Journal of Reproduction and Development, Vol. 50, No. 6, 2004

—Original—

Long-Term Treatment Effects of Pueraria mirifica Phytoestrogens on Parathyroid Hormone and Calcium Levels in Aged Menopausal Cynomolgus Monkeys

Hataitip TRISOMBOON1,2), Suchinda MALAIVIJITNOND2), Juri SUZUKI3), Yuzuru HAMADA3), Gen WATANABE4,5) and Kazuyoshi TAYA4,5)

1)Biological Science Ph.D. Program, Faculty of Science, Chulalongkorn University, Bangkok 10330,2)Primate Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand, 3)Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, 4)Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, 5)Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan

Abstract. To determine the effect of Pueraria mirifica (PM) on serum parathyroid hormone (PTH) and calcium levels on aged menopausal monkeys (Macaca fascicularis), subjects were treated with 10, 100, or 1,000 mg/day of PM. Blood samples were collected every 5 days for 30, 90, and 60 days during pre- treatment, treatment, and post-treatment periods, respectively. Sera were assayed for PTH, estradiol, and calcium levels. PM-1,000 had the strongest effect on the decrease in PTH (0.001estrogen deficiency. Key words: Pueraria mirifica, Phytoestrogen, PTH, Calcium, Aged menopausal monkey (J. Reprod. Dev. 50: 639–645, 2004)

enopausal osteoporosis is a disorder of the the systemic regulation of bone resorption. bone characterized by the progressive loss of Overproduction of PTH leads to an increase in bone bone tissue, and is caused by estrogen deficiency in resorption compared with bone formation and both natural and surgical menopause [1]. Increased contributes to general skeletal demineralization. parathyroid hormone (PTH) secretion contributes Increased PTH levels have been found to be to an increase in bone resorption and osteoporosis, concomitant with decreased bone mass in aging which is related to estrogen deficiency [2, 3]. people [4]. One pathogenetic mechanism of Although the exact mechanism has not been osteoporosis involves chronic loss of calcium elucidated yet, PTH is a major factor involved in balance caused by intestinal and renal calcium handling. This mechanism is characterized by an Accepted for publication: August 17, 2004 Correspondence: S. Malaivijitnond increase in PTH concentration, and is generally (e-mail address: [email protected]) though to be a secondary response to a small 640 TRISOMBOON et al. reduction of serum calcium levels. menopausal cynomolgus monkeys were used in Several recent reports have indicated that soy, a this study as a representative of menopausal rich source of isoflavone genistein and daidzein, women, because the physiological systems of has a beneficial effect, reducing bone loss cynomolgus monkeys, including hormonal associated with ovarian hormone deficiency [5–7]. patterns and reproductive function, are similar to Soy isoflavone treatment induced a large increase those of humans [21, 22]. in bone mineral density (BMD) in ovariectomized rats [8] and mice [5]. Postmenopausal women with habitually high intakes of dietary isoflavones had Materials and Methods significantly lower levels of serum PTH and higher BMD [6]. In an in vitro study, the decrease in bone Animals calcium content induced by bone resorbing factors, Aged menopausal monkeys (Macaca fascicularis, PTH, and prostaglandins E2 was inhibited n=9) with a complete cessation of menstruation for completely by genistein [9]. Moreover, genistein at least one year, age more than 25 years, and blocked both the inactivation of acid phosphatase weighing from 4.0–6.5 kg, were selected. The and the activation of alkaline phosphatase due to menopausal state of the monkeys was confirmed PTH in bone tissue, resulting in reduced bone and checked daily by vaginal swabbing before and resorption in rats [10]. The evidence strongly during treatment. The monkeys were housed suggests that phytoestrogens play a role in separately in individual cages at the Primate preventing bone loss caused by estrogen deficiency Research Unit, Department of Biology, Faculty of in women as well as female animals, possibly Science, Chulalongkorn University, Bangkok, through the reduction of PTH levels. Thailand. Lighting conditions in the animal room Pueraria mirifica (PM), known as White Kwao were controlled (12:12 h light to dark cycle). Krua, is an indigenous Thai plant that has long Temperature and humidity fluctuated slightly been used as a rejuvenating drug. The chemical depending on the season. The monkeys were fed content of its tuberous roots has been analyzed by daily with monkey chow (Pokaphan Animal Feed high performance liquid chromatography and Co., Ltd., Bangkok, Thailand) in the morning many phytoestrogenic substances were found, (09:00–10:00 h) and were given fresh fruits in the including miroestrol [11, 12], deoxymiroestrol, afternoon (14:00–15:00 h). The experimental kwakhurin [13], coumestrol, and isoflavones protocol was approved by the ethics committee in (genistein and daidzein) [14, 15]. Several accordance with the guide for the care and use of investigators have studied PM’s estrogenic effect laboratory animals prepared by the Primate on the reproductive organs and their functions [16– Research Unit, Chulalongkorn University. 18]. Prior research found a PM effect using an animal model [16–18]. Treatment with a Experimental design suspension of PM at various doses influenced The nine aged menopausal monkeys were serum gonadotropin levels in both aged divided into three groups. The monkeys in each menopausal and adult cyclic cynomolgus monkeys group (n=3) were fed daily with a suspension of [18, 19]. One study found that administration of PM at doses of 10, 100, or 1,000 mg/individual/day crude extract of PM improved menopause-related between 08:00–08:30 h. (hereafter abbreviated as symptoms in women such as hot flushes, PM-10, PM-100, and PM-1,000) The treatment frustration, sleep disorder, skin dryness, high blood schedule was separated into 3 periods: pre- cholesterol, and amenorrhea with no change in treatment, treatment, and post-treatment for 30, 90, blood cells, or liver or renal functions [20]. No and 60 days, respectively. During the pre- research, however, has studied the effects of PM on treatment and post-treatment periods, monkeys bone, calcium, or PTH levels. This is of interest for were fed with 5 ml of distilled water. Blood female animals and menopausal women who face samples, 3 ml, were collected from the femoral vein bone disorder problems. The aim of this study, without anesthetization between 08:30–09:30 h therefore, was to investigate the estrogenic effect of every 5 days, then centrifuged 1,700 × g at 4 C, for long-term treatment of PM on serum levels of PTH 20 minutes and stored at –20 C until PTH, estradiol, and calcium in aged menopausal monkeys. Aged and calcium were assayed. PUERARIA MIRIFICA ON PTH AND CALCIUM 641

P. mirifica suspension preparation were described in a previous report [25]. The fresh tuberous roots of PM were sliced, desiccated in a hot air oven at 70 C, and Statistical analysis subsequently ground into 100 mesh powder. Then, Serum levels of hormones were expressed as the powdered stock was kept in a dark desiccator mean ± S.E.M. Analysis of variance (ANOVA) until preparation of the suspension with distilled followed by the LSD test was applied to determine water. The PM suspension was kept in a dark the significance of difference among the three bottle at 4 C until feeding time periods of the experiment, and among the three groups. Differences were considered significant at Hormonal analyses P<0.05. Serum total calcium levels were measured by an atomic absorption spectrophotometer (AAS) according the method of Zettner and Seligson using Results a Roche/Hitachi system [23]. After extraction with ether, the serum level of estradiol was determined Characteristics of hormonal pattern in aged by RIA with 3H-labeled radioligands as described menopausal monkeys by the established method of the World Health The menopausal state of the monkeys was Organization (WHO) [24]. Serum PTH levels was confirmed by lower levels of serum estradiol (14.71 assayed by a PTH-C radioimmunoassay kit (Eiken ± 1.18 pg/ml) and higher levels of LH (5.85 ± 0.80 Chemical Co.Ltd. Bunkyo-ku, Tokyo, Japan). The ng/ml) compared to normal cyclic monkeys in the procedure of the PTH assay and parallel checks early follicular phase of the menstrual cycle in our

Fig. 1. Relationship between basal serum levels of PTH and calcium with estradiol in aged menopausal monkeys. Fig. 2. Changes in serum PTH, calcium, and estradiol levels during the pre-treatment (A), treatment (B), and post- treatment (C) periods in monkeys treated with PM-10 (n=3). The horizontal bar indicates the treatment period. Stars show significant differences compared to pre-treatment levels (P<0.05). 642 TRISOMBOON et al.

Fig. 3. Changes in serum PTH, calcium, and estradiol levels during the pre-treatment (A), treatment (B), and post- Fig. 4. Changes in serum PTH, calcium, and estradiol levels treatment (C) periods in monkeys treated with PM- during the pre-treatment (A), treatment (B), and post- 100 (n=3). The horizontal bar indicates the treatment treatment (C) periods in monkeys treated with PM- period. Stars show significant differences compared 1,000 (n=3). The horizontal bar indicates the treatment to pre-treatment levels (P<0.05). period. Stars show significant differences compared to pre-treatment levels (P<0.05).

colony (27.54 ± 4.31 pg/ml for estradiol and 0.51 ± cessation of PM treatment, the PTH level was 0.03 ng/ml for LH, unpublished data). Figure 1 quickly returned to the pre-treatment levels. Serum shows the relationship between basal levels of calcium levels decreased significantly on day 75 estradiol and PTH as well as calcium. There was a (P=0.01) during the treatment period but gradually slight positive correlation between the basal levels returned to the basal levels thereafter. Serum of calcium and estradiol (r=0.46, P=0.001), but no estradiol levels significantly but sporadically correlation between the levels of PTH and estradiol decreased (days 30, 55, 65, 70, and 90 during the (r=–0.02, P=0.88). treatment period; 0.01estrogen receptor (ER) period. Estradiol levels were significantly lower was found in parathyroid cells, and estradiol, than the pre-treatment levels on day 65 (P=0.03) of treatment caused a decrease in their basal DNA the treatment period and on day 20 (P=0.01) of the synthesis [30], suggesting that estrogen may have a post-treatment period. direct effect on PTH secretion. We consider that phytoestrogens in P. mirifica behave as an estrogen and decrease the PTH levels, Discussion since phytoestrogens compete with estradiol to bind to estrogen receptors, which are found in the Healthy menopausal monkeys have low levels of renal, gastrointestinal tract, and bone [31, 32]. serum estradiol and there is a positive correlation Phytoestrogens may effect these organs, improving between estradiol levels and calcium levels in their calcium absorption, resulting in a secondary serum. It has been proposed that changes in decrease in the PTH level. Based on the findings of estradiol levels influence intestinal calcium Wong et al. [30] described above, we hypothesize absorption and increase serum calcium levels as that PM phytoestrogens have a direct action on found in healthy humans [26]. However, we did decreasing PTH secretion from the parathyroid not find the correlation between estradiol levels gland. and PTH levels because of vary estradiol levels. It is known that the mechanism of PTH in Our study is the first to report on the effect of regulating calcium balance is very complex. phytoestrogens from the medicinal plant, PM, on Normally, it acts directly on the bone and kidney to altering serum levels of PTH and calcium in increase calcium influx into the blood circulation. It estrogen deficient animals. The results clearly also stimulates indirectly calcium absorption by the demonstrate that aged menopausal monkeys intestine. The overall effect of PTH is to increase treated with PM had reduced in PTH levels which the circulating calcium level [2]. Administration of were followed by a decline in serum calcium levels. PTH increased the serum calcium level in patients The highest dose (PM-1,000) seems to have been with hypoparathyroidism [33]. Low levels of more effective for the decrease of PTH and calcium serum PTH induced a reduction of serum calcium levels than the lowest dose (PM-10). The effect, level in the blood circulation [2]. The results of the however, did not depend on dose, because a present study show that long-term treatment with significant decrease was not observed in the PM- PM can suppress the serum PTH level, followed by 100 group. The reason why there was no effect of a decrease in the serum calcium level. PM-100 on serum PTH and calcium levels in this Although the long-term treatment of PM led to a study remains unknown. decrease in serum calcium, serum calcium levels Similar to our result, post-menopausal women during the treatment period in all monkey groups consuming high isoflavones from a soy diet had (9.91 ± 0.13 mg/dl) stayed in a narrow range and lower levels of serum PTH [6]. The decrease of remained within the normal range (10.57 ± 0.22 PTH levels was associated with increased BMD in mg/dl) of aged monkeys. After the cessation of PM the lumbar spine and Ward’s triangle, suggesting treatment, serum calcium levels returned to the that high intake of phytoestrogens may help to pre-treatment level. Calcium is of fundamental improve the state of hyperparathyroidism in importance to all biological activities, and it is also postmenopausal women resulting in both lower a vital component, not only in the mechanism of rates of bone turnover and bone loss [6]. hormone secretion, but also in hormone action and Estrogen administrations have been is involved in neurotransmission and muscle demonstrated to decrease serum levels of PTH and contraction. For this reason, it is vital that the calcium in postmenopausal women [27, 28]. It has serum calcium concentration is kept within a been suggested that estrogen directly inhibits bone narrow range. The fact that aged monkeys treated 644 TRISOMBOON et al. with PM for 90 days, did not have greatly lowered In summary, the long-term treatment of 1,000 serum calcium levels outside their normal range, mg/day of PM can decrease the serum levels of means that the long-term treatment of PM, which PTH and calcium in aged menopausal monkeys. contains phytoestrogens, does not have an adverse The present study suggests that 1,000 mg/day of effect on calcium homeostasis in the physiological PM has a beneficial effect on preservation of system. calcium content in the bone by reducing PTH Although our study cannot completely explain secretion. However, to determine the appropriate the mechanism of PM on the decrease of PTH and dose of PM to reduce bone loss in menopausal calcium levels, at the very least it can be said that women additional studies are necessary. the effects on PTH and calcium levels were not caused by endogenous ovarian estradiol but by PM phytoestrogens. This was confirmed by the low Acknowledgments estradiol levels seen throughout the study period in all monkey groups. During PM treatment, serum This work was supported by The Thailand estradiol levels were even lower than those in the Research Fund (TRF) contract no. PHD/0014/2544 pre-treatment period, which is considered to have for the Royal Golden Jubilee Program, (RGJ been caused by a reduction in peripheral network) and no. BGJ/15/2544, Chulalongkorn conversion of estrone and testosterone to estradiol. University Research Grant and a Grant-in-Aid for Previous reports showed that both genistein and Scientific Research (The 21st Century Center-of- coumestrol reduced conversion of estrone to Excellence-Program, E-I) from the Ministry of estradiol [34, 35], and of androstenedione and Education, Culture, Sports, Science and Technology testosterone to estradiol in human granulosa luteal of Japan. cells [36].

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