Antioxidant enriched fraction from Pueraria tuberosa alleviates ovariectomized-induced osteoporosis in rats, and inhibits growth of breast and ovarian cancer cell lines in vitro

Pueraria tuberosa (P. tuberosa), known as Indian Kudzu belongs to family Fabaceae and it is solicited as “Rasayana” drugs in Ayurveda. In the present study, we analyzed the efficacy an antioxidant enriched fraction (AEF) from the tuber extract of P. tuberosa against menopausal osteoporosis and breast and ovarian cancer cell lines. The AEF from P. tuberosa was identified by determining phenolic composition (total phenolic and flavonoid amount). Antioxidant property (in vitro assays) was also carried out followed by analysis of the AEF for its antiosteoporotic and anticancer potentials. Antiosteoporotic activity of AEF was investigated in ovariectomy-induced osteoporosis in rats and in vitro anticancer activity by MTT assay. Also, the GC/MS analysis of AEF was performed to determine various phytoconstituents. A docking analysis was performed to verify the interaction of bioactive molecules with estrogen receptors (ERs). Ethyl acetate fraction of the mother extract was proved as the AEF. AEF significantly improved various biomechanical and biochemical parameters in a dose dependent manner in the ovariectomized animals. AEF also controlled the increased body weight and decreased uterus weight following ovariectomy. Histopathology of femur revealed the restoration of typical bone structure and trabecular width in ovariectomized animals after AEF and raloxifene treatment. AEF also exhibited in vitro cytotoxicity in breast (MCF-7 and MDA-MB-231) and ovarian (SKOV-3) cancer cells. Further, genistein and daidzein exhibited a high affinity towards both estrogen receptors (α and β) in docking study revealing the probable mechanism of the antiosteoporotic activity. GC/MS analysis confirmed the presence of bioactive molecules such as stigmasterol, β-sitosterol, and stigmasta-3,5-dien-7-one. The observations of this study vindicate the potency of AEF from P. tuberosa in the treatment of menopausal osteoporosis and cancer.


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World Health Organization defines osteoporosis as a decrease of bone mineral density (BMD) to 51 greater than 2.5 standard deviations of the standard reference for BMD in young health women 52 [1]. Osteoporosis deteriorates BMD, bone architectural structure and enhances the risk of 53 fracture. In addition, osteoporosis causes severe problems to human's quality of life, such as 54 disability, loss of living ability, and even death [2]. Variation in bone forming (osteoblastic) and 55 bone resorbing (osteoclastic) cell function causes osteoporosis [3]. Osteoporosis has the highest 56 prevalence in senile people and severely affects about 50% of menopausal women worldwide.

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The expected adult population over 60 years in India by 2050 would be 315 million signifying 58 more incidence of osteoporosis compared to 26 million in 2003 [4]. A decrease in the level 59 estrogen is the key contributing feature for menopausal osteoporosis (MO) in women. The 60 reduced estrogen causes diminished bone formation, enhanced bone resorption, and elevated 61 production of proinflammatory cytokines such as IL-1, . The osteoclasts produce reactive oxygen species like superoxides and rise in malondialdehyde level 70 in blood. These oxidative stresses also contribute to bone loss in osteoporosis [4]. Antioxidants 71 can be useful in the management of MO by normalizing the altered osteoblastic and osteoclastic 72 functions [10]. 73 Several drugs, such as estrogens, biphosphonates, and parathyroid hormone analogs are 74 used for the inhibition and management of osteoporosis. They promote bone formation or 75 decrease bone resorption or both [11]. However, these treatments comprise serious concerns 76 related to their safety and efficacy. Estrogen therapy is not preferred in patients with hepatopathy 77 and venous embolism. Also, the possibility of cancers (breast, cervical, ovary), heart disease, and 78 stroke are high in long-term use of estrogen [12]. Long-term application of biphosphonates 79 shows adverse effects such as osteonecrosis of the jaw and atypical femoral fractures [13]. 80 Parathyroid hormone analogs are costly, with patients needing daily injection, and may cause 81 adverse consequence like osteosarcoma [14]. Therefore, it is important to develop drugs from 82 plant origin with that have a protective effect on bone loss with fewer side effects. These plant-83 derived estrogenic compounds are known as "Phytoestrogens" and are accepted worldwide as 84 safe treatments [7]. The phytoestrogens mostly include isoflavones, isoflavanones, coumestans, genistein, and daidzein [22]. Despite the significant pharmacological and phytochemical 97 potential, the antiosteoporotic activity of P. tuberosa has not been explored. Our objective was to 98 identify an antioxidant enriched fraction (AEF) from the tubers of the plant, and to investigate 99 the preventive effect of AEF in menopausal osteoporosis and anticancer activity.

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The following chemicals in high grade were obtained commercially or as a gift: 3-(4,5-  Ltd., India) were procured. Resources, Regional Station, Phagli, Shimla, India. A voucher specimen has been preserved in 115 the Institute of Pharmacy, GGU, Bilaspur for future references. The fresh tubers were cut into 116 small pieces and dried under shade, then coarsely powdered and stored in an air-tight container 117 until further use. The coarse powder material was extracted with ethanol using soxhlet apparatus.

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The extract was concentrated under reduced pressure using a rotary vacuum evaporator. The 119 concentrated extract was suspended in distilled water and successively fractionated by liquid-120 liquid partitioning with n-hexane, ethyl acetate and n-butanol. Finally, the remaining aqueous 121 fraction was also prepared. All the fractions were dried and stored in air tight container until 122 further use.  Acute oral toxicity study 206 An OECD 423 guideline was employed to determine the acute oral toxicity of AEF. The limit 207 test was performed as per the guidelines on female rats (three rats per step) at a dose of 2000 208 mg/kg, orally and monitored for 14 days. The AEF was suspended in carboxy methyl cellulose

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(1.0%). Neither mortality nor any signs of moribund status were found at this dose (2000 mg/kg).

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Therefore, the LD 50 cut-off is 5000 mg/kg (category 5 in the Globally Harmonized Classification Group I: Sham-operated and received 1% CMC (Sham control).

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Antiosteoporotic activity 317 The antiosteoporotic potential of AEF of P. tuberosa was evaluated in ovariectomized-induced 318 osteoporosis in female rats by determining the following parameters:

Effect of AEF on biochemical parameters
320 Phosphorous (P) and calcium (Ca) level were analyzed both in serum and urine (Fig 1 and 2). Both ALP and TRAP levels were reduced significantly and dose dependent after AEF treatment 326 (versus OVX). Serum ALP and TRAP level were also reduced significantly after raloxifene 327 treatment (Fig 1). OVX caused significant increase in the level of urine hydroxyproline (HP) 328 compared to sham control. However the level of HP in raloxifene and AEF (100 and 200 mg/kg) 329 treated groups was distinctly lowered (p<0.001) compared to the OVX group (Fig 2). Level of 330 TC and TG increased significantly (p<0.001) in the OVX group compared to the sham control 331 group (Fig 1). These increased TC and TG level was markedly lowered by AEF and raloxifene 332 treatment. TG levels in raloxifene and AEF-200 groups are comparable with the sham control, 333 and AEF exhibited better effect over raloxifene. compared to the sham control group. Significant increase in all these parameters (Fig 3 and 4) 349 was observed with AEF and raloxifene administration. Furthermore, OVX caused a significant 350 reduction of femur density, and treatment with AEF showed a substantial improvement of femur 351 density. Treatment with raloxifene and AEF restored the breaking strength of 4 th lumbar 352 vertebrae caused by ovariectomy (Fig 4).

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## p < 0.01, ### p < 0.001 significantly different from OVX group. 363 A significant (p<0.001) increase in body weight (BW) was observed due to OVX though there 364 was no variation at the start of the study. Treatment with AEF and raloxifene markedly reduced 365 the increased BW (Fig 5) as well as the final and initial BW difference compared to OVX. OVX 366 caused a marked reduction in uterus weight. In comparison, administration of raloxifene, and 367 AEF significantly increased uterine weight compared to OVX (Fig 5).  absorption of calcium in intestine, as reported in previous studies [4,7]. ALP and TRAP (bone 441 turnover markers) activity are signs of bone osteoblast functioning and factors of bone formation.

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OVX increased these markers in serum because of the reduction in the estrogen level. HP is a 443 commonly accepted biochemical parameter associated with bone metabolism, and its level is a strength. In the current study, the breaking strength of femur and 4 th lumbar vertebrae increased 459 substantially by AEF of P. tuberosa proving the defensive effect of AEF against menopausal 460 osteoporosis which are comparable to earlier reports [4,7]. The phytoestrogens of AEF might 461 have an estrogen like activity that manages osteoclast activity and reduces bone turnover.

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The reduction of estrogen level in OVX animals causes increase in energy intake and to estradiol, which is also supported by earlier studies as daidzein and genistein showed high 482 affinity into estrogen receptors [43]. Therefore, these two compounds might be mainly 483 responsible for the antiosteoporotic property, which has been reported earlier [44,45].

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Phytoestrogens have been used as an alternative therapy for the management of