Philippine Journal of Science 135 (1): 39-48, June 2006 ISSN 0031 - 7683

Estrogenic Activity of phaseoloides Roxb. Benth Evaluated in Ovariectomized Rats

Reginald R. Cordial*, Bella Marilou Baxa-Daguplo*, Paul Michael S. Fermanes, Abigail S. Garcia, Rod Mark M. Clavel, Malou Ombac-Herradura, Joselito C. Javier and Ricardo R. Santos

Department of Biochemistry and Nutrition, Fatima College of Medicine Our Lady of Fatima University, Valenzuela City, Philippines

This study investigated the potential estrogenic bioactivity of tropical (Pueraria phaseoloides Roxb. Benth) crude extracts. Three- to five-week old Sprague-Dawley rats were ovariectomized (OVX) and randomly treated 10 days post-OVX with extract at 3 doses (1050, 787.5, 525 mg/kg•d) with a positive control of 17ß-estradiol (50µg/kg•d) in corn oil as vehicle for 15 days. Estrogenic activity was determined by analyzing body and uterine weight changes, vaginal cell cornification, uterine morphometry, and histopathology characteristic of estrogen-induced responses. The plant produced a dose-dependent increase in uterine weight and epithelial cell heights. The 2 higher doses of the plant induced differentiation of vaginal cornification in the OVX rats. Estrogenic potency of the plant crude extract used was significantly lower than 17ß-estradiol. Data suggested that the plant crude extract exhibited weak estrogenic effects in immature ovariectomized rat model.

Key Words: Pueraria phaseoloides, estrogenic activity, isoflavones, phytoestrogens, vaginal cornification, uterotrophic bioassay

Introduction data have shown that a diet rich in isoflavones, such as those in soy, may reduce the risk of cardiovascular Phytoestrogens, specifically isoflavones, are receiving disease and breast and endometrial cancer (Adlercreutz great commercial interest at present. Isoflavones 1995). It has also been suggested that the consumption are found almost exclusively in . Due to the of food containing phytoestrogens contributes to a lower structural similarity they possess with estrogenic steroids occurrence of hormone-related cancers in Japanese present in the human body, they mimic the effects of and Chinese women compared with women in Western naturally occurring estrogenic compounds in the body. countries (Adlercreutz and Mazur 1997; De Kleijn et In fact, a variety of phytoestrogens have already been al. 2001; Xu et al. 2004). Phytoestrogen might also β α identified that bind stronger to the ER than ER , which help prevent the onset of hormonal carcinogenesis by can induce estrogen-like actions (Davis et al. 1999; decreasing genotoxic estrogen metabolites (Dixon and Nikov et al. 2000). Specifically, the biological effects Ferreira 2002). associated with ingesting isoflavones indicate that dietary supplements rich in these compounds might be useful for Known phytoestrogen compounds have already alleviating menopausal health concerns. Epidemiological been isolated from some Pueraria species (Morris 1999; Lee et al. 2001; Kim et al. 2003). Pueraria *Corresponding authors: [email protected] mirifica, an indigenous Thai herb, also contains [email protected] phytoestrogenic substances including miroestrol,

39 Philippine Journal of Science Cordial et al.: Estrogenic Activity of Pueraria Vol. 135 No. 1, June 2006 phaseoloides Roxb. Benth

puerarin, deoxymiroestrol, kwakhurin, and other plant relatives positive for phytoestrogens, this study phytoestrogens that belong to the isoflavone and hypothesized that P. phaseoloides also contains coumestrol class (Bounds and Pope 1960; Ingham et some still-unidentified phytoestrogenic substances. al. 1988; Ingham et al. 1989; Chansakaow et al. 2000). Moreover, no research so far has been conducted to It was observed that P. mirifica plant greatly influences assess its potential estrogenic properties that may be menstrual cycles and may suppress ovulation by tapped as a locally available alternative compound for lowering serum levels of gonadotropins in cynomolgus HRT. Thus, the present study aimed to evaluate the monkeys (Trisomboon et al. 2005). The plant showed estrogenic activity in P. phaseoloides with immature, different effects to FSH and LH in ovariectomized ovariectomized rat using a four-fold parametric and gonadectomized rats (Malaivijitnond et al. 2004). criterion: uterotrophic assay, vaginal cornification, Clinical trial of crude drug derived from the plant powder morphometric and histopathological analysis. showed effectiveness of treatment of menopausal symptoms. It is believed that p h y t o e s t r o g e n s contained in the plant, especially isoflavones, support female sexual characteristics including breast and skin MATERIALS AND METHODS appearances (Muangman and Cherdshewasart 2001). Estrogenic activity of the plant chemicals was related Animals to metabolic activation (Lee et. al. 2002). Toxicity Three- to five-wk old female Sprague-Dawley rats test of the plant powder and extract did not show any weighing 28-47 g were obtained from the Bureau of significant toxicity in both animal and human tests Food and Drugs Rat Breeding Laboratory (Alabang, (Cherdshewasart 2003). High concentration of the Muntinlupa, Philippines). The animals were transported plant extract showed antiproliferation to HeLa and and housed at the Laboratory Animal Room of MCF-7 cells (Cherdshewasart 2004a,b). Currently, Limnological Station, University of the Philippines phytoestrogenic compounds from P. mirifica are Los Baños (UPLB) (College, Laguna, Philippines). marketed as cosmetic and dietary supplement with Experimental condition was maintained at temperature indication for pre- and post-menopausal women. of 23 +± 2°C, and relative humidity of 55 ± 5% on an Tropical kudzu, Pueraria phaseoloides (Roxb.) approximate conventional 12-h light/dark cycle. Rat Benth, is the counterpart leguminous weed species that cages were randomly arranged to limit variation based naturally grows in the Philippines. This forage on temperature and light. is used traditionally as a cattle feed and ground cover The animals were bilaterally ovariectomized for many agricultural crops. Its usage can be traced following standard rodent ovariectomy procedure. back to century-old Chinese herbal medicine where A period of 10 d was allotted for wound healing and kudzu root has been indicated to reduce addiction to acclimatization. alcohol (Keung and Vallee 1998; Shebek and Rindone 2000; Lukas et al. 2005) similar to that of Chinese kudzu, Pueraria lobata (Lin et al. 1996). To date, Pueraria phaseoloides this plant is a noxious weed in the Philippines having P. phaseoloides was collected at Mt. Makiling, Los overgrown roadside landscapes, uncultivated, and Baños, Laguna, Philippines over the period of September abandoned lots. Moreover, in the Philippines, which to December, 2004. The voucher specimen of the currently encourages herbal medicine research through plant was properly identified by plant systematists the Traditional and Alternative Medicine Act of 1997 from UPLB Museum of Natural History. The stem (Republic Act No. 8423), no plant indicated to alleviate and leaves were separated and placed in brown paper post-menopausal conditions is presently included in the bags for oven drying at 60°C for 2-3 h. Dried stem and National Integrated Research Program for Medicinal leaves were combined and then grinded using a Thomas- (NIRPROMP) priority list for pharmacologic and Wiley mill until a fine substance was achieved. The clinical studies. NIRPROMP is a Philippine Council pulverized material was then soaked in 95% ethanol for for Health Research and Development - Department 3 d, which was then occasionally swirled and agitated. of Science and Technology (PCHRD-DOST) governed The resulting mixture was filtered and the supernatant body tasked to spearhead herbal medicine research in that was collected concentrated under vacuum reduced the Philippines. pressure through the rotatory evaporator. The resultant thick syrupy mass was kept under 4°C, and then used to In the light of studies above and the evidence of compute for different doses based on a constant volume their direct affinity with their now-acknowledged per body weight basis.

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Food and Treatment Groups from entirely leucocytes (indicating a diestrus stage) To minimize the amounts of phytoestrogens, the to entirely cornified (indicating an estrous stage). The conventional rat palletized food was replaced with a guide from Maligalig (2001) was used to evaluate mixture of pulverized corn, mungbean, and sunflower staging of smears. seeds. Vitamin and mineral premix was also given to ensure adequate nutrient supplementation. Access Preparation of Tissues for Histopathology to food was unrestricted and water was administered Collected uteri were immediately fixed in 4% formaldehyde through an automatic watering system. in 0.1M phosphate buffered saline for 10 h. Collected Three treatment groups were assigned to receive tissue samples were processed through a series of 3 different treatment preparation of the plant extract: alchohols and xylene, infiltrated with Paraplast high dose (1050 mg/kg•d), middle dose (787.5 mg/ paraffin under vacuum, and embedded in paraffin. Five-micron sections of cross and longitudinal sections kg•d), and low dose (525 mg/kg•d) based on LD50 of the plant (5250 mg/kg•bw) and initial body weight. of the mid-uterine body and tip of uterine horns were 17ß-estradiol (17ß-E2) was given on a 50 µg/kg•d body cut on a rotatory microtome and mounted on slides. weight basis for positive control, which was within the Uterine sections were stained primarily to measure treatment range used in the study by Padilla-Blanks changes in the luminal and glandular epithelium by et al. (2001) resulting to significant increase in body morphometric analyses (Grunert et al. 1987; Tinwell et weight and about 3-fold increase in uterine wet weight. al. 2000). Tissue sections were then sent to a veterinary Corn oil was used as the vehicle for crude kudzu extract pathologist for histopathological evaluation. and 17ß-E2. Only corn oil was used for the negative control (Yamasaki et al. 2001a). All groups received Morphometric Analyses: Luminal and Glandular treatment via gastric gavage at 0.5 ml per 50-g body Epithelial Development of Uterus weight, daily for 15 d to cover at least 3 reproductive Quantitative measurements were done to morphometrically cycles in the animal. assess the variations in epithelial cell heights. Histologic Using a triple beam balance, rat body weight was responses were quantified using a Nikon Microscope measured initially before treatment and then every three interfaced with an image analysis system (Computer days for the 15-d duration of the treatment. Imaging Laboratory, Institute of Biological Sciences, UPLB) using the program SCapture and ImageJ 1.33 (National Institute of Health, USA). Following Markey Uterotrophic Assay: Uterine wet weight et al. (2001) and Padilla-Blanks (2001) protocols, the determination following parameters were performed. Rats were sacrificed by cervical dislocation on day 15 of the study. But prior to this, the body weight and Epithelial cell height was measured in areas of vaginal smear of each rat were obtained. The horns and epithelium in which the nucleus and basement membrane body of the uterus were excised and their wet weights of single cuboidal/columnar epithelial cells are seen. determined. Absolute uterine weights were calculated To determine luminal cell height, 20 measurements as dividing the uterine wet weight by the body weight from each animal for three animals (60 measurements and multiplying by 100 (Padilla-Blanks et al. 2001). per group) were randomly obtained. Measurements were done in longitudinally embedded tissues, always from the middle of the uterus and away from Vaginal Cellular Differentiation the oviduct or cervical segments. In determining cell Vaginal smears were taken and scored daily to monitor height of glandular epithelium, 10 measurements from cellular differentiation. A smear was also performed each animal for 3 animals (30 measurements per group) on all rats before dosing to establish a baseline, and to were randomly obtained. The mean for each animal confirm that all ovariectomized animal smears showed was calculated and these animal means were used to no cornification. Vaginal smears were taken daily determine the mean ± SD for each treatment group. using a Pasteur pipette containing 8.5g/L of saline, placed on slides and observed under a light microscope using a 10X eyepiece and 10X objective. The same Statistical Analysis technician, unblinded, read smears immediately and Values were expressed as the mean ± SD. Data were cells were identified as leucocytes (PMNs), nucleated, assessed using Student’s t test and analysis of variance or cornified epithelial cells. A raw score on a scale of (ANOVA). Results were considered significant at 1 through 5 was assigned for cell populations ranging p<0.05.

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Results of leukocytes, advancing to nucleated cells, and terminating as fully cornified cells. Figure 1 shows the changes in vaginal cytology during the 15-d exposure Body weight and uterine weight changes period to doses of kudzu crude extract and 17ß-E2. As No evidence of overt toxicity and abnormal clinical expected, exposure to 17ß-E2 resulted in a persistent signs were observed in any of the groups during the 15-d estrous endocrine status, which was reflected by the treatment period. Although rat mortalities were noted appearance of cornified epithelial cells in vaginal due to impacted and non-healing wounds at the post- smears from OVX rats within 3 days following the OVX incision area prior to treatment (n=2) and on the initial dose and showed total cornification of cells sixth treatment day (n=1). Results of the uterotrophic in 5 days. Some cornified cells were also observed bioassay in rats are shown in Table 1. Treatment with following exposure to high dose of KCE (1050 mg/ various doses of P. phaseoloides as well as 17β-E2 caused kg•d) after 8 days of treatment, but failed to reach to

Table 1. Body weight and uterine weight of immature ovariectomized SD rats treated with tropical kudzu crude extract (KCE), 17β-estradiol or vehicle for 15 daysa Initial Body Final Body Weightb Uterine Wet Weight Treatment n Weight Absolute Uterine Weight (g) (g) (g) 1050 mg/kg KCE 5 37.94 ± 8.28 64.58 ± 7.54c 0.17 ± 0.02d***e 0.26 ± 0.01de*** 787.5 mg/kg KCE 6 35.73 ± 4.00 55.40 ± 7.19c 0.11 ± 0.05d***e 0.20 ± 0.09d**e 525 mg/kg KCE 6 35.98 ± 4.55 49.17 ± 5.15 0.08 ± 0.05de 0.16 ± 0.10de 17β-estradiol 5 43.88 ± 3.16 77.44 ± 2.60c 0.24 ± 0.03d 0.31 ± 0.03d Corn Oil 5 39.50 ± 2.70 51.82 ± 4.64 0.02 ± 0.00e 0.03 ± 0.01e aAll values represented as means ± SD. bStatistically different from the initial body weight [p < 0.001]. cChange in BW statistically significant compared with negative control (corn oil) [p<0.01]. dStatistically significant compared with negative control (corn oil) at p<0.05 [ * p<0.01; ** p<0.001; *** p<0.0001]. eStatistically significant compared with positive control (17β-E2) at p<0.05.

27-43% increase in body weight (BW) compared to initial recordings in all treatments (p<0.001). The increase in BW compared with untreated group (corn oil) was significant with 17β-E2 (50 µg/kg•d) and at the 2 higher doses of kudzu crude extract (KCE), i.e., 1050 and 787.5 mg/kg•day (p<0.005) but not with the low-dose KCE. However, the increase in BW with 3 doses of KCE was significantly lower than 17β-E2 treatment. Similar dose response increase in the uterine weight was observed with increasing doses of plant extract. Data of uterine weights were expressed as uterine wet weight and absolute uterine weight (Table 1). P. phaseoloides extract administered to OVX rats at 525, 787.5, and 1050 mg/kg dose-dependently increased both wet (p<0.05, 0.005, 0.0005) and absolute uterine weights (p<0.05, 0.001, 0.0001)], producing 7.6-, 6.9- and 4.8- Figure 1. Vaginal cellular differentiation induced in ovariectomized fold increases in absolute uterine weight, respectively. immature rats in the 5 treatments. (Scale used: 1-completely However, the response was significantly less than that undifferentiated leucocyte, 2-leucocytes and some observed in 17ß-E2 -treated rats (9.2 – fold), indicating nucleocytes, 3-predominantly nucleocytes, 4-nucleocytes and lower potency (p<0.01). some cornified cells, 5-totally cornified cells). Significant cellular differentiation of cells compared with the negative control was observed in 1050 and 787.5 mg/kg•d doses of Vaginal Cellular Differentiation KCE at particular days within the 15-d treatment period (* p<0.05). None of the KCE-treated groups was able to achieve Vaginal cells exhibited a distinct pattern of maturation total cornification of cells as observed significantly after 3 in response to 17ß-E2, beginning as a population days with 17β-E2-treated rats.

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the extended estrous stage. Middle dose KCE (787.5 mg/kg•d) induced a significant cellular differentiation compared with the negative control on the 12th day of exposure, but was not effective to induce cornification of cells until the last day of treatment period. The lowest dose of KCE did not stimulate differentiation relative to controls.

Uterine histologic observation: Luminal and glandular epithelial cell height The uterus exhibited an increase in epithelial cell height in response to 17ß-E2 and KCE (Fig. 2 to 4). Figure 2 shows the results of luminal epithelial cell height measurements. The increase in cell height was highest in rats treated with 17ß-E2, which induced a significant increase of 205% relative to the control group Figure 3. Glandular epithelial height in ovariectomized rats treated with crude kudzu extracts, 17-β-estradiol, and corn oil alone. (p<0.005). A dose-dependent increase in epithelial Values represent the mean ± standard deviation (SD) of 10 height was observed in rats treated with increasing dose measurements from 3 animals (total of 30 counts/group). All of P. phaseoloides crude extract inducing 11, 23, and treatment doses of crude kudzu extract induced significant 116% increase at 525, 787.5, and 1050 mg/kg•d KCE, increase (*) on glandular epithelial cell height compared with corn oil-treated rats (p<0.05 ). The increase in cell height respectively. However, the increase in cell height was observed in all the treatment groups was significantly lower significant relative to the negative control only at the (**) than 17-β-E2. highest concentration of KCE (p<0.01), and not with the middle (p= 0.07) and low doses (p= 0.56) of the plant extract. 169% were exhibited by the 3 doses of KCE and found to be statistically significant compared to the negative control (p<0.05, p<0.01). 17β-E2 was able to increase the glandular epithelial cell height by as much as 252% of the negative control (p<0.001). Any increase in the epithelial cell height induced by the plant extract, either luminal or glandular, was found to be statistically lower than that of 17ß-E2 treatment. Histopathological findings showed varying degrees of endometrial epithelial thickening, which is consistent to the dose used, i.e. the higher the dose the greater the degree of thickening and proliferation of cells in the stroma (Fig. 4). Uterine tissues demonstrated the characteristic hyperplasia and hypertrophy maximally seen in rats treated with 17ß-E2 becoming lesser in Figure 2. Luminal epithelial cell height in ovariectomized rats treated degree in the dose of tropical kudzu extract used. Thin with crude kudzu extracts, 17-β-estradiol, and corn oil alone. layer of endometrium with very few blood vessels in the Values represent the mean ± standard deviation (SD) of 20 measurements from 3 animals (total of 60 counts/group). stratum vasculare was observed in the uterine tissues Treatment with 1050 mg/kg•d of KCE induced a significant of corn oil-treated rats. Signs of actively functioning increase (*) on luminal epithelial cell height compared with uterus like thick endometrium with numerous glands, corn oil-treated rats (p< 0.05). The increase in cell height distinct vascularization of the stratum vasculare were observed in all the treatment groups was significantly lower (**) than 17-β-E2 noted on 17ß-E2 treatment resembling findings with higher dose of the plant crude extract. Low- and middle-dose treatments had normal microscopic appearance. Similarly, significant difference in glandular epithelial cell heights was observed as a response of the uterus with varying treatment doses of KCE (Fig. 3). A relative increases in glandular cell height of 52, 62, and

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Figure 4. Photomicrograph showing the luminal epithelium and stroma of the uterus from immature ovariectomized rats exposed for 15 days to corn oil (A), 50 µg/kg•d 17-β-estradiol (B), and different doses of Pueraria phaseoloides (Roxb.) Benth. (C- 525 mg/kg•d, D- 787.5 mg/kg•d, E-1050 mg/kg•d). (A) The unstimulated uterus from immature OVX rat shows a single layer of cuboidal cells or low columnar cells (e) lining the uterine lumen with minimal differentiation in the stroma (s). (B) The stimulated uterus treated with 17-β-estradiol shows tall columnar epithelial cells. Notice the dose-dependent increase in epithelial thickness among the treatment groups (C-E) compared to controls. Magnification at low power (100x), stained in H&E

Discussion The effect of estrogen on immature rat uterus has established the importance of this organ for the bioassay The estrous cycle involves several physiological and of estrogen-like substances (Sonnenchein and Soto morphological changes in the uterus, ovaries, and vagina. In 1998; Yamasaki et al. 2001b). In the uterotrophic assay, rats, the cycle is completed and recurs at 4- to 5-d intervals laboratory animals were exposed to a chemical substance (Maligalig 2001). The present experiment evaluated the believed to have an effect on the uterus. Afterwards, effect of P. phaseoloides through gastric gavage in some the uterus was collected and its weight assessed. This reproductive parameters of ovariectomized rats. Gastric procedure was an indirect measure of the proliferative administration of the test substance in rats at the organism effects of estrogen reflected by the increase in uterine level simulates the most possible means by which the crude weight (Sonnenchein and Soto 1998). As expected, extract can be consumed by humans. Besides, this in vivo exogenous administration of 17ß-E2 caused a significant experiment replicated the myriad of pharmacokinetic and increase in uterine weight. pharmacodynamic interactions that likely influence the estrogenic activity of the crude extract (Sutter 1995), which Uterotrophic assay revealed that gastric administration may not be demonstrated by in vitro assays. It should also of P. phaseoloides had a significant effect in increasing be noted that a reliance on in vitro assays for predicting the absolute uterine weight. The effect of P. phaseoloides in vivo disrupter effects may generate false-negative and in the uterus of ovariectomized rats strongly indicates false positive results (Chearskul et al. 2004). Therefore, in that some phytoestrogens of the plant led to an increase vivo assay must be conducted to test the in-vitro-generated in uterine weight. This finding was consistent to the hypotheses (Vos 2003). In vivo studies indicated that 15-d report of Tinwell et al. (2000) and Padilla-Blanks et uterotrophic assay in prepubertal rats or mice is an efficient al. (2001) that immature female rats and mice fed with method for determination of an estrogenic activity (Laws phytoestrogen coumestrol-containing diet can cause a et al. 2000). marked increase in uterine weight. However, the uterine

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weight increase was significantly lower than that of while ERβ prevails in ovaries (Couse et al. 1997; 17ß-E2-treated group, indicating that the plant exhibited Kuiper et al. 1997). Buchanan et al. (1998) found that only weakly estrogenic at high doses. Other studies with vaginal epithelial proliferation was mediated indirectly genistein and daidzen, the 2 major isoflavones found through stromal ERα, and both epithelial and stromal in leguminous plants, have also demonstrated weak ERα was required for estrogen-induced cornification estrogenic effects as reflected by changes in uterine and stratification. Phytoestrogens compete stronger weight (Santell et al. 1997; Picherit et al. 2000). with 17β-E2 for binding to ERβ than to ERα and trigger their estrogenic-like physiological responses Estrogen is known to elicit uterine growth response by mainly acting through ERβ (Kuiper et al. 1998). in a non-genomic mechanism (Grunert et al. 1987; This could probably explain the weak estrogenic Sonnenchein and Soto 1998), which involves inducing activity exhibited by P. phaseolides crude extract on changes such as increase in vascular permeability, water the ovariectomized rat. imbibition, and cellular infiltration (Rockwell et al., 2002). This sequence of events subsequently leads to Several mechanisms of action have been proposed to an increase in uterine weight. In ovariectomized rats, underlie the purported beneficial effects of phytoestrogens circulating level of estrogen is very low because of on menopausal symptoms. This includes weak estrogenic the removal of ovary, which is the primary source of action on the release of pituitary hormones (Murkies et the steroid hormone. Furthermore, immature rats were al. 1998). Phytoestrogens also stimulate the production found to be most sensitive to increase in circulating of sex-hormone binding globulin (SHBG) by the liver estrogen in the blood as its reproductive system totally (Pino et al. 2002). Higher SHBG levels result in more depends on estrogen for growth and differentiation. The bound and thus less free estradiol, reducing the amount study was conducted in ovariectomized, immature rat to of estrogens available for binding with estrogen rule out the effects of ovarian estrogen on uterotrophic receptors. Because of their weak estrogenic potential response patterns. Hence, the increase in uterine weight (≤0.1% that of estradiol), phytoestrogens did not elicit a in uterotrophic assay can very well be attributed to the strong estrogenic response and thus have anti-estrogenic effects of P. phaseoloides. Corn oil cannot cause the property that inhibits the growth and proliferation of increase in uterine weight since it was found to be the estrogen-dependent cancer cells (Makela et al. 1995; most appropriate vehicle for substances that measures Chearskul et al. 2004). This action is contrary to the reproductive organ weights in rats, i.e. it does not cause known fact that estrogen was typically involved, at least an increase in uterine weight (Yamasaki et al. 2001a). as a permissive agent, in initiation and progression of neoplasias of estrogen target organs (such as mammary Estrogen also promotes cornification of the gland, endometrium, vagina, and cervix) (Buchanan vaginal epithelial cells. Safranski et al. (1993) found et al., 1998). Davis et al., (1999) reported that this that vaginal estrus characterized by full cornification anti-estrogenic property functions in in vivo tests by of vaginal epithelial cells requires a higher surge of protecting estrogen receptor activation by the more estrogen level. This could explain the abrupt appearance potent steroidal agonists such as endogenous estradiol. of fully cornified cells at day 4 in smears of rats treated with 17ß-E2. Rats treated with 1050 mg/kg of the The implication of these preliminary findings may be crude extract showed an increase in this differentiation clinically significant. From the above results and previous pattern. This finding suggests the presence of some studies (Shi et al. 2003, Kintzios et al. 2004), it confirms components of P. phaseoloides, which can induce that the isoflavone content in P. phaseoloides (Roxb.) estrogen-related changes. However, this effect did not Benth has the estrogenic activity, as related subspecies match the pattern seen in rats treated with exogenous also exhibited the same effects. Local postmenopausal 17β-E2 where high dose of the plant extract was unable women, especially folks in the countryside where the plant to reach full cornification in the whole 15-d duration naturally abounds, may benefit from taking P. phaseoloides of the experiment. Gray and Ostby (1998) reported extracts. However, the safe dose is yet to be firmly vaginal mucification and vaginal histologic alterations established clinically as more experiments are needed to in ovariectomized rats after 10-wk exposure to weak settle other pharmacodynamic aspects of drug approval estrogenic compounds. Thus, a longer exposure period such as metabolism, toxicology, and isoflavone analysis with this weak estrogenic crude extract dose may be of the plant samples. In addition, there is also a need to able to achieve full cornification of cells. screen other wild varieties of the plant that contain the The uterus and ovaries are the most sensitive tissues highest concentration of the active principle. The high- to estrogenic regulation, and both tissues express ERα yielding variety is then selected or harvested from the and ERβ, ERα being the predominant isoform in uterus plant invasion areas or set up a controlled-cultivation for

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large-scale commercial production. Future formulation and chemical properties of miroestrol, the estrogenic of commercially prepared drug forms (e.g. as tablet or substance of Pueraria mirifica. J Chem Soc 739: syrup) may be underway as the active principle of the 3196-3705. plant is identified. In the end, this study hopes to share its BUCHANAN DL, KURITA T, TAYLOR JA, LUBAHN contribution in fulfilling the mandate of health sector in DB, CUNHA GR, and COOKE V. 1998. Role providing safe, effective, and affordable pharmaceutical of Stromal and Epithelial Estrogen Receptors in products derived from commonly available plants. Vaginal Epithelial Proliferation, Stratification and Cornification. Endocrinol 139: 4345-4352. Chansakaow S, Ishikawa T, Sekine Conclusion and K, Okada M, Higuchi Y, Kudo M and Recommendation Chaichantipyuth C. 2000. Isoflavonoids from Pueraria mirifica and their estrogenic activity. This study has shown that P. phaseoloides exhibited weak Planta Medica 66: 572-575. estrogenic effects in vivo in the rat uterus and vaginal cells. Also, the phytoestrogen(s) in P. phaseoloides CHEARSKUL S, KOOPTIWUT S, CHATCHAWALVANIT has low potency for estrogenic action. Short-term S, ONREABROI S, CHURINTRAPUN M, moderate consumption of tropical kudzu is unlikely to SARALAMP P and SOONTHORNCHAREONNON cause physical problems due to its estrogenic effect. N. 2004. Morinda citrifolia has very weak estrogenic However, long term exposure to higher doses may cause activity in vivo. J Physiol Sci 17: 22-29. pharmaceutical benefits or risks to humans. CHERDSHEWASART W. 2003. Toxicity tests of a The chemical nature of phytoestrogens in P. phaseoloides phytoestrogen-rich herb Pueraria mirifica. J Sci is still unknown and therefore warrants further investigation. Res (Chulalangkorn University) 28: 1-12. In addition, there is also a need to screen other wild varieties CHERDSHEWASART W, CHEEWASOPLIT W and of the plant that may be capitalized on for large-scale PICHA P. 2004a. The differential anti-proliferation commercial production. effect of white (Pueraria mirifica), red (Butea superba), and black (Mucuna collettii) Kwao Krua plants on the growth of MCF-7 cells. J Ethnopharmacol 93: 255-260. Acknowledgements CHERDSHEWASART W, CHEEWASOPLIT W and The authors are grateful to Our Lady of Fatima University PICHA P. 2004b. Anti-proliferation effects of white Administration and First Year Medicine, Section A2, (Pueraria mirifica), red (Butea superba), and black SY 2004-2005 for the financial assistance; Dr. MDG (Mucuna collettii) Kwao Krua plants on the growth Maligalig, Dr. EB Rodriguez and Prof. N. Sabino for the of HeLa cells. J Sci Res (Chulalangkorn University) use of laboratory facilities at University of the Philippines 29: 27-31. Los Baños; to Drs. PP Ocampo, Prof. JM Desamero and COUSE JF, LINDZEY J, GRANDIEN K, GUSTAFFSON Prof. RM Neyra for stimulating discussions, and; to Beth JA and KORACH KS. 1997. Tissue distribution Lontoc for providing excellent technical assistance in the and quantitative analysis of estrogen receptor-α whole project duration. (ERα) and estrogen receptor-β (ERβ) messenger ribonucleic acid in the wild-type and ERα-knockout mouse. Endocrinol 138: 4613-4621. References DAVIS SR, DALAIS FS, SIMPSON ER and MURKIES AL. 1999. Phytoestrogens in health and disease. Rec ADLERCREUTZ H. 1995. Phytoestrogens: Prog Hormone Res 54: 185:210. Epidemiology and a possible role in cancer protection. Environmental Health Perspectives 103 DE KLEIJN MJJ, VAN DER SCHOUW YT, WILSON (suppl 7): 103-112. PWF, ALDERCREUTZ H, MAZUR W, GROBBEE DE and JACQUES PF. 2001. Intake of Dietary ADLERCREUTZ H and MAZUR W. 1997. Phyto- Phytoestrogens is low in postmenopausal women estrogens and Western Diseases. Ann Med 29: in the United States: The Framingham Study. J Nutr 95-120. 131: 1826-1832. BOUNDS DG and POPE SG. 1960. Light absorption

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DIXON RA and FERREIRA D. 2002. Genistein. L E E Y S , PA R K L S , C H O S D , S o n J K , Phytochem 60: 205-211. CHERDSHEWASART W and KANG KS. 2002. Requirement of metabolic activation for estrogenic GRAY LE JR and OSTBY J. 1998. Effects of activity of Pueraria mirifica. J Vet Sci 3: 273-277. pesticides and toxic substances on behavioral and morphological reproductive development: endocrine LEE K, SOHN I, KIM Y, CHOI J, PARK H, ITOH versus non-endocrine mechanisms. Toxicol Ind Y and MIYAMOTO K. 2001. Tectorigenin, an Health 14: 159-184. Isoflavone of Pueraria thunbergiana Benth., Induces Differentiation and Apoptosis in Human GRUNERT G, NEUMANN G, PORCIA M and Promyelocytic Leukemia HL-60 cells. Biol Pharm TCHERNITCHIN AN. 1987. The estrogenic Bull 24: 1117-1121. responses to Clomiphene in the different cell types of the rat uterus: Morphometrical evaluation. Biol LIN RC, GUTHRIE S, XIE CY, MAI K, LEE DY, Reproduct 37: 527-538. LUMENG L and LI TK. 1996. Isoflavonoid compounds extracted from Pueraria lobata suppress INGHAM JL, TAHARA S and DZIEDEC SZ. 1988. alcohol preference in a pharmacogenetic rat model Coumestans from the root of Pueraria mirifica. of alcoholism. Alcohol Clin Res 20: 659-663. Zeitschrift fur Naturforschung C 43c: 5-10. LUKAS SE, PENETAR D, BERKO J, VICENS L, INGHAM JL, TAHARA S and DZIEDEC SZ. 1989. PALMER C, MALLYA G, MACKLIN EA and LEE Minor isoflavones from the root of Pueraria mirifica. DY-W. 2005. An extract of the Chinese herbal root Zeitschrift fur Naturforschung C 44c: 724-726. kudzu reduces alcohol drinking by heavy drinkers KEUNG WK and VALLEE BL. 1998. Kudzu root: an in a naturalistic setting. Alcoholism: Clin Expe Res ancient Chinese source of modern antidipsotropic 29:756-762. agents. Phytochem 47: 499-506. MAKELA SI, PYLKKANEN LH, SANTTI RSS and KIM HY, HONG JH, KIM DS, KANG KJ, HAN SB and ADLERCREUTZ H. 1995. Dietary soybean may be LEE EJ et al. 2003. Isoflavone content and estrogen antiestrogenic in male mice. J Nutrit 125: 437- 445. activity in Arrowsoot Puerariae Radix. Food Sci MALAIVIJITNOND S, KIATTHAIPIPAT P, Biotechnol 12: 1-7 CHERSDSHEWASART W, WATANABE G and KINTZIOS S, MAKRI O, PISTOLA E, MATAKIADIS TAYA K. 2004. Different effect of Pueraria mirifica, T, SHI HP and ECONOMOU A. 2004. Scale –up an herb containing phytoestogens, on LH and FSH production of puerarin from hairy roots of Pueraria secretion in gonadectomized female and male rats. phaseoloides in an airlift bioreactor. Biotechnol Lett J Pharmacol Sci 96: 428-435. 26: 1057-1059. MALIGALIG MDG. 2001. Laboratory Techniques in KUIPER GGJM, CARLSSON B, GRANDIEN K, Reproductive Physiology. College, Laguna: Institute ENMARK E, HÄGBLADD J, NILSSON S AND of Biological Sciences, College of Arts and Sciences, GUSTAFSSON J-A. 1997. Comparison of the ligand University of the Philippines at Los Banos. College, binding specificity and transcript tissue distribution Laguna. 18 pp. of estrogen receptor α and β. Endocrinol 138, 863- MARKEY CM, MICHAELSON CL, VESON EC, 870. SONNESCHEIN C and SOTO AM. 2001. The KUIPER GGJM, LEMMEN JG, CARLSSON B, mouse uterotrophic assay: A Reevaluation of its CORTON JC, SAFE SH, VANDER SAAG PT, validity in assessing the estrogenicity of Bisphenol VARDER BURG B and GUSTAFSSON J-A. A. Environ Health Perspect 109(1): 55-60. 1998. Interaction of estrogenic chemicals and MORRIS JB. 1999. Legume genetic resources with novel phytoestrogens with estrogen receptor β. Endocrinol “value added” industrial and pharmaceutical use. In: 139, 4252-4263. Perspectives on new crops and new uses. Janick J ed. LAWS SC, CAREY SA, FERRELL JM, BODMAN Alexandria, VA: ASHS Press. p. 196–201. GJ and COOPER RL. 2000. Estrogenic activity MUANGMAN V and CHERDSHEWASART W. of octylphenol, nonylphenol, bisphenol A and 2001. Clinical trial of the phytoestrogen-rich herb, methoxychlor in rats. Toxicol Sci 54: 154-167. Pueraria mirifica as a crude drug in the treatment of symptoms in menopausal women. Siriraj Hospital Gazette 53: 300-309.

47 Philippine Journal of Science Cordial et al.: Estrogenic Activity of Pueraria Vol. 135 No. 1, June 2006 phaseoloides Roxb. Benth

MUHR L, TARAWALI SA, PETER SM and SCHULTZE- SONNENSCHEIN C and SOTO AM. 1998. An updated KRAFT R. 1999. Forage legumes for improved review of environmental estrogen and androgen fallows in agropastoral systems of subhumid West mimics and antagonists. J Steroid Biochem Mol Africa. Trop Grasslands 33: 234-244. Biol 65: 143-150. MURKIES AL, G WILCOX and DAVIS SR. 1998. SUTTER TR. 1995. Molecular and Cellular Approaches Phytoestrogens. J Clin Endocrinol Metabol 83: to Extrapolation for Risk Assessment. Environ 297-303. Health Perspect. 103: 386-389. PADILLA-BLANKS E, JEFFERSON WN and TINWELL H, SOAMES AR, FOSTER JR and ASHBY NEWBOLD RR. 2001. The immature mouse is a J. 2000. Estradiol-type activity of coumestrol in suitable model for detection of estrogenicity in the mature and immature ovariectomized rat uterotrophic uterotropic bioassay. Environ Health Perspect 109: assay. Environ Health Perspect 108 (7): 631-634. 821-826. t r IS O MB o o n H, MALAIVIJI t n o n d S , NIKOV GN, HOPKINS NE, BOUE S and ALWORTH WATANABE G and TAYA K. 2005. Ovulation block WL. 2000. Interactions of dietary estrogens with by Pueraria mirifica: A study of its endocrinological human estrogen receptors and the effect on estrogen effect in female monkeys. Endocrine 26: 33-40. receptor-estrogen response element complex VOS JG. 2003. Risk assessment of endocrine active formation. Environ Health Perspect 108: 867-872. compounds. The All ChemE Seminars ‘Where PICHERIT C, DALLE M, NELIAT G, LEBECQUE science meets society’. Retrieved August 15, 2004 P, DAVICCO MJ, BARLET JP and COXAM V. from http://www.allchemeseminars.org. 2000. Genistein and daidzein modulate in vitro rat XU WH, ZHENG W, XIANG YB, RUAN ZX, CHENG uterine contractile activity. J Steroid Biochem 75: JR, DAI Q, GAO YT and SHU XO. 2004. Soya food 201-208. intake and risk of endometrial cancer among Chinese PINO AM, VALLADARES LE, PALMA MA, women in Shanghai: population based case-control MANCILLA AM, YANEZ M and ALBALA C. 2000. study. Brit J Med 328:1285-1289. Dietary isoflavones affect sex hormone-binding YAMASAKI K, SAWAKI M, NODA S and TAKATUKI globulin levels in postmenopausal women. J Clin M. 2001a. Effects of olive, corn, sesame or peanut oil Endocrinol Metabol 85: 2797-2800. on the body weights and reproductive organ weights ROCKWELL LC, PILLAI S, OLSON CE and KOOS of immature male and female rats. Exp Animal 50: RD. 2002. Inhibition of vascular endothelial growth 173-177. factor/vascular permeability factor action blocks YAMASAKI K, SAWAKI M, NODA S and TAKATUKI estrogen induced uterine edema and implantation M. 2001b. Effects of age and weaning on the immature in rodents. Biol Reprod 67: 1804-1810. rat uterotrophic assay using ehtynylestradiol. Exp SAFRANSKI TJ, LAMBERSON WR and KEISLER D. Animal 50: 87-89. 1993. Correlations among three measures of puberty in mice and relationships with estradiol concentration and ovulation. Biol Reprod 48: 669-673. SANTELL RC, CHANG YC, NAIR MG and HELFERICH WG. 1997. Dietary genistein exerts estrogenic effects upon the uterus, mammary gland and the Hypothalamic-Pituitary axis in rats. J Nutrit 127: 263-269. SHEBEK J and RINDONE JP. 2000. A pilot study exploring the effect of kudzu root on the drinking habits of patients with chronic alcoholism. J Alternative and Complementary Med 6: 45-48. SHI HP and KINTZIOS S. 2003. Genetic transformation of Pueraria phaseoloides in hairy roots. Plant Cell Rep 21: 1103-1107.

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