Anti-osteoporosis Effect of Fisetin against Ovariectomy Induced Osteoporosis in Rats: In silico, in vitro and in vivo Activity.

Osteoporosis is a bone related disease that is characterised by bone loss that further increases the susceptibility to bone fractures and bone frailty due to disturbances in the micro-architecture of bone tissue. Fisetin (flavonoids) exhibited anti-inflammatory and antioxidative stress effects against various diseases. In this protocol, we make an effort to comfort the anti-osteoporosis effect of fisetin against ovariectomy (OVX) induced osteoporosis. A docking study of fisetin and alendronate on the estrogen (α and β) and vitamin D receptors was carried out. SaOS-2 (osteoblast like human) cells were used for the estimation of cell proliferation. The OVX induced OVX model was used and three doses of fisetin and alendronate was given to rats till 16 weeks. The hormone levels, bone turnover markers and biochemical parameters were estimated. Fisetin was docked into estrogen (α and β) and vitamin D receptors, resulting in stable complexes with lower binding scores. Fisetin significantly (p < 0.001) exhibited the induction of cell proliferation against the SaOS-2 cells. OVX induced osteoporosis rats exhibited a suppression of body weight and uterus index, after the Fisetin treatment. Fisetin treatment significantly (p < 0.001) improved the level of bone mineral content (BMC), bone mineral density (BMD) and biochemical parameters such as energy, maximum load, stiffness, young modules, maximum stress and reduced the level of 1,25(OH) 2 D3 and E 2 . Fisetin treatment significantly (p < 0.001) declined the level of phosphorus (P), calcium (Ca) and boosted the level of VitD. Fisetin treatment significantly (p < 0.001) reduced the malonaldehyde (MDA) level and enhanced the glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) level in the bone, intestine and hepatic tissue. Fisetin treatment suppressed the cytokines, RANKL/OPG ratio, receptor activator of nuclear factor-κB ligand (RANKL) and improved the level of osteoprotegerin (OPG). The findings suggest that fisetin could be a beneficial phytoconstituent for the treatment and prevention of postmenopausal osteoporotic complications.


Introduction
Osteoporosis is a chronic and multifactorial bone disease that expends unnoticeably until the advanced stage with complex pathophysiology 1 . Osteoporosis is the major common health problem in the geriatric population worldwide 2,3 . Every year, 1.24 million fractures caused by osteoporosis are reported worldwide. In the next 30 years, the incidence of hip fractures in both men and women will increase by 240 and 310 , respectively. The incidence of osteoporosis is increasing in Asia day by day, especially in China. A clinical study showed that postmenopausal women have osteoporosis and osteopenia 2,5,6 . The incidence of osteoporosis increases in men at the age of 20 to 89 years old. Osteoporosis is mainly categorised by the suppression of bone mass and density, which results in enhanced fragility and fracture of bones 2,3,7 . It is a major health problem in women, especially the elderly and postmenopausal women. Menopause in those women is related to an enhanced risk of osteoporosis, which creates an imbalance between the osteoclast resorption and formation of bone due to loss of hormones such as estrogen E 2 3, 8, 9 . The reduction of E 2 plays a crucial role in the inhibition of bone density loss and ensuing the osteoporosis.
The current available therapies for osteoporosis in women elderly and postmenopausal are estrogen replacement therapy ERT and hormone replacement therapy HRT 2,9,10 . However, there are limitations to the therapy due to adverse/side effects, such as long-term HRT therapy increasing the risk of endometrial, ovarian, and breast cancer 9,11,12 . The levels of vitamin D and calcium are altered during osteoporosis, and studies suggest that avoiding smoking, alcohol, caffeine, and regular exercise can help prevent the development of osteoporosis. Another thing, regular intake of a controlled diet rich in vitamin D and Ca helps in reducing bone density and loss. Bone loss occurred in postmenopausal women due to increased oxidative stress and inflammatory reactions, which started due to the continuous production of free radicals and modulated the level of endogenous antioxidants and boosted the inflammatory parameters 13 . Oxidative stress promotes the osteoclastic function and bone differentiation of bones and starts bone loss 2,3 .
The treatment of osteoporosis was very tedious a few years ago. Now, with the advancement of the science, there is an easy way to treat osteoporosis. Moreover, the recent treatments have limitations such as long-term safety and e f f i c a c y 1,14 . C u r r e n t l y, h o r m o n e s a n d e s t r o g e n replacement therapy have various nonskeletal effects, including stroke, breast cancer and heart disease. Long term bisphosphonate treatment is used for the treatment of osteoporosis, but the treatment has adverse effects such as atypical fractures and osteonecrosis of the jaw. Parathyroid hormones newer anabolic agents are used for the treatment of osteoporosis 15 . Furthermore, the cost of the treatment and daily need for the injection have a limit for prolonged and widespread use. Moreover, some plants or herbs have a protective effect against bone loss due to its estrogen like effects but also contain a little amount of estrogen, it may also hint at the dangers of unidentifiable, unknown estrogen 2 . Therefore, the researchers continue searching for a cheaper and effective drug with less or no side effect to improve upon the present therapies still show a definite need.
Excessive osteoclast resorption and formation are considered as the significant pathological alteration in OVX induced osteoporosis. Osteoclast precursor cells express the receptor activator of nuclear factor-κB NF-κB ligand RANKL , which binds to RANKL receptors on the surface of osteoclast precursors. RANKL-osteoprotegerin OPG isolated from osteoblasts and marrow stomal cells MSCs , opposed the effect. Estrogen deficiency enhance the OPG production and suppress the RANKL source, thereby arbitrating osteoclast function and formation 16 . Estrogen also boost the production of osteoclastogenic cytokines, which act as pro-resorptive factors via boosting the expression of RANKL in osteoblast lineage cells 17 . Flavonoids are the low molecular weight polyphenolic phytoconstituent commonly synthesized in lot of plants 18,19 . Fisetin IUPAC name-3,3 ,4 ,7-tetrahydroxyflavone , flavonoid, commonly found in various vegetable and fruits such as grape, cucumber, strawberry, onion and persimmon 20,21 . Fisetin involved in balancing the various oxidative stress aspects viz., anti-lipoperoxidation effect or scavenge the free radicals in the biological system 20,22 . The capability of fisetin to scavenge the free radicals exhibited its antioxidant effect and biological effects. Fisetin exhibited the wide range of pharmacological effects such as antioxidant, neuroprotective, anti-tumor, antiinflammatory and neurotrophic 18,20,23 . Fisetin also suggest the neuroprotective and chemotherapeutic effect in human and mice. As hydrophobic agent, fisetin easily infiltrate into cell membranes and accumulate in cells to exert its neuroprotective, neurotrophic and antioxidant effects 18,20,21 . Previous report suggests that fisetin treatment could relieve inflammation, cell apoptosis and oxidative stress via suppression the receptor for advanced glycation end products RAGE /nuclear factor-κB NF-κB signaling 24,25 . Thus, this experimental protocol scrutinizes the antiosteoporosis effect against SaOS-2 osteoblast like human cells and ovariectomy induced osteoporosis model in female rats and explore the underlying mechanism.

Chemicals
Fisetin 98 , alendronate sodium salt, trichloroacetic acid TCA and 2-thiobarbituric acid TBA were purchased from the Sigma Aldrich St. Louis, USA . All the reagents and chemicals used in this experimental study was analytical grade.

In silico study
We used molecular docking simulations at the Estrogen Alpha receptor catalytic ligand binding site to better understand the binding mode of Fisetin at the molecular level PDBID: 5wgd . The docking of fisetin was done with Maestro, a Schrödinger software suite programme, version 9.6. Using the build panel, the ligand was sketched in 3D format and prepped for docking using the ligprep tool. The protein for the docking investigation was obtained from the Protein Data Bank PDB ID: 5wgd and synthesised by removing the solvent, adding hydrogen, and then using the Protein Preparation Wizard to minimise energy. The catalytic domain was used to create grids for molecule docking. Glide extra-precision XP mode was used to dock fisetin, with up to three postures preserved per molecule.

MM/GBSA study
MM/GBSA energy calculations implemented in the Prime module of the Schrödinger molecular modelling package were used to investigate the free binding energy of complexes of Fisetin and Alendronate with Estrogen Alpha.

ADMET analysis
Schrödinger s QikProp Version 3.5 was used to perform ADMET analysis on fisetin and alendronate. It provides comparison ranges for comparing a molecule s attributes to those of 95 of known medications. The partition coefficient, human oral absorption, CNS central nervous system activity, and gut-blood barrier permeability were the descriptors calculated.

In vitro model 2.5.1 Cell proliferation
The effect of Fisetin and ALN on the cell proliferation was estimated on the human SAOS-2 osteosarcoma cell lines osteoblast like effect . Briefly, the cells were cultured in the McCoy s medium contained foetal bovine serum 15 . The cells cultured in the medium was incubated under standard condition such as temperature 37 and humidified CO 2 atmosphere 5 13 . 2.5.2 MTT assay MTT assay was used for the determination of cell viability effect of Fisetin on metabolic activity of SAOS-2. The cells 100 μl/well were harvested and seeded in 96-well plates with a density of 1.5 10 5 /mL and cells were kept for incubation for 24 h. After the incubation, the different concentration of Fisetin and ALN was treated with the cells 13 .

Experimental animals
Sprague-Dawley aged-3 month old, sex-female; weight 200-230 g were procured from the departmental animal house and housed under the controlled laboratory condition such as 20 5 temperature, 65 relative humidity and 12/12 h light/dark cycle. The rats were fed with standard pellet diet and water ad libitum. Before start the experimental protocol, the rats were kept in the 7 days for acclimatization for adopting the laboratory condition.

Experimental design
The rats were divided into 6 groups and each groups contains 6 rats. Table 1 showed the experimental groups. The rats were received the oral administration of above treatment for 8 weeks, respectively.
The rats were starved overnight at the end of the experiment, and urine from all groups was collected via micturition induced by manual pressure and stored at 20 for further analysis. The rats were anaesthesia using the ketamine and blood was collected from the dorsal aorta in the pre-incubated tubes and centrifuged at 1000 g for 10 min to separate the serum. The absolute weight of uterine and thymus was estimated and normalized with body weight using the following formula:

Relative weight of uterus Weight of uterus per 100 g of body weight
The femoral neck was further processed for performing the mechanical testing. The left femur and vertebra bone were used for estimation the mineral content 26 .

Bone marker parameters
The osteocalcin EIA kit was used to calculate the amount of osteocalcin OC based on the manufacturer s instructions Xinqidi Bio Technology, Inc, China . The urine deoxypyridinoline DPD level was determined using a competitive enzyme immunoassay in a microassay stripwell model Quidel, Mountain View, CA, USA as directed by the manufacturer. Acid phosphatase ACP and Beta-CrossLaps β-CTx were estimated using the immunoassay analyser Cobas-Roche, Basel, Switzerland following the Table 1 List of experimental groups.

S. No
Group Treatment

Urine parameters
Urine calcium UCa and Urine phosphorus UP level was estimated using the commercial colorimetrically kits Boehringer Mannheim GmbH following the manufacture description.

Hormone estimation
The serum hormones E 2 was estimated using the radioimmunoassay Subio, Inc., China .

Serum phosphorus P and calcium Ca level
Ammonium molybdate and arsenazo-3 dye colorimetric model was used for the estimation of P and Ca level using the previous method with minor modification 26 .

Plasma enzymes
The level of Bone specific alkaline phosphatase BALP , Cross-linked C-terminal telopeptides of type I CTX and Tartrate resistant acid phosphatase TRAcP 5b were estimated using the nitrophenol based method using the previous reported method with minor modification 26 .

BMD and BMC
Lunar prodigy advance dual energy X ray absorptiometry was used for the estimation the level of BMD and BMC with minor modification of previous reported method 26 .

Statistical analysis
The data were showed as mean S.E.M. Comparisons between the groups were made using the two-way analysis of variance using the Dunnett s. Statistical analysis was carried out using the Graphpad Prism 7 St. Louis, USA . The level of significance was showed as p 0.05.   Fisetin docking studies were carried out in order to gain a better understanding of the VitD enzyme potency at the molecular level and to throw light on the interactions in the active site of the VitD enzyme. On the VitD enzyme, docking investigations were carried out utilising the Glide module of the Schrödinger-9.6 programme PDB id: 1DB1 .

Molecular docking of the vitamin D receptor VDR and oestrogen receptors ERs
The re-docking of the reference ligand Alendronate into the active site of the VitD enzyme indicates that it binds to the same binding pocket, indicating that the current docking methodology is valid. The molecule fisetin binds securely in the active region of the VitD enzyme, according to docking results. This molecule fills the receptor cavity completely and forms a hydrogen bond with Ser237. In addition, when compared to normal alendronate, it forms hydrogen bonds with Ser237 and Tyr143 Fig. 3 .

MM/GBSA study
The stability of the ligands in the binding pocket of the Estrogen Alpha receptor was investigated using molecular mechanics/generalized born surface area MM/GBSA . MM/ GBSA tests were performed on compound fisetin. Fisetin has a binding free energy of 7.52 kcal, whereas the reference chemical Alendronate has a binding free energy of 2.21 kcal, demonstrating that Fisetin is stable in the oestrogen alpha receptor pocket Table S1 .
The stability of the ligands in the binding pocket of the VitD receptor was investigated using molecular mechanics/ generalized born surface area MM/GBSA . MM/GBSA tests were performed on compound fisetin. Fisetin has a binding free energy of 2.15 kcal, whereas the reference chemical Alendronate has a binding free energy of 0.145 kcal, demonstrating that Fisetin is stable in the VitD receptor pocket Table S2 .

ADMET analysis
The ADMET studies of fisetin and alendronate were performed using Schrödinger s Qikprop programme. Table  S3 summarises the findings.
The ADMET studies of fisetin and alendronate were performed using Schrödinger s Qikprop programme. Table  S4 summarises the findings. Figure 4 shows the effect of fisetin and ALN on the cell viability of SAOS-2 celllines. ALN standard drugs did not show any impact on the cell proliferation. On the other hand, fisetin treatment significantly showed the alteration in a dose dependent manner on metabolic activity. Fisetin higher doses exhibited the most pronounced inhibitory effects and showed the potential effect on osteoblastic activity.

Body weight and uterus index
Body weight of the control group increases in a normal pattern. OVX induced osteoporosis rats exhibited enhanced body weight as compared to their initial body weight and normal and treated rats. OVX treated rats received fisetin and ALN treatment significantly suppressed their body weight as compared to the OVX control group. The body weight of fisetin and ALN treated    Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and *** p 0.001 extreme significant . Fig. 6 The effect of fisetin alendronate on the level of BMC and BMD in OVX induced osteoporosis. Treatment groups mention in Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and *** p 0.001 extreme significant . BMC were estimated g and BMD g/cm 2 . Where BMC; Bone mineral content, BMD; Bone mineral density, ALN; Alendronate.

Fig. 7
The effect of fisetin alendronate on the level of biochemical parameters in OVX induced osteoporosis. Treatment groups mention in Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and *** p 0.001 extreme significant . Energy N.mm , Maximum load N , Stiffness N/mm , Young modulus MPa , Maximum stress MPa . OVX; Ovariectomy, ALN; Alendronate.
rats was boosted as compared to their initial body weight Fig. 5a . The uterus index was increased in the osteoporosis control rats, but it was significantly reduced p 0.001 in the fisetin and ALN treated rats Fig. 5b .

BMC and BMD
OVX induced osteoporosis rats showed a decreased level of BMC and BDM. Fisetin treatment significantly p 0.001 increased the level of BMC and BMD. Fisetin 30 mg/ kg showed the maximum up-regulation of BMC and BMD levels. ALN treatment showed the up-regulation of BMC and BMD levels Fig. 6 .

Biomechanical parameters
Biomechanical parameters such as energy, maximum load, stiffness, young modules and maximum stress Fig.  7 . OVX rats exhibited a reduction in the biomechanical parameters and fisetin treatment significantly p 0.001 increased the level of biomechanical parameters.

Ca, P and Vit D
During osteoporosis, calcium and phosphorus levels decreased while vitamin D levels increased. OVX induced group rats showed similar results as compared to normal and other treated group rats. Fisetin significantly p 0.001 enhanced the level of Ca, P and reduced the level of Vit D Fig. 9 .

BALP, CTX and TRAcP5b
OVX induced rats presented an augmented level of Fig. 8 The effect of fisetin alendronate on the level of E 2 and 1,25 OH 2 D 3 in OVX induced osteoporosis. Treatment groups mention in Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and *** p 0.001 extreme significant . E 2 ng/L and 1,25 OH 2 D 3 ng/mL . OVX; Ovariectomy, ALN; Alendronate.

Fig. 9
The effect of fisetin alendronate on the level of Ca, P and vit D in OVX induced osteoporosis. Treatment groups mention in Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and *** p 0.001 extreme significant . Ca mmol/L , P mmol/L , Vit. D nmol/L . Ca; Calcium, P; Phosphorus, Vit.D; Vitamin D, OVX; Ovariectomy, ALN; Alendronate.
BALP, CTX and TRAcP5b as compared to other treated and non-treated group rats. Fisetin and ALN treated group rats significantly p 0.001 declined the level of BALP, CTX and TRAxP5b Fig. 10 .

Urine parameters
Urine parameters such as Sca, SP, OC, ALP, Uca/Cr, DPD/Cr and UP/Cr presented in Fig. 11. OVX induced osteoporosis rats displayed an augmented level of urine parameter and fisetin treatment significantly p 0.001 reduced the level of urine parameters Fig. 11 . Figure 12 showed the antioxidant parameters MDA, SOD, GSH and CAT in the bone, liver and intestine. OVX induced rats presented an augmented level of MDA Fig.  12a and reduced level of SOD Fig. 12b , GSH Fig. 12c and CAT Fig. 12d in the bone, liver and intestine. Fisetin treatment significantly p 0.001 reduced the level of MDA and increased the levels of SOD, GSH and CAT in the bone liver and intestine. Figure 13 exhibited the effect of fisetin on the level of inflammatory cytokines. OVX induced osteoporosis rats exhibited an increased level of inflammatory cytokines and fisetin treatment significantly p 0.001 declined the level of inflammatory cytokines.  Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and *** p 0.001 extreme significant . Where BALP pg/mL , CTX pg/mL and TRAcp pg/ mL . BALP; Bone specific alkaline phosphatase, CTX; Cross-linked C-terminal telopeptides of type I, TRAcP 5b; Tartrate resistant acid phosphatase, OVX; Ovariectomy, ALN; Alendronate.

Fig. 11
The effect of fisetin alendronate on the level of urine biomarker in OVX induced osteoporosis. Treatment groups mention in Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and ** 3.14 RANKL, OPG and RANKL/OPG ratio OVX rats showed the increased level of RANKL, reduced level of OPG, and augmented the RANKL/OPG ratio

Discussion
According to a previous study, ovariectomy reduces blood flow by lowering endothelial dysfunction and erythropoietic marrow levels 27 . Reduced blood flow appears to play a key role in lowering BMD, according to both experimental and clinical evidence 27 . Reduced estradiol levels in postmenopausal women impede bone growth and eventually contribute to osteoporosis. It is well documented that hypoxia significantly promotes the size and number of osteoclasts which results in enhanced  Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and *** p 0.001 extreme significant . MDA; Malonaldehyde, SOD; Superoxide dismutase, GSH; Glutathione, CAT; Catalase, OVX; Ovariectomy, ALN; Alendronate.

Fig. 13
The effect of fisetin alendronate on the level of proinflammatory cytokines in OVX induced osteoporosis. Treatment groups mention in Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and *** p 0.001 extreme significant . Where TNF-α pg/mL , IL-1β pg/mL and IL-6 pg/ mL . TNF-α; Tumor necrosis factor-α, IL-1β; Interleukin-1β, IL-6; Interleukin-6, OVX; Ovariectomy, ALN; Alendronate.
inhibition and resorption of osteoblast activity 13 . Furthermore, plant drugs and plant isolated constituents that boost the blood supply to the bone tissue might be beneficial for the treatment of osteoporosis. In this experimental study, we used fisetin a well-known phytoconstituent to scrutinize the bone protective effect against the ovariectomy induced osteoporosis model in female rats. Body weight increase during aging-related osteoporosis and women menopause is the main phenomenon 2, 4 . A similar result was observed in the osteoporosis control group rats. OVX rats exhibited enhanced body weight and reduced uterine index as compared to the control group rats. Previous research suggests that the decreased uterine weight in OVX-induced rats may be due to E 2 deficiency 2, 4 . Fisetin treatment significantly eased the excess gain in body weight and enhanced the uterine index in OVX induced osteoporosis rats. Fisetin treatment demonstrated a protective effect against fat accumulation, visceral fat and body weight gain, which correlates with our current findings.
OC, a biomarker of bone formation which is synthesised by osteoblasts and directly corresponds to their specific functions 6,14 . Upregulated or boosted level of bone formation show the osteoporosis in postmenopausal or menopausal women and our findings coincide with this view 3, 28. In this study, the boost level of OC due to osteoblast formation in order to compensate for the bone loss induced due to E 2 deficiency.
Osteoporosis is a condition caused by a balance or homeostasis between the formation and resorption of bones 2,3 . In this experimental study, we scrutinised the inflammatory markers and bone turnover in the serum of experimental rats. In this study, we discovered that ovariectomy causes increased ROS deposition, which leads to an increase in inflammatory cytokines accumulation and oxidative stress, which increases bone loss and osteoclast generation. In postmenopausal women, the deterioration of bone function and composition, bone architecture is a common problem that further leads to the induction of osteoporosis. As people age, their bone structure, composition, and function often become impaired, leading to osteoporosis 2 5 . Bone is highly prone to the hormones dysfunction and age related loss, the current research focus is on scrutinising the protective effect of fisetin against OVX deficiency induced osteoporosis. During the postmenopausal period, the deficiency of E 2 ovary hormones acts as a significant risk factor in the expansion of osteoporosis. Ovariectomy is a well-developed model for osteoporosis and it shows that the bone loss is similar to that observed in postmenopausal conditions 4,5,28 . This model is the most recommended for estimating the safety and effectiveness of therapies involved in the treatment of osteoporosis. OVX rats exhibited significant altered uterine, bone density, BMC, biochemical and other biochemical parameters due to E 2 deficiency 2, 3 .
Calcium is the essential nutrient for the growth of bone and teeth. Calcium plays a significant role in the maintenance and expansion of bone health and around 99 calcium is commonly present in the bones 2 14 . Some concentration of calcium is observed in the circulation, but the amount is low and stable. According to the previous research, the calcium concentration was around 0.85 mmol/L and 1.27 0.02 mmol/L in adult Wistar rats 13 . In this experimental study, we have found that the calcium concentration is almost near the previous studies. The calcium concentration was altered in the OVX group rats due to enhanced bone turnover following deprivation of estrogen. This is the important reason for induction the hypercalcemia. Another explanation for increasing calcium levels could be increased intestinal calcium absorption due to less antioxidant enzymes 1, 2 .
The absorption of intestinal calcium is influenced by Fig. 14 The effect of fisetin alendronate on the level of RANKL, OPG and RANKL/OPG ratio in OVX induced osteoporosis. Treatment groups mention in Table 1. All the data showed as mean SEM. The comparison was performed between the OVX control and tested group rats using the Dunnett s multiple comparison test. Where * p 0.05 significant , ** p 0.01 more significant and *** p 0.001 extreme significant . Where RANKL ng/mL and OPG ng/mL . RANKL; Receptor activator of nuclear factor-κB, OPG; Osteoprotegerin, OVX; Ovariectomy, ALN; Alendronate.
GSH levels, according to previous research. GSH shortage can reduce intestinal calcium absorption by changing the mechanism and molecules involved in its transfer 1,5,29 .
Flavonoids have been shown in some studies to promote intestinal absorption and hence increase the bioavailability of micronutrients that are poorly or minimally absorbed, such as calcium. Some reported studies exhibit that flavonoids might enhance the body capacity to absorb the micronutrient calcium . The result of present investigation showed that fisetin significantly boosts the concentration of GSH in the intestine and hepatic tissue, which in turn might enhance the calcium intestinal intake 1, 6, 30 .
It is generally known that adequate vitamin D and calcium intake lessens the risk of osteoporosis. Cholecalciferol D 3 is essential for the absorption of P and Ca in the body, which is required for bone expansion. OVX induced osteoporosis rats exhibited altered levels due to deficiency of E 2 2, 3, 31 . The deficiency of E 2 is a common problem that arises during the aging process and postmenopausal related disorder 2, 3, 6 . 1,25 OH 2 D 3 is considered as the key regulator of Ca homeostasis in the body. Figure 8 showed the reduction of PTH due to the observed hypercalcemia in the osteoporosis group, which further reduced the formation of 1,25 OH 2 D 3 in the renal tissue 1,14 . A vitamin D deficiency is defined as a 25 OH D level less than 25 nmol/L. In this experimental study, we observed that reduced levels of Vit D in the serum and fisetin treatment significantly increased the level of vitamin D.
The in silico target prediction by Schrödinger software exhibited that fisetin could affect estradiol synthesis or Ca absorption and bind to Vit D receptor 32,33 . The binding capability of fisetin to the Vit D receptor was confirmed by the molecular docking study. The bone turnover marker includes CTx ALP and OC was boosted in the OVX group due to expansion of osteoporosis. Fisetin treatment significantly suppressed the level of bone turnover marker in the serum due to oestrogenic and antioxidant effects 13 . Fisetin suppressed the level of bone turnover marker due to its estradiol like effects. The molecular docking study of fisetin on VDR, ERα and ERβ exhibited that fisetin binds in a similar way as estradiol. Fisetin exhibited a lower score on the VDR, ERα and ERβ due to a lower number of hydrogen bonds with these receptors. Despite these in silico connections, the overall effects of fiestin were found to be inconsequential. Fisetin showed a protective effect against osteoporosis due to its antioxidant effect with a beneficial estrogenic effect, vit D and boosting the absorption of intestinal calcium. The RANKL/OPG axis is well established as a significant pathway in the regulation of homeostatic balance between bone resorption and formation. RANKL/OPG plays a significant role in arbitrating osteoclast differentiation 34 . It is well known that OPG generated through osteoclast linkage and has a repressive effect on bone formation and RANKL is involved in bone fracture, which results in the formation of osteoclast 35 . So, the ratio between the RANKL and OPG is necessary to estimate the bone integrity and density. We m e a s u r e d t h e l e v e l s o f R A N K L a n d O P G i n t h e experimental rats, and found that OVX-induced rats had higher levels of RANKL and lower levels of OPG. Fisetin treatment significantly reduced the RANKL level and boosted the OPG level almost to control group rats. Consequently, the result suggests that the fisetin treatment considerably showed a protective effect against the OVX induced bone loss via restoration of the level of RANKL/ OPG.
It is well proven that postmenopausal women have higher levels of pro-inflammatory cytokines than women undergoing ERT. Previous research suggests that increased levels of vitamin D and E 2 initiate the production of cytokines 16 . The boosted level of inflammatory cytokines, boost the production of free radicals and reduces bone formation and resorption. During osteoporosis, cytokines play a significant role in the regulation of bone turnover via increasing bone resorption 36,37 . OVX induced osteoporosis rats exhibited an enhanced level of cytokines IL-1β, TNF-α and IL-6 and fisetin treatment significantly suppressed the production of cytokines.

Conclusion
The current research showed the anti-osteoporosis effect of fisetin against OVX induced osteoporosis. An in silico study showed that fisetin docked to VDR Vit D receptor and estrogen receptors such as estrogen-α and estrogen-β. The docking study projected the VDR binding affinity and boosted the absorption of Ca. Fisetin treatment significantly maintains the level of phosphorus and calcium. Fisetin treatment significantly reduced the level of MDA and increased the level of SOD, CAT, GSH in the bone, liver and intestine. Fisetin improved the status of endogenous antioxidants and also helped with the absorption of intestinal Ca. Fisetin significantly reduced the level of inflammatory cytokines and inflammatory mediators, suggesting an anti-inflammatory effect. Fisetin also reduces osteoclast activity and suggesting an anti-inflammatory effect.

Author Contributions
Peng Feng performed the experimental protocol. Shijun Shu and Feifei Zhao analysed the biochemical data and performed the docking study. Feifei Zhao design the experimental protocol and supervised the experimental study. All the authors equally contributed in drafting and proof reading the manuscript.

Supporting Information
This material is available free of charge via the Internet at doi: 10.5650/jos.ess21252