Wichai Cherdshewasart

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.

The estrogenic effect of Pueraria mirifica on gonadotrophin levels in aged 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.

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.

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.

Effects of Pueraria mirifica, an herb containing phytoestrogens, on reproductive organs and fertility of adult male mice.

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 μg/kg BW/d of synthetic estrogen diesthylstibestol (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.

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.

Differential binding with ERα and ERβ of the phytoestrogen-rich plant Pueraria mirifica

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) hERα and hERβ and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the β-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the β-galactosidase activity (EC50) of the tuber extracts in relation to 17β-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERα and hERβ, respectively, with a higher relative estrogenic potency with hERβ than with hERα. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17β-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.

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.

The mutagenic and antimutagenic effects of the traditional phytoestrogen-rich herbs, Pueraria mirifica and Pueraria lobata

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.

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 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 (EC50) 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.