Effects of Pueraria mirifica on vaginal artery vascularization in postmenopausal women with genitourinary syndrome of menopause.

OBJECTIVE: To compare the effects of 6% Pueraria mirifica vaginal gel with those of placebo gel on vaginal blood flow, vaginal maturation index (VMI), vaginal health index (VHI), endometrial thickness and genitourinary symptoms in postmenopausal women. STUDY DESIGN: In a randomized, double-blinded, placebo-controlled study (TCTR20200624007), 72 postmenopausal women were randomized into the P. mirifica or the placebo gel group. Both groups were followed up at week 4 and week 12. MAIN OUTCOME MEASURE: Doppler ultrasonography pulsatility index (PI) and resistance index (RI), VMI, VHI, endometrial thickness and genitourinary symptoms were evaluated at baseline, at week 4 and week 12 of treatment. RESULTS: Sixty-three participants completed the study. After 4 and 12 weeks of treatment, PI and RI had significantly decreased in the P. mirifica group compared with the placebo group. At week 12, PI in the P. mirifica group and in the placebo group were 3.03 + 1.09 and 6.88 + 2.16, respectively (p = 0.002). Similar changes were also demonstrated in the resistance indices. The P. mirifica group demonstrated a markedly higher mean VMI at week 12 compared with the placebo group, 55.19 ± 18.53 and 20.29 ± 28.46 (p = 0.012). In addition, all parameters of VHI based on the vaginal physical findings at week 12 in the P. mirifica group were significantly higher than in the placebo group (p < 0.001). CONCLUSION: In this study, 6% P. mirifica vaginal gel for 12 weeks in postmenopausal women with GSM appeared to increase vaginal artery circulation and restore atrophic vaginal tissue. CLINICAL TRIAL REGISTRATION NUMBER: TCTR20200624007.

Improved yeast-screening assay for phytoestrogens.

A yeast estrogen screening (YES) assay was improved to increase sensitivity for detection of phytoestrogens. New yeast strains minus one or the other of transporters Pdr5 or Snq2 and harboring yEGFP as a reporter gene were developed. The new strains showed 2-100-fold improvement in sensitivity for detection of standard estrogens and antiestrogens. In addition, the assay time (1 h) using the newly developed strains was shorter than that (4 h) previously reported. Furthermore, the snq2-minus strains were most effective for detection of estrogenic activity while the pdr5-minus strains were most effective for detection of antiestrogenic activity. The efficacy of the new methods was evaluated and confirmed by testing with 23 Thai medicinal plant species. The new strains were also tested for detection of xenoestrogens. The results revealed that the newly developed YES methods were specific and rapid and suitable for simple high-throughput screening or detection of estrogen-like compounds.

Comparison of the effects of Pueraria mirifica gel and of placebo gel on the vaginal microenvironment of postmenopausal women with Genitourinary Syndrome of Menopause (GSM).

OBJECTIVE: 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. STUDY DESIGN: 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. MAIN OUTCOME MEASURE: Vaginal Nugent score, fungal culture, pH, VHI, and genitourinary symptoms were evaluated at baseline and after 12 weeks of treatment. RESULTS: 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). CONCLUSION: 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.

Comparison of Pueraria mirifica gel and conjugated equine estrogen cream effects on vaginal health in postmenopausal women.

OBJECTIVES: To compare the effects of a 12-week treatment with Pueraria mirifica (PM) gel or a conjugated estrogen cream on vaginal maturation index, vaginal symptom score, vaginal health assessment score, and vaginal flora in postmenopausal women. METHODS: In a prospective, noninferiority, randomized controlled study, 82 postmenopausal women with at least one vulvovaginal symptom, that is, dryness, soreness, irritation, dyspareunia, or discharge, were recruited. Participants were randomly assigned into two groups for treatment with either PM gel or conjugated estrogen cream (CEE). The treatment regimen consisted of application of 0.5 g of product intravaginally daily for 2 weeks, and then decreased to three times per week for 10 weeks. Vaginal maturation index, vaginal symptoms score, and vaginal health assessment score were evaluated before the beginning of the study and at 6 weeks and 12 weeks after treatment with PM or CEE. RESULTS: The vaginal maturation index increased significantly in both treatment groups at week 12, from 22.44 ±â€Š22.28 to 47.56 ±â€Š29.00 and from 27.07 ±â€Š26.12 to 66.90 ±â€Š22.42 in the PM and the CEE groups, respectively (P < 0.05). A significantly higher effect was, however, observed in the CEE group (P < 0.05). Vaginal symptom scores decreased significantly after treatment in both groups, from 4.37 ±â€Š2.79 to 0.95 ±â€Š1.88 and from 4.15 ±â€Š2.16 to 0.68 ±â€Š1.64 in the PM and the CEE groups, respectively (P < 0.05), with no statistically significant different between the two groups (P > 0.05). CONCLUSIONS: The use of a 12-week treatment regimen with Pueraria mirifica gel was shown to be efficacious and safe for the treatment of vulvovaginal atrophy. Conjugated estrogen cream was found to be more effective compared to Pueraria mirifica gel in improving signs of vaginal atrophy and restoring vaginal epithelium at 6 and 12 weeks.

Comparative protein profiles of Butea superba tubers under seasonal changes.

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.

Proteome analysis of Pueraria mirifica tubers collected in different seasons.

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.

Pueraria mirifica leaves, an alternative potential isoflavonoid source.

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 hERß). Their estrogenic activity was higher when determined by the YES system containing hERß 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.

Estrogenic and anti-estrogenic activities of the Thai traditional herb, Butea superba Roxb.

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 hERß and the human transcriptional intermediary factor 2 or human steroid receptor coactivator 1, respectively, together with the ß-galactosidase expression cassette as the reporter. The relative potency, effectiveness and relative inductive efficiency were evaluated by determining the ß-galactosidase activity (EC(50)) of each tuberous extract in relation to that induced by 17ß-estradiol. Six pure compounds isolated from B. superba were tested in parallel and exhibited a maximum relative potency compared to 17ß-estradiol of 15.5% and 5.27% in the respective hERα and hERß assays. Eighteen and seventeen plant extracts were respectively found to interact with the hERα and hERß receptors in the YES assays with higher relative potency and relative inductive efficiency with hERß 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.

Mutagenic and antimutagenic effects of the traditional herb used for treating erectile dysfunction, Butea superba Roxb.

Butea superba is a traditional tuberous Thai plant enriched with flavonoids that is used for treating erectile dysfunction. We investigated the mutagenic and antimutagenic potentials of a B. superba extract by using the pre-incubation method of the Ames test. Salmonella typhimurium strains TA 98 and TA 100 were applied as the tester strains in the presence and absence of an S9 mixture. Prior to the mutagenic and antimutagenic tests, the survival of the tester strains was measured by treating with the B. superba extract. The results show that the B. superba extract exhibited dose-dependent cytotoxic effects. Data from the Ames test revealed that the B. superba extract to be non-mutagenic in the presence and absence of the S9 mixture. In contrast, the B. superba extract showed antimutagenic potential towards either or both of the tested mutagens: 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) and benzo(a)pyrene (B(a)P) in the respective presence and absence of the S9 mixture, respectively. The plant antimutagenic activity was confirmed by a rec assay. A further study by micronucleus test demonstrated that the B. superba extract at the maximum loading volume could induce acute micronucleus formation in the tested animals. The in vitro mutagenic and antimutagenic assays confirmed the safe consumption of B. superba products at low dose (not more than 781.25 microg/ml of the plant extract), but the in vivo genotoxic assay demonstrated the unsafe consumption at a high dose (300 mg/kg of the BW plant extract or 16 g/kg of the BW plant powder).

Androgenic activity of the Thai traditional male potency herb, Butea superba Roxb., in female rats.

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.

Variance of estrogenic activity of the phytoestrogen-rich plant.

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, 200microg/100g BW 17beta-estradiol (E2) or tuberous powder of PM-I, PM-II and PM-V at dosages of 100, or 1000mg/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.

Androgen disruption and toxicity tests of Butea superba Roxb., a traditional herb used for treatment of erectile dysfunction, in male rats.

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.

Preventive effects of Pueraria mirifica on bone loss in ovariectomized rats.

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 0.1mg/kg B.W./day of 17-alpha-ethinylestradiol (EE), 0, 10, 100 and 1000mg/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.

Metabolic activation promotes estrogenic activity of the phytoestrogen-rich plant.

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 ERalpha-positive human mammary adenocarcinoma cell line for 3 days at dosages of 0.1, 1, 10, 100 and 1000microg/ml. These data were compared with the major plant isoflavonoids puerarin, daidzin, genistin, daidzein and genistein and with 17beta-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.

Determination of the estrogenic activity of wild phytoestrogen-rich Pueraria mirifica by MCF-7 proliferation assay.

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.

Antimutagenic potential of the Thai herb, Mucuna collettii Lace.

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.

Major isoflavonoid contents of the 1-year-cultivated phytoestrogen-rich herb, Pueraria mirifica.

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.

Pretreatment with phytoestrogen-rich plant decreases breast tumor incidence and exhibits lower profile of mammary ERalpha and ERbeta.

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 ERalpha and ERbeta. RESULTS: Pretreatment of 1000 mg/(kgBWday) of P. mirifica tuberous powder resulted in decreasing of the virulence of rat tumor development. The mammary tumor tissues exhibited lower profile of ERalpha and ERbeta as well as ERalpha/ERbeta. 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 ERalpha and/or synthesis suppressor of ERalpha.

Assessment of fertility and reproductive toxicity in adult female mice after long-term exposure to Pueraria mirifica herb.

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.

Assessment of urinary gonadotropin and steroid hormone profiles of female cynomolgus monkeys after treatment with Pueraria mirifica.

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.