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international journal of phytomedicine

International Journal of Phytomedicine 2 (2010) 363-372 Research article
Phytoestrogens of Pachyrhizus erosus prevent Bone Loss in an
Ovariectomized Rat Model of Osteoporosis
Arief Nurrochmad1*, Fransiska Leviana2, Caecilia Govita Wulancarsari3, Endang Lukitaningsih4
*Corresponding author:
Abstract
The effects of the etyl acetate extract of root of Pachyrhizus
Arief Nurrochmad
erosus (L) Urb (EPE) on bone loss and in ovariectomized (ovx) rats model of osteoporosis were investigated. Forty-two 6-weeks- old female Sprague–Dawley rats were randomly assigned to six groups as followed, sham-operated, OVX, OVX-Estradiol (2 μg/day), OVX-EPE 200 mg/kg BW, OVX-EPE 400 mg/kg BW, OVX-EPE 800 mg/kg BW for 4 weeks. The administration of EPE was given orally using a stomach tube. The results demonstrated that the administration EPE 200, 400, and 800 mg/kg BW significantly prevented bone loss in OVX rats which these effect equivalent to estradiol. These effects were described in increased length of femur and tibiae, bone density, and mineral content of calcium and phosphorous in bone ash. EPE also significantly prevented OVX-induced uterine atrophy and increased in body weight gain. The femur mechanical testing significantly increased the ultimate load and stiffness of femurs of ovariectomized-rats that its effect was greater than OVX or sham- operated rats. Increased bone density may lead to enhanced bone strength, reducing the risk of fracture, which is evident in the administration of EPE due to high content of mineral density and content and increase the ultimate load. This effect seems to be pro-estrogenic compound, which suppress bone resorption by directly acting on estrogen receptor in bone sites. This study suggest that phytoestrogen compound from Pachyrhizus erosus may offer a potential alternative therapy for the treatment of health problems such as osteoporosis in post-menopausal women. Keywords: phytoestrogen, Pachyrhizus erosus, ovariectomized-
Introduction
Americans are at risk of osteoporosis by having low bone mineral contents and densities. Ten Osteoporosis is one of the major health problem, and expected to increase dramatically in the osteoporosis and the majority of these patients are women. Recent epidemiological studies have Foundation reported that about 44 million doi:10.5138/ijpm.2010.0975.0185.02051 arjournals.org, All rights reserved. Nurrochmad et al. International Journal of Phytomedicine 2 (2010) 363-372 suggested that the incidence of osteoporosis is a Recently, much attention has been focused on complex interaction due to many factors such as phytoestrogens, especially isoflavones, as a variety of genetic, geographic, and ethnic factors potential safe alternative for pharmaceutical HRT [1-3]. Estrogen deficiency is generally not one of [12]. Phytoestrogen is one of the natural alternatives that appear to offer the most potential osteoporosis, but it is indirect and strongly related for the prevent bone loss. Phytoestrogen is non- with the many recognized osteoporosis risk factors especially in women such as thin, structurally similar to estrogen and possesses advanced age, postmenopausal, amenorrhea, and both weak estrogenic and antiestrogenic effects [13,14]. Previous study in animals showed that Several line of evidence reported the importance phytoestrogen had a protective effect against of estrogen in bone remodeling and metabolism. bone loss due to estrogen deficiency. The Furthermore, the evident from the clinical used consumption of natural phytoestrogen from that the administration of hormone replacement soybean instead of a casein-based diet had been therapy (HRT) in a dose dependent manner effectively prevents bone loss in postmenopausal ovariectomized (OVX) rats [15,16]. Similarly, women [4,5] and reduces the incidence of genistein, a phytoestrogen found predominantly osteoporosis [6-9]. Unfortunately, the use of HRT in soybean, prevented bone loss in OVX rats [17- for long term caused several unwanted side effect associated with these powerful steroids and Several line of evidences reported that estrogen increased risk for breast and endometrial cancers receptors ERα and ERβ are presence in bone [10,11]. Therefore, further exploration of [20,21]. Both in vitro and in vivo studies have alternatives and/or adjunctive approaches that can shown that daidzein, genistein, and their produce clinically relevant prevent bone loss like glycosides exert a weak estrogenic effect [22]. In in osteoporosis would be interest. Non-hormonal addition, raloxifene shown the positive effects of theraphy or natural product therapy may more selective estrogen receptor modulators in animals acceptable for the treatment and prevent [23] and humans [24]. Because of their similarity to raloxifene in conformational binding to Fig 1. Structure of phytoestrogen compounds from root of Pachyrhizus erosus (Lukitaningsih, 2009)
Nurrochmad et al. International Journal of Phytomedicine 2 (2010) 363-372 estrogen receptors [25], genistin have selective The dried powders of roots of Pachyrhizus erosus were extracted by soxhlet using petroleum ether demonstrated that human dietary studies shown in order to separate phytosterol. Further, the the effects of isoflavone-rich soy protein diets on residue that obtained extracted again using markers of bone turnover and preventing bone methanol. The methanol extracts evaporated to loss as measured from bone mineral density obtain concentrated extract. This extract further (BMD) and content [28,29]. Recent studies dissolved in water and partition with ethyl indicate that oral administration of daidzin, acetate. Subsequently, the ethyl acetate fractions genistin, genistein and their succinyl derivatives obtained were dried at 60°C on steam bath significantly prevents bone loss in an ovx model followed by a freeze dried to obtain dried extracts from Pachyrhizus erosus (EPE). The extractive Pachyrhizus erosus (L) Urb or bengkoang is one value of ethyl acetate from dried powders was of the natural plant contain some phytoestrogens. calculated as % w/w yield and was found to be Many years, bengkoang root known and use for traditional cosmetic as sunscreen and whitening. At least four phytoestrogen compounds have been isolated and identified at least 4 phytoestrogen compounds in root of Pachyrhizus erosus (L) Urb such as daidzein, daidzein-7-O-β-glucopyranose, (8,9)-furanyl-pterocarpan-3-ol, and 5-hydroxy-daidzein-7-O-β-glucopyranose (Fig.1) [31]. However, the estrogenic effect of phytoestrogen from Pachyrhizus erosus (L) Urb have not been investigated especially the novel compound, (8,9)-furanyl-pterocarpan-3-ol. Because of that, there is a great interest to investigate the effect and action of phytoestrogen of root of Pachyrhizus erosus (L) Urb on bone and uterine tissue in this osteoporosis model. Materials and Methods

Plant and Chemical Materials
Pachyrhizus erosus (L) Urb used in the present
study were collected from commercial market
and authenticated at the Laboratory of
Pharmacognosy, Department of Pharmaceutical
Biology, Gadjah Mada University, Yogyakarta,
Indonesia. Ethanol 96%, methanol p.a, ethyl
acetate p.a and petroleum ether p.a. were
obtained from Sigma (St. Louis, MO, USA). Estradiol were purchased from Sigma (St. Louis, Figure 2. Effect of phytoestrogen of EPE on femoral mechanical
MO, USA). All other reagents were of analytical strenght [ultimate load (A), Stiffness (B)] in ovariectomized
(OVX)-rat model of osteoporosis. Each column represents mean
± S.E.M. of 5 rats. *p<0.05, **p<0.01 significantly different with
OVX-rats group.
Preparation of Ethyl Acetate Extract of
Pachyrhizus erosus
(EPE)
Nurrochmad et al. International Journal of Phytomedicine 2 (2010) 363-372 and tibiae were also removed immediately for bone analyses. Female Sprague–Dawley rats, aged 42 days, were purchased from Animal Laboratory Center Unit Serum and Urine Analysis
(The Laboratory of Research and Assessment, Blood samples were centrifuged at 2000 × g for Gadjah Mada University, Yogyakarta, Indonesia). 15 min to obtain serum, after standing for 30 min polyacrylic cages with not more than five animals laboratory conditions (temperature 25 ± 2°C) spectrophotometrically, using commercial kits with dark and light cycle (12/12 h) and allowed from DiaSys International (Holzheim, Germany). free access to commercial pellet diet (PT. Urinary excretion of creatinine, calcium, and Multipala Agrinusa, Indonesia) and water ad phosphorus were measured with a commercial kit from DiaSys International (Holzheim, Germany). Bone Length, Density and mineral content
Administration Procedure
The femurs were also removed immediately after Rats were acclimatized to laboratory condition sacrified for bone analyses. The right and left for 1 week before commencement of experiment. femurs were freed of soft tissue. The removed right femurs were freed of soft tissue using small accordance with Guideline for Care and Use of scissors, tweezers and cotton gauze. The length of Animals Laboratory of Faculty of Pharmacy, each femur was measured with a Vernier caliper. Gadjah Mada University. At 50 days of age, Following the same method as in the previous bilaterial ovariectomy was performed via a dorsal report [30] bone volume and density were midline incision under ether anesthesia. Upon measured by applying Archimedes’ principle [32] recovery from anesthesia, animals were assigned Then the bones were dehydrated and defatted in to experimental groups, normal (sham-operated), acetone and anhydrous ether, dried for 12 h at OVX, OVX-estradiol, OVX-EPE 200, OVX-EPE 110°C and reweighed to obtain the dry bone weights. Bone calcium and phosporus content in animals per group, per experiment. Twenty days ash bone were determined by atomic absorbtion after ovariectomy, all the rats were allowed controlled access to a commercial standard pellet and free access to deionized water for 20 days. Femoral Mechanical Testing
Normal (sham-operated) and OVX rats were sacrified under light anesthesia to determine the Bone Strength (Breaking Force) was measured baseline at 70 days. From 70 days, estradiol (2 according to Yao et al. (2005) [33] by means of a µg/day), EPE (200, 400 and 800 mg/kg BW/day) three-point bending test on an universal test was given orally using a stomach tube for 4 instrument of the Instron type (Tokyo Testing weeks. The food intake of all rats was measured every 3 d. At day 28 after first dosing, the urine reported previously. The three-point bending test of each rat was collected over 24 h, using a was performed at a displacement rate of 0.05 mm.min-1. The load-displacement curve was recorded simultaneously during the test. The On the day after the last dose, the rats were blood ultimate load and the stiffness of the femoral collected from orbital plexus after and sacrified were measured from the load-displacement curve. under light anesthesia. The uterus was removed and the wet weight, was determined. The femurs Nurrochmad et al. International Journal of Phytomedicine 2 (2010) 363-372
Statistical Analysis
Table 1. Effect of phytoestrogen of EPE on body weight
and uterus in ovariectomized (OVX)-rat model of
osteoporosis

expressed as the mean ± S.E.M. The statistical significance of differences between the groups were assessed with a one-way ANOVA, followed by Bonferroni or LSD post-hoc test analysis using rel 13.0 software SPSS (Chicago, IL, USA). p values of less than 0.05 were considered Body and Uterine Weights
The effect of EPE on average body weight gain and uterus are presented in table 1. As decribed in table 1, ovariectomi caused atrophy of uterus (p<0.001). This effect was prevented by the administration of EPE 200 and 400 mg/kg BW, Values are means±S.E.M., n=6−8 rats. Within a column, values with a superscript are significantly different: ## p,0.01, ### ovariectomy increased average daily body weight p,0.001 compared with sham rats; *p<0.05; **p<0.01; ***p<0.001 compared with OVX rats. gain (p<0.001). This effect also was prevented by the administration of EPE 200, 400 and 800 Table.2 Effect of phytoestrogen of EPE on femoral and
tibiae lenght, bone density and bone mineral content in

mg/Kg BW. The effect of EPE 800 mg/kg BW ovariectomized (OVX)-rat model of osteoporosis
Femoral Length, Density, and Calcium and
Phosphorus Content
content of
phosphorus
The activities of EPE on bone were demonstrated in table 2. The result demonstrated that OVX caused bone loss which determined by decreased bone density (p<0.01), content of calcium bone (p< 0.05) and phosphorous (p<0.05) (Table. 2). The results shown that the administration of EPE 200, 400 and 800 mg/kg BW for 28 days capable to increase the length of femur, tibiae, bone density and calcium content in bone (Table 2). These effect of all dose of EPE were greater than estradiol for length of femoral and tibiae. The effect of EPE 400 mg/kg BW on bone density was equivalent with estradiol. Furthermore, the administration of all doses of EPE and estradiol Values are means±S.E.M., n=6−8 rats. Within a column, values with a superscript are significantly different: ## p<0.01, ### could restore the calcium content loss to the sham- p<0.001 compared with sham rats; *p<0.05; **p<0.01; group, but only 400 mg/kg could restore to the sham- ***p<0.001 compared with OVX rats. group for phosphorous content loss and this effect was Nurrochmad et al. International Journal of Phytomedicine 2 (2010) 363-372 Table.3 Effect of phytoestrogen of EPE on serum calcium, phosphorus and alkaline
phosphatase (ALP) in ovariectomized (OVX)-rat model of osteoporosis

Serum phosphorus
Alkaline phosphatase
Serum calcium (mg/dl)
ALP (U/l)
Values are means±S.E.M., n=6−8 rats. Within a column, values with a superscript are significantly different: *p<0.05; **p<0.01; ***p<0.001 compared with OVX rats. Table 4. Effect of phytoestrogen of EPE on urinary calcium and phosphorus in ovariectomized (OVX)-rat
model of osteoporosis

Creatinine
Urinary calcium
Calcium/
Urinary phoshorus
Phosphorus/
creatinine
Creatinine
Values are means±S.E.M., n=6−8 rats. Within a column, values with a superscript are significantly different: ## p<0.01 compared with sham rats. *p<0.05; **p<0.01; ***p<0.001 compared with OVX rats Nurrochmad et al. International Journal of Phytomedicine 2 (2010) 363-372
Femoral mechanical testing
circulation of daidzein in the blood circulation. In OVX rats, ovariectomy slightly reduced Previous study shown that isoflavone genistin ultimate load, an indicator of the material properties of bone, compared with sham rats (Fig. ovariectomized rats [30]. Other study reported that daidzein more effective preventing bone loss administration of EPE 200, 400, and 800 mg/kg in ovariectomy- induced bone loss in rats [19]. BW significantly increased the ultimate load and The present study was investigated the potential stiffness of femurs of ovariectomized-rats that its preventive effects of ethyl acetate exctract of effect was greater than OVX or Sham (Fig 1 and Pachyrhizus erosus (EPE) which contain daizein 2). EPE 400 mg/kg BW is the optimum dose to or its glycoside and the novel compound, (8,9)- achieved greatest ultimate load and stiffness of furanyl-pterocarpan-3-ol on bone loss in animal femoral and its effect was equivalent to estradiol. model of osteoporosis. The administration of EPE prevented OVX-induced increase average body Serum and Urinary Calcium and Phosphorus
weight gain in rats. This results also support by The effect of EPE on the mineral serum and previous study that daidzin prevented OVX- induced uterine atrophy and increases in body administration of EPE 200, 400 and 800 mg/kg BW decreased the concentration of calcium in cholesterol and triglyceride [27]. In addition, serum that indicated the bone formation. This other study also reported that soybeans-rich effect is greater than estradiol (Table 3). isoflavones dietary interventions effectively Similarly, the clearance of urinary calcium decreased by the administration of EPE 200, 400, increased cholesterol serum in rats [15]. and 800 mg/kg BW and estradiol. The clearance According with previous report, rats in the OVX group had lower densities of the right femur and administration of EPE 200, 400 mg/kg BW but tibiae because of reducing the ovariectomy- induced increase in bone resorption [15,19,32]. The administration of EPE 200, 400 and 800 Discussion
mg/kg BB effectively prevented OVX-induced The main objective of this study was to evaluate lowering bone density and increased lenght of whether ethyl acetate extract of Pachyrhizus femure and tibiae. These observations are erosus (EPE) is effective in preventing bone loss supported by previous study that isoflavones due to ovariectomy and compare with estradiol. daidzin, genistin and glycitin significantly Ovariectomized rats are classically used as an prevented bone loss in OVX rats, like estrone [27]. In addition, the result also demonstrated that postmenopausal bone loss [32]. Furthermore, the rats receiving EPE shown greater load strain they may provide a useful model for investigating than sham and OVX. Increased bone density may the biological effect of EPE on bone loss in rat- lead to enhanced bone strength, reducing the risk ovariectomized. Ethyl acetate extract from of fracture, which is evident in the administration Pachyrhizus erosus at least contain isoflavone of EPE due to high content of mineral density and such as daidzein, daidzein-7-O-β-glucopyranose, content and increase the ultimate load. The 5-hydroxy-daidzein-7-O-β-glucopyranose and preventive effect of EPE may be due to enhanced (8,9)-furanyl-pterocarpan-3-ol [31]. Isoflavones intestinal absorption. Although we did not assess daidzein in Pachyrhizus erosus form in conjugate intestinal calcium absorption in this study, the enhanced intestinal absorption of calcium along microflora, which influences their bioavailability. hormone and renal function [34]. This finding indicated that the administration of EPE for 28 Nurrochmad et al. International Journal of Phytomedicine 2 (2010) 363-372 days increased the ovariectomy-induced rate of 2. Ross PD, Fujiwara S, Huang C, Davis JW, bone formation. Previous study proposed that daidzein act as proestrogenic compounds based Melton LJ Vertebral fracture prevalence in on bone loss and uterine in OVX rats which that women in Hiroshima compared to Caucasian may be tissue-specific 27]. Both in vitro and in vivo studies have shown that daidzein, genistein, and their glycosides exert a weak estrogenic 3. Lau EMC, Cooper C. The epidemiology of effect [22]. In fact, estrogen receptors are osteoporosis: the oriental perspective in a presence in bone [20,21]. In accord similarity of world context. Clin Orthop 1996; 323:65–74. isoflavone content and structure, we propose that 4. Lindsay R, Hart DM, Aitken JM, MacDonald the mechanism of preventing bone loss in ovariectomy rats through the binding of its prevention of postmenopausal osteoporosis compounds in Pachyrhizus erosus (Fig.1) to by oestrogen. Lancet 1976;1:1038–1041. In summary, we have demonstrated that the administration of ethyl acetate extract from Pachyrhizus erosus prevents bone loss in an ovariectomy rat model of osteoporosis. This 6. Gordon GS, Picchi J, Roof BS. Antifracture effect seems to be proestrogenic compound, which suppress bone resorption by directly acting on estrogen receptor in bone sites. Isolation and 7. Hutchinson TA, Polansky SM, Feinstein AP. Pachyrhizus erosus may offer a potential Postmenopausal oestrogens protect against alternative therapy for the treatment of health fractures of hip and distal radius. Lancet 8. Michaëlsson K, Baron JA, Farahmand BY, phytoestrogen compounds of Pachyrhizus erosus on bone in OVX rats appear to be similar to that of estradiol. Further studies are needed to replacement therapy and risk of hip fracture: investigate the efficacy of that phytoestrogen in population based case-control study. The Acknowledgements
9. Blank RD, Bockman RS. A review of clinical trials of therapies for osteoporosis using This work was supported in part by Providing fracture as an end point. J Clin Densitom Research Grants on Herbal Medicine Indonesia, Managing Higher Education For Relevance and 10. Writing Group for the Women’s Health Initiative Investigators. Risks and benefits of MHERE/III/10, recipient AN). We also thank to Prof. Dr. Edy Meiyanto for suggestions to References
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