1-3 were orally treated with a dose of 0 (PM-0), 10 (PM-10) or 100 mg/kg BW/day PM (PM-100), and group 4 was subcutaneously injected with 200 mug/kg BW/day of synthetic estrogen diethylstilbestrol (DES). The treatment schedule was separated into treatment and post-treatment periods. The duration of each period was 8 weeks. The PM-10 mice exhibited regular estrous cycles, while the PM-100 and DES treatments induced prolonged estrous cycles. Although no changes were observed in the uterus and ovary weights of the mice after the PM-100 and DES treatments, hyperplasia of the uterine endothelium and a decrease in the number of growing ovarian follicles were detected. The changes in the ovarian histologies of the PM-100 and DES mice were related to reductions in the levels of LH and FSH, which subsequently caused a decrease in mating efficiency. Once the PM mice were able to copulate, they were capable of successfully becoming pregnant and mothering offspring. No abnormalities were observed in the external morphologies and reproductive organ weights of the 50-day-old offspring. In conclusion, our results suggest that long-term exposure to 100 mg/kg BW of PM has adverse effects on the mating efficiency and reproduction of adult female mice and that administration of 10 mg/kg BW of PM does not induce any changes in the hypothalamic-pituitary-ovarian-uterine axis.

Objective Phytoestrogens have been reported to exhibit antiproliferation to human breast cancer cells in vitro . We tested the phytoestrogen-rich, Pueraria mirifica against rat breast cancer induction in vivo . Methods The weanling female Spargue–Dawley rats were pretreated with P. mirifica tuberous powder at a dosage of 0, 10, 100 and 1000 mg/kg BW/day for four consecutive weeks. Mammary tumor development was then induced with a single dose of 7,12-DMBA, 80 mg/kg BW, followed by a weekly examination for size and multiplicity of mammary tumors for 20 weeks and finally a necropsy. Mammary tissues were investigated for the virulence of tumor and also monoclonal antibody stained against ERα and ERβ. Results Pretreatment of 1000 mg/(kg BW day) of P. mirifica tuberous powder resulted in decreasing of the virulence of rat tumor development. The mammary tumor tissues exhibited lower profile of ERα and ERβ as well as ERα/ERβ. Conclusion P. mirifica exhibited prevention of 7,12-DMBA-induced rat mammary tumors, with a proposed mechanism of strong competitive binding of its phytoestrogens to ERα and/or synthesis suppressor of ERα.

The aim of this study was to evaluate the estrogenic activity of tuberous samples of phytoestrogen-rich Pueraria mirifica collected from 25 of 76 provinces in Thailand by vaginal cornification assay. Tuberous powders were prepared and administered to ovariectomized rats for 14 consecutive days at dosages of 10, 100 and 1,000 mg/kg BW respectively, and were compared with a daily treatment with 2 mg/kg BW 17beta-estradiol (E(2)). Rats treated with 10 mg/kg BW Pueraria mirifica showed no vaginal cornification. Treatment with 100 mg/kg BW Pueraria mirifica from 13 out of 25 plant samples resulted in development of vaginal cornification. The cell count percentages of the vaginal smeared cells for the treatment with the 2 plant samples that exhibited the fastest vaginal cornification revealed large variation in their estrogenic activities. Treatment with 1,000 mg/kg BW Pueraria mirifica from all plant samples produced vaginal cornification with the mean value for the period (day) of first appearance of cornified cells being 4.08 days compared to 2 days with 2 mg/kg BW E(2). The overall appearance period (day) of cornified cells during the treatment and post-treatment period with 1,000 mg/kg BW per day Pueraria mirifica was shorter than treatment with 2 mg/kg BW E(2). The results demonstrate that the plant population shows differential estrogenic activity as evaluated by vaginal cornification assay.

Pueraria mirifica tubers collected from 28 out of 76 provinces of Thailand and Pueraria lobata tubers collected from Guangzhou province, China were submitted to HPLC analysis with the established gradient system comprising 1.5% acetic acid and acetonitrile. Five major isoflavonoids, including puerarin, daidzin, genistin, daidzein and genistein, were adopted as authentic standards. P. mirifica tubers showed intra- as well as inter-provincial differences in isoflavonoid and total isoflavonoid contents. The difference in both cases should be mostly influenced by genetic and environmental factors. In comparison with P. lobata, P. mirifica population exhibited differences only with a lower amount of daidzein.

A carpin (3-hydroxy-9-methoxypterocarpan) (Medicarpin) (1) and four isoflavones, 7-hydroxy-4'-methoxy-isoflavone (Formononetin) (2); 7,4'-dimethoxyisoflavone (3); 5,4'-dihydroxy-7-methoxy-isoflavone (Prunetin) (4) and 7-hydroxy-6,4'-dimethoxyisoflavone (5) were isolated from the tuber roots of Butea superba Roxb. Compounds 2 and 4 showed moderate cytotoxic activity on KB cell lines with IC(50) (microM) values of 37.3+/-2.5 and 71.1+/-0.8 and on BC cell lines with IC(50) (microM) values of 32.7+/-1.5 and 47.3+/-0.3, respectively.

To assess the estrogenic activities of synthetic estrogen, synthetic phytoestrogen, Pueraria lobata and three distinct cultivars of Pueraria mirifica, a phytoestrogen-rich herb, a vaginal cytology assay in ovariectomized rats were used. Rats were ovariectomized and treated with DW, estradiol valerate (1 mg/kg BW), genistein (0.25-2.5 mg/kg BW), Pueraria lobata and Pueraria mirifica (10-1,000 mg/kg BW) for 14 days. The vaginal cytology was checked daily and the uteri were dissected and weighed at the end of treatment or post-treatment periods. The treatments of DW, genistein and Pueraria lobata did not influence the vaginal epithelium, but the injection of estradiol valerate induced a vaginal cornification from day-3 of treatment to day-14 of post-treatment period. The occurrence of vaginal cornification after treatment and the recovery after the cessation was dependent on dosages and cultivars of Pueraria mirifica. The increments of uterus weight in all rats agreed with the cornification of vaginal epithelium. Although both uterotropic and vaginal cytology assays can be used to assess the estrogenic activity of phytoestrogen-rich herb, however, using vaginal cytology assay has two advantages: (1) we do not need to kill the animals and (2) we can follow up the recovery after the cessation of treatment.

The differential anti-proliferation effect of white (Pueraria mirifica), red (Butea superba) and black (Mucuna collettii) Kwao Krua plant extracts on the growth of MCF-7 cells was evaluated after 4 days of incubation. The percent cell growth comparison was based on protein determination of the harvested cells in parallel with the control group and Pueraria lobata treatment group. Pueraria lobata led to no proliferation and a mild anti-proliferation effect on the growth of MCF-7 cells. Pueraria mirifica caused proliferation at 1 microg/mL and an anti-proliferative effect on the growth of MCF-7 cells at 100 and 1000 microg/mL with an ED50 value of 642.83 microg/mL. Butea superba led to no proliferation and an anti-proliferation effect on the growth of MCF-7 cells at 10, 100 and 1000 microg/mL with an ED50 value of 370.91 microg/mL. Mucuna collettii led to no proliferation and an anti-proliferation effect on the growth of MCF-7 cells at 100 and 1000 microg/mL with an ED50 value of 85.36 microg/mL. The results demonstrated that only Pueraria mirifica showed an estrogenic effect on MCF-7 cell growth and a clear antagonistic effect with E2 at high concentration. Butea superba and Mucuna collettii exhibited only anti-proliferation effects on the growth of MCF-7 cells in relation with a possible anti-estrogen mechanism or a potent cytotoxic effect.

Red kwao krua, Butea superba Roxb., is a herbal leguminous plant endemic in Thailand whose tuberous roots are used for male rejuvenation and the prevention of erectile dysfunction. Different populations from different provinces vary in their bioactivities, raising the need to evaluate the causes prior to optimal population selection and growth. Morphometric analysis of leaves collected from 34 populations from 24 provinces throughout Thailand using nine parameters for factor and cluster analyses were correlated with latitude and longitude revealing a clinal pattern. From the North to the South, leaf length increased in size in factor 1 but decreased in size in factor 2. Genetic analysis on the same samples, using DNA sequence analysis from the chloroplast rbcL, trnLF-cd, and trnLF-cf regions and maximum parsimony and neighbour- joining phylogenetic analysis, revealed essentially no genetic variation in the rbcL region, as expected. However, some between-population genetic variation was revealed by trnLF-cd and trnLF-cf sequences, suggesting potentially considerable genetic polymorphism. This was supported by preliminary RAPD analyses using five primers which indicated high genetic variation within and between populations.