Antiosteoporotic effect of hesperidin against ovariectomy‐induced osteoporosis in rats via reduction of oxidative stress and inflammation

Qiang Zhang1 | Xiaoxia Song2 | Xiaoming Chen1 | Ruizhong Jiang1 |
Ke Peng3 | Xinqiao Tang1 | Zhong Liu1

1Department of Orthopedics, Xiangtan Central Hospital, Xiangtan, Hunan, China

2Department of Respiratory Medicine, Xiangtan Central Hospital, Xiangtan, Hunan, China
3Department of Spine Surgery, Xiangtan Central Hospital, Xiangtan, Hunan, China

Correspondence
Zhong Liu and Xinqiao Tang, Department of Orthopedics, Xiangtan Central Hospital, 120 Heping Rd, Xiangtan, 411413 Hunan, China. Email: [email protected] and xtszxyytxq@ 163.com

Abstract
Osteoporosis is a serious health problem, especially in geriatric patients. Worldwide, it affects 8.9 million people every year. Oxidative stress and inflammation expand the osteoporosis reaction. Hesperidin supplement helps to decrease inflammation and oxidative stress. In this study, we estimated the antiosteoporotic effect of hesperidin against the ovariectomized (OVX) rat model of osteoporosis. Hesperidin was orally administered at 5, 10, and 20 mg/kg to OVX rats for 10 weeks. Different biochemical parameters, such as alkaline phosphatase (ALP), osteocalcin (OC),
phosphorus (P), calcium (Ca), and antioxidant parameters, were also estimated. The three‐point bending test, bone mineral density (BMD), and histomorphometric features of the femoral bone were also scrutinized. Hesperidin significantly de-
creased body weight and increased uterine weight. Hesperidin significantly reduced the ALP, OC, acid phosphatase, and β‐isomerized C‐terminal telopeptides levels in
OVX rats. Hesperidin considerably increased BMD and dose‐dependently reduced
the pixel density. Hesperidin considerably increased the maximum load, energy, stiffness, maximum stress, and young modulus. Hesperidin significantly (p < 0.001)
reduced the levels of thiobarbituric acid reactive substance and increased the level of superoxide dismutase, glutathione, glutathione peroxidase, catalase in OVX‐ induced rats. Hesperidin significantly diminishes the cytokine levels, such as tumor necrosis factor‐α, interleukin‐6 (IL‐6), and IL‐1β, and inflammatory mediators such
as nuclear factor‐kappa B. It significantly reduced the level of Ca, P, and increased
the level of vitamin D in OVX rats. Hesperidin significantly (p < 0.001) reduced the expression of sirtuin 1. Collectively, we can conclude that hesperidin exhibited better protection against osteoporosis by enhancing the bone density and bone mineral content in addition to biomechanical parameters.

KEYWOR DS
biochemical parameter, bone loss, hesperidin, inflammation, osteoporosis, ovariectomy

Abbreviations: ACP, acid phosphatase; ALP, alkaline phosphatase; AN, alendronate; BMD, bone mineral density; Ca, calcium; CAT, catalase; CMC, carboxymethyl cellulose; ERT, estrogen replacement therapy; GPx, glutathione peroxidase; GSH, glutathione; HRT, hormones replacement therapy; IL‐6, interleukin‐6; IL‐1β, interleukin‐1β; NF‐κB, nuclear factor‐kappa B; OC, osteocalcin; OVX, ovariectomized; P, phosphorus; PTH, parathyroid hormone; RT‐PCR, reverse transcription polymerase chain reaction; SIRT1, sirtuin 1; TBARS, thiobarbituric acid reactive substance; TNF‐α, tumor necrosis factor‐α; β‐CTx, β‐isomerized C‐terminal telopeptides.

J Biochem Mol Toxicol. 2021;e22832. wileyonlinelibrary.com/journal/jbt © 2021 Wiley Periodicals LLC | 1 of 9

1 | INTRODUCTION

Osteoporosis is considered a major health burden, especially in the geriatric population.[1] Osteoporosis is a metabolic disease categor- ized via damaged bone formation resulting in suppressed bone mass and microstructure dysfunction of bone tissue.[2] Fractures are the main complication of osteoporotic patients occurring after insignif- icant traumas and suppress the quality of life. According to reports, every year more than 8.9 million fractures are caused by osteo- porosis.[3] In the last few decades, hip fracture incidence increased 240% in women and 310% in men globally.[4,5] In China, osteoporosis induces a deformity that is equal to or higher than that caused by various diseases, including rheumatoid arthritis, bronchial asthma, and heart disease.[6] In Egypt, a report suggests that a fraction of postmenopausal women have osteoporosis (28.4%), osteopenia (53.9%), and osteopenia (53.9%); even as males at the ages between 20 and 89 years have 21.9% osteopenia. Moreover, timely treatment and prevention of osteoporosis are the best approach to reduce fracture risk and bone resorption in postmenopausal women.[4]
The available treatment of osteoporosis requires long‐term
treatment; due to long‐term treatment inducing serious side effects such as enhanced cardiovascular disease risk and initiates the ex-
pansion or development of various types of cancer.[2,7] The anti-
resorptive oral bisphosphonates including alendronate (AN) are the first‐line drugs for treating women postmenopausal osteoporosis.[2] However, AN has limitations due to side effects such as impairment
of liver function and induces organ injury. The scientific focus on exploring alternative remedies from natural sources as a non- therapeutic approach to improving bone health and reducing the risk of fractures has recently modified these constraints of conventional osteoporosis drugs.[8,9]
Previous studies suggest that various factors such as endocrine disorders, nutritional factors, and genetic disorders are involved in the expansion of osteoporosis.[8,9] Recently, various pharmacological agents have been explored for the treatment of osteoporosis. However, few pharmacological agents have effectiveness against osteoporosis with few side effects or organ toxicity.[10,11] The major side effects of these pharmacological agents on osteoporosis are organ toxicity. Given the aging population worldwide, it is becoming increasingly important to develop strategies for osteoporosis pre- vention rather than treatment to improve human health and reduce medical expenditure.[2]
Hormone replacement therapy (HRT) and estrogen replacement therapy (ERT) have been confirming their beneficial effect against bone loss in elderly and postmenopausal women.[12,13] Un- fortunately, continuous use of HRT has serious side effects such as the development of breast, endometrial, and ovarian cancers. Pre- vious reports suggest that maintaining the level of vitamin D and calcium (Ca) helps in the prevention of osteoporosis.[11,14] In addi- tion, avoiding caffeine, alcohol, smoking, and regular exercise also acts as a potential deterrent against the development of osteo-
porosis. It is well‐documented that regular intake of nutrients in the
diet, along with vitamin D and Ca helps in suppressing bone density

and loss.[15,16] Postmenopausal women showed the loss of bone due to increased oxidative stress, which is commonly increased due to overproduction of free radicals and reduction of endogenous anti- oxidants and inflammation.[13,15] Oxidative stress enhances the os- teoclastic function of bones and bone differentiation, thus resulting in bone loss.[8,9]
Hesperidin (hesperetin‐7‐rhamnoglucoside) flavanone glycoside
is commonly present in lemons and sweet oranges. Hesperidin pos- sesses various pharmacological activities such as analgesic, anti‐ inflammatory, antihypercholesterolemic, anticarcinogenic, and anti-
fungal activity.[17,18] Its deficiency has been connected to abnormal pain and capillary leakiness in the extremities, inducing weakness, night leg cramps, and aches. Hesperidin supplementation helps de- crease inflammation and oxidative stress.[17,18] The radical scaven- ging and antioxidant properties of hesperidin have been reported via various studies, using various assay systems.
Due to this, the current investigation scrutinizes the efficacy and mechanism of hesperidin using ovariectomy‐induced osteoporo- tic rats.
2 | MATERIAL AND METHODS

2.1 | Animals

Sprague‐Dawley rats (2–3 months old) 170–195 g weight; sex female
were used for the current protocol. The rats were obtained from the institutional animal house and kept under standard laboratory con- ditions. The rats were acclimatized for 7 days before the experi- mental protocol. The rats received average food and water ad
libitum. The rats were either sham‐operated (Sham; n ¼ or ovar-
iectomized (OVX) (n ¼ 40). All experimental surgical procedure was conducted under anesthesia conditions using pentobarbital (50 mg/kg).

2.2 | Experimental protocol

In this current experimental protocol, the rats were divided after the surgical procedure (5 weeks) into five groups and each group con- tained eight rats. The groups as follows:

Group I: normal control (treated with carboxymethyl cellulose [CMC] 1% suspension),
Group II: OVX control,
Group III: OVX + HP (5 mg/kg, body weight), Group IV: OVX + HP (10 mg/kg, body weight), Group V: OVX + HP (20 mg/kg, body weight), Group VI: OVX + AN (2.5 mg/kg, body weight).

All the group rats received the vehicle (1% CMC), HP, and AN were given to all rats every morning via oral gavage as presented above. The treatment was started after the fourth week of surgery and lasted for 12 weeks. At the end of the experimental study, each rat was kept in the metabolic cage (24 h) before the sacrifice and we collected the urine samples and acidified them with HCl. Before sa- crifice, blood samples of all group rats were collected from the ab- dominal aorta and centrifuged to separate the serum for estimation of biochemical parameters. The urine and serum samples were kept at −20°C for further estimation. The uterus of all group rats was dissected immediately and weighed and we calculated the uterine index as uterine weight/body weight. Femurs were removed, wrap- ped, and finally stored at −20°C for estimation of structural and biomechanical analysis.[2,7]

2.3 | Ca homeostasis marker

The Ca homeostasis markers were estimated on Days 35 and 71.
Phosphorus (P) and Ca were estimated using the available com- mercial kits (Biochemical Analyzer, Mindray). 25‐OH Vit D was es- timated using the electrochemiluminescence immunoassay kits.
2.4 | Estimation of bone density

For estimating bone density, the femur bone was scanned at
0.5 × 0.5 mm resolution with a speed of 1 mm/s. The bone mineral density (BMD) was estimated at milligrams per square centimeter (mg/cm2).
2.5 | Estimation of bone turnover markers

Bone turnover markers such as bone‐specific alkaline phosphatase (ALP) and acid phosphatase (ACP) activity were estimated using
commercially available kits (Boehringer Mannheim GmbH). Serum osteocalcin (OC) was scrutinized by a one‐step sandwich enzyme‐ linked immunosorbent assay using streptavidin technology (Boeh-
ringer Mannheim GmbH).
2.6 | Biomechanical estimation

For estimating biomechanical strength, the left femurs of the rats
were used. A material testing machine was used for estimating the three‐point bending test (MTS Corp.). Briefly, the length of each femur bone was estimated using the digital caliper and its midpoint was marked before executing the three‐point test. Bone samples were positioned on two support points 19 mm apart in a similar
orientation and a force was applied to the femoral midshaft by a crosshead moving at a constant speed of 1 mm/min. The displace- ment and central load were recorded until the fracture occurred in the bone. The Young’s modulus (MPa), the maximum stress (MPa), maximum load (N), and energy absorption (N/mm) were estimated using the previously reported method with minor modification.[4,5,19]

2.7 | Antioxidant enzymes and bone oxidative stress assay

For estimating antioxidant and bone oxidative stress enzyme para- meters, all group rats left femur was removed and homogenized in cold NaCl (0.9%) to make up to homogenate (10%). Finally, the homogenate was centrifuged at 15,000 rpm to get the clear super- natants for estimating glutathione (GSH), thiobarbituric acid reactive substance (TBARS), glutathione peroxidase (GPx), catalase (CAT),
glutathione‐S‐transferase (GST), and glutathione reductase (GR)
levels.[11,20]
2.8 | Proinflammatory cytokines

Proinflammatory cytokines including interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), and IL‐6 were estimated using the ELISA kits procured from NeoBioscience Biological Technology Co., Ltd.

2.9 | Real‐time polymerase chain reaction

A commercially available kit was used for isolation of cDNA (Prime
Script RT) and real‐time polymerase chain reaction (PCR) was per- formed using reverse transcription‐PCR. GAPDH was used as a housekeeping gene. The primer sequences are presented in Table S1.

2.10 | Statistical analysis

One‐way analysis of variance was followed by an LSD test to com- pare various groups with each other. Results were presented as mean ± standard deviation and p < 0.05, p < 0.01, and p < 0.001 were
considered as significant, more significant, and extremely significant, respectively.
3 | RESULTS

3.1 | Effect of hesperidin on body and tissue weight

Normal rats showed increased body weight during all experimental study periods. OVX‐induced group rats showed increased body weight compared to control group rats . OVX rats trea- ted with hesperidin demonstrated increased body weight compared
to the initial body weight. OVX rats treated with hesperidin (20 mg/kg) showed increased body weight almost similar to the normal control. OVX rats treated with the showed a similar pattern as presented in normal rats.  S1b demonstrates the weight variance of the different groups of rats.
S2 shows the uterus index of different groups of rats. Normal rats showed increased uterus index compared to the other group of rats. OVX‐induced rats illustrated the reduction of the uterus index compared to other group rats. Dose‐dependent treat- ment of hesperidin significantly (p < 0.001) increased the uterus in-

dex. AN showed improvement in the uterus index compared to OVX‐ induced rats.

3.2 | Effect of hesperidin on biomechanical parameters

During osteoporosis, bone biomechanical parameters such as the maximum load, energy, stiffness, maximum stress, and young mod- ulus were reduced due to bone deformity. A similar result was ob-
served in the OVX‐induced group rats. OVX‐induced rats treated
with hesperidin significantly (p < 0.001) increased the level of max- imum load, energy, stiffness, maximum stress, and young modulus in a dose‐dependent manner.

3.3 | Effect of hesperidin on biochemical parameters

 2 shows the effect of hesperidin on the biochemical

parameters of OVX‐induced rats. increased levels of the bio- chemical parameters such as OC, ALP, ACP, and β‐isomerized C‐terminal telopeptides (β‐CTx) were observed in the OVX‐ induced rats at 35 days. Hesperidin significantly (p < 0.001) re-
duced the level of OC, ALP, ACP, and β‐CTx in a dose‐dependent manner.
S3 shows the effect of hesperidin on the biochemical parameters of OVX‐induced rats after the 71‐day treatment. OVX‐
induced group rats showed increased levels of OC, ALP, ACP, β‐CTx,
and hesperidin significantly (p < 0.001) reduced the level in a dose‐ dependent manner.

3.4 | Effect on BMD

S4 shows the pixel intensity and BMD on the different ex- perimental groups of rats. OVX‐induced rats showed increased pixel intensity and reduced BMD compared to normal and treatment group rats. Hesperidin‐treated group rats showed the down- regulated pixel intensity  and upregulation in the level of
BMD .

3.5 | Effect of hesperidin on Ca, P, and 25‐OH Vit D

During the bone deformity, Ca, P level boosted and 25‐OH Vit D level reduction was observed. OVX‐induced rats showed increased levels of Ca , P , and reduced levels of 25‐OH Vit D  compared to normal rats. Hesperidin significantly
(p < 0.001) decreased the level of Ca, P, and increased the level of 25‐OH Vit D compared with OVX group rats after 35 days of treatment. The AN group of rats showed similar effects.
S5a–c showed a similar effect of hesperidin against the OVX‐induced group of rats.

3.6 | Effect of hesperidin on the antioxidant marker and oxidative stress parameters

Antioxidant parameters such as TBARS boosted and GSH, GST, GR, GPx, CAT reduced during the bone deformity. OVX‐induced group rats showed an increased level of TBARS and reduced levels of GSH,
GST, GR, GPx, CAT compared with normal group rats. Hesperidin significantly (p < 0.001) reduced the level of TBARS and increased
levels of GSH, GST, GR, GPx, CAT in a dose‐dependent manner.

 1 Exhibited the effect of hesperidin on the biomechanical parameters of ovariectomy (OVX)‐induced osteoporosis in rats. (A) maximum load, (B) energy, (C) stiffness, (D) maximum stress, and (E) Young’s modules. Values are showed as mean ± standard error of the mean (analysis of variance). *p < 0.05, **p < 0.01, and ***p < 0.001 versus OVX

 2 Exhibited the effect of hesperidin on the biochemical parameters of ovariectomy (OVX) induced osteoporosis in rats after 35 days. (A) Osteocalcin (OC), (B) alkaline phosphatase (ALP), (C) acid phosphatase (ACP), and (D) β‐isomerized C‐terminal telopeptides (β‐CTx). Values are showed as mean ± standard error of the mean (analysis of variance). *p < 0.05, **p < 0.01, and ***p < 0.001 versus OVX

 3 The effect of hesperidin on the Ca, P, and 25‐OH Vit D parameters of ovariectomy (OVX)‐induced osteoporosis in rats after 35 days. (A) Ca, (B) P, and (C) 25‐OH Vit D. Values are shown as mean ± standard error of the mean (analysis of variance). *p < 0.05, **p < 0.01, and

***p < 0.001 versus OVX

3.7 | Effect of hesperidin on proinflammatory cytokines

Inflammatory reaction plays an important role in the expansion of bone deformity. OVX‐induced group rats showed increased

levels of proinflammatory cytokines such as IL‐6, IL‐1β, and TNF‐ α compared to control group rats. Hesperidin‐treated group rats showed significantly (p < 0.001) reduced the level of proin-
flammatory cytokines such as IL‐6, IL‐1β, and TNF‐α .

 4 The effect of hesperidin on the antioxidant parameters of ovariectomy (OVX)‐induced osteoporosis in rats after 71 days. (A) Thiobarbituric acid reactive substance (TBARS), (B) glutathione (GSH), (C) glutathione‐S‐transferase (GST), (D) glutathione peroxidase (GPx), (E) glutathione reductase (GR), and (F) catalase (CAT). Values are showed as mean ± standard error of the mean (analysis of variance). *p < 0.05,

**p < 0.01, and ***p < 0.001 versus OVX

 5 The effect of hesperidin on the procytokines parameters of ovariectomy (OVX)‐induced osteoporosis in rats after 71 days. (A) Interleukin‐1β (IL‐1β), (B) interleukin‐6 (IL‐6), and (C) tumor necrosis factor‐α (TNF‐α). Values are showed as mean ± standard error of the mean (analysis of variance). *p < 0.05, **p < 0.01, and ***p < 0.001 versus OVX

3.8 | Effect of hesperidin on SIRT1 expression

OVX‐induced group rats showed increased expression of SIRT1 com- pared to control group rats. Hesperidin and the AN‐treated group rats showed a reduction in the expression of SIRT1

4 | DISCUSSION

It is well‐documented that the level of estradiol during menopause leads to reduce bone formation and later contributes to osteo-
porosis.[21] The experimental and clinical investigation demonstrated

that decreased blood flow is the contributing factor to suppress BMD and occurs concurrently.[2] Previous studies suggest that the
decreased level of endothelial dysfunction and erythropoietic mar- row after OVX reduced blood flow.[2,21] It is well‐proved that hypoxia strongly boosts the size and number of osteoclasts, reducing the
osteoclast activity and enhancing bone resorption. Osteoporosis is considered a bone metabolic disease categorized via dysregulated bone resorption and formation, which quickly expanded the danger
of bone fractures.[9,11] Moreover, plant‐based phytoconstituents or
drugs that enhance blood supply into the bone might be useful for the treatment of osteoporosis. The current study shows the anti- osteoporotic effect of hesperidin against the OVX model of

 6 The effect of hesperidin on the messenger RNA expression of ovariectomy (OVX)‐induced osteoporosis in rats after 71 days. (A) Nuclear factor‐kappa B (NF‐κB) and (B) SIRT1. Values are shown as mean ± standard error of the mean (analysis of variance). *p < 0.05,

**p < 0.01, and ***p < 0.001 versus OVX osteoporosis rats. In this study, we estimated the bone, urine, and serum turnover markers of rats.

Previously, studies showed that the OVX model demonstrated increased body weight and reduced uterine weight in rats.[20,22] A similar result was observed in our experimental study. In this ex- perimental study, the OVX group showed increased body weight and hesperidin treatment reduced the body weight and uterine weight.
Bilateral OVX is a widely accepted model for scrutinizing post- menopausal osteoporotic complications and is also used for the evalua- tion of naturally occurring or synthetic compounds on osteoporotic treatment.[23] Previous studies suggest that the reduction in estradiol level is considered a significant mechanism leading to reduced bone mass, boosted body mass, and altered oxidative stress.[23,24] In the current experimental study, we have observed a reduced level of estradiol in the experimental group of rats (data not shown). We assume that the in- sufficiency of estradiol in OVX rats boosted body weight and hesperidin reduced the estradiol level reduced body weight.[2] Previous studies suggest that oxidative stress plays an important role in the expansion of osteoporosis.[2] According to a previous study, reactive oxygen species
(ROS) mainly involved in estrogen deficiency‐induced bone loss via en-
hanced osteoblast activity leading to a difference between the resorption and formation of bones.[23] In this experimental study, we found in- creased levels of TBARS (LPO indicator) and reduced levels of en- dogenous antioxidants such as superoxide dismutase, CAT, GPx and confirmed the oxidative stress formation. According to a previous study, natural phytoconstituents such as thymol may reduce osteoclast activity and consequently reduce the bone resorption process via various mechanisms.
In this current experimental protocol, the marker of bone homeostasis and Ca estimated at different time intervals (35 and 71
days). At the start of the experimental protocol (35 days), OVX‐
induced rats showed an increased level of Ca and bone home- ostasis.[2] Hesperidin treatment normalized bone formation, dis- cerned via amelioration of bone turnover markers, and finally reduced oxidative stress and inflammation. The result confirms the potential effect of hesperidin could be due not to its osteogenic effect but also to its effects on the circulation system. Hesperidin also showed its vasoconstrictive effect by improving the blood sup- ply in the body and boosting blood supply to the bones.

It is well‐proved that Ca plays a significant role in the expansion, maintenance, and growth of bone health. As we know, 99% of Ca is
found in the body bone. The Ca content is low in the blood maintained at a stable concentration. Osteoporosis is created due to homeostasis or imbalance, which occurs between bone formation and resorption.[2,7] In this experimental study, we examined the inflammatory markers and bone turnover in the serum of experimental rats. OVX induces enhanced ROS accumulation resulting in boosting the production of inflammatory cytokines and oxidative stress that uphold bone loss and osteoclast generation.[25] Cholecalciferol D3 plays a prime role in the absorption of P and Ca in the entire body, which is necessary for the expansion of bones. P and Ca also play a significant role as bone mineralization mar- kers. According to a previous study, the Ca level in adult Wistar rats was
0.85 mmol/L and the normocalcarmic level in Wistar rats is 1.27 mmol/ L.[26] In this protocol, the Ca level observed in the OVX group was higher in 35 and 71 days. Evidently, the boosted bone turnover after estrogen deficiency after OVX is one of the prime reasons for inducing hy- percalcemia. We have observed that increased Ca level might be due to enhanced intestinal Ca absorption due to the altered endogenous antioxidant status of the gut, which was modulated by hesperidin treatment.[2] Previous studies suggest that intestinal Ca absorption depends on the GSH level. During the disease, GSH deficiency can re- duce intestinal Ca absorption by altering the pathways and molecules involved in its transfer.[27] Previous literature suggests that flavonoids could increase intestinal absorption and boost the bioavailability of poorly or insufficiently absorbed micronutrients include Ca.[28] The re- sults of the current experimental study suggest that the hesperidin treatment significantly boosted the endogenous antioxidant levels and reduced the Ca level.
It is well known that 1,25(OH)2D3 (Vitamin D active form) and parathyroid hormone regulate Ca homeostasis inside the body. In this study, we observed hypercalcemia due to boosted level of Ca,
which further reduces the secretion of 1,25(OH)2D3 in the renal tissue.[29,30] It is well‐documented that the level of 25(OH)D levels less than 25 nmol/L in circulation shows vitamin D deficiency. In the
current experimental protocol, we observed the vitamin D level less than 25 nmol/L in the OVX group which confirms the vitamin D deficiency and hesperidin treatment increased the level of vitamin D and it reached near the normal control level.

The bone turnover parameters such as OC, ALP, β‐CTx, and ACP boosted during bone deformity. In this experimental protocol, we observed an increased level of bone turnover markers such as OC,
ALP, β‐CTx, and ACP at different time intervals (35 and 71 days). Hesperidin treatment significantly reduced the level of OC, ALP, β‐CTx, and ACP. Hesperidin showed the effect due to its estradiol‐ like, osteogenic, and antioxidant effect.
Procytokines play a significant role in the formation and re- sorption of bone.[11,31] Proinflammatory cytokines boosted during osteoporosis and these cytokines, especially in regulating bone turnover by enhancing bone resorption. Previous studies have con- firmed that the levels of proinflammatory cytokines are found to be high in postmenopausal women than those who are undergoing ERT.[11,14,32] Enhanced levels of proinflammatory cytokines boosted the secretion of free radicals and suppresses bone formation and encourages bone resorption. Proinflammatory cytokines including
TNF‐α, IL‐6, and IL‐1β levels were considerably boosted in the OVX
group rats. Our results were compared with the previous reports, which showed an increased level of proinflammatory cytokines in the
serum of OVX rats compared to normal control. OVX‐induced rats
that received the treatment of hesperidin showed attenuation in the level of proinflammatory cytokines, which might be due to an anti- osteoporotic effect of hesperidin. Previous studies suggest that the
nuclear factor kappa B (NF‐κB) plays an important role in the ex-
pansion of the osteoporosis effect. Osteoporosis conditions boosted the level of NF‐κB, which further causes the weakness of cellular differentiation, mineralization, and bone proliferation. Osteoclast formation needs NF‐κB signaling to extend the disease.[2] During
osteoporosis, the level of NF‐κB boosted due to disease, and re-
duction of the level of NF‐κB boosts the mineralization. Thus, the reduction of signal molecules might reduce osteoclast differentiation, proliferation and suppress the process of bone resorption.[2,7] OVX‐
induced rats showed the boosted level of NF‐κB and hesperidin
considerably reduced the NF‐κB, suggesting a bone protective effect. Previous studies suggest that SIRT1 plays an important role in
bone metabolism and mass. Histone had the capability to decrease the OC, ALP levels during the osteoblasts and maintains the bone
formation. It also reduced the proliferation and differentiation during the osteoblasts. Plant‐based drugs and their constituents have a potential effect on osteoporosis and they also increase proliferation
and differentiation.[33] Previous research has suggested a reduction of the SIRT1 expression, increasing the BMD in the rodent (old age). Researchers are targeting SIRT1 to treat osteoblasts.[33] In the cur- rent experimental protocol, we have found that increased expression of SIRT1 and hesperidin considerably reduced the expression of SIRT1 and suggested an antiosteoporosis effect Hesperadin

5 | CONCLUSION

Our finding clearly suggests the antiosteoporotic, antioxidant, and anti‐inflammatory effect of hesperidin in OVX‐induced osteoporotic rats by maintaining P and Ca homeostasis, by reduction of

antioxidant potential and anti‐inflammatory effects that augments BMD and bone markers formation. The bone protective effect of hesperidin could be attributed to stimulating bone formation and
osteoblastogenesis. It also boosted endogenous antioxidants espe- cially intestinal antioxidants by maintaining Ca intestinal absorption. Hesperidin inhibited the NF‐κB and proinflammatory cytokines such
as IL‐1β, IL‐6, TNF‐α and is suggested to have anti‐inflammatory potential.

ACKNOWLEDGMENTS
Xiangtan Central Hospital is acknowledged for guidance and support. This study was funded by the Hunan Provincial Natural Fund Youth Fund Project (Grant no. 2020JJ5556) and the 2019 Xiangtan Science
and Technology Bureau Guiding Science and Technology Plan Project (Grant no. zdx‐sf2019004).

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

ETHICS STATEMENT
The whole experimental protocol was approved by the Institutional Animal Use and Care Committee and all institutional procedures and protocols (NIH guidelines) regarding animal handling and care were followed.

DATA AVAILABILITY STATEMENT
All the data with the corresponding author.

ORCID
Xinqiao Tang https://orcid.org/0000-0001-7023-1910
Zhong Liu http://orcid.org/0000-0003-3067-1967
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