Chemical Characterization and Ameliorating Effect of Centratherum anthelminticum Extract against Polycystic Ovary Syndrome in Wistar Rats

Polycystic ovary syndrome (PCOS) in females is an endocrine pathological condition of reproductive age which is usually caused by insulin resistance, hyperlipidemia, and oxidative stress. This research was aimed at evaluating the therapeutic effect of the Centratherum anthelminticum seed extract (CA) against PCOS in rodents as it is traditionally used to treat diabetes, inflammation, and gynecological problems. The CA was chemically characterized by high-performance liquid chromatography-diode array detection (HPLC-DAD). For the induction of PCOS, a high-fat diet (HFD) was given to all female Wistar rats for nine weeks except the normal control group, which was given a normal chow diet. Estradiol valerate was given to all rats except normal control. After the induction of PCOS, oral metformin (300 mg/kg) was given to the standard group, while CA was orally administered to diseased rats at 250, 500, and 750 mg/kg/day for 28 days. HPLC-DAD analysis revealed that kaempferol-3-pcoumaroylglucoside was present in the highest amount (146.8 ± 1.8 mg/g) of the extract followed by ferulic acid and malvidin-3-(6-caffeoyl)-glucoside. The in vivo results revealed a marked reduction in cholesterol and triglyceride levels in CA treatment groups. A significant rise was observed in progesterone and follicle stimulating hormone with a decrease in luteinizing hormone in the treatment groups as compared to disease control, which indicated normalization of the estrus cycle. The decrease in insulin resistance was characterized by low serum insulin levels in treatment groups. Treatment with CA also reduced inflammatory markers, such as IL-6 and NF-κB in PCOS rats. NrF2 and oxidative stress markers such as catalase, superoxide dismutase, malondialdehyde, and reduced glutathione were also improved by CA in the ovary of diseased rats. Histopathological examination showed the different developmental stages of normal follicles in CA-treated diseased rats which were indicative of a normal fertile estrous cycle. Overall, the results confirmed the efficacy of CA against PCOS in treating estradiol-HFD-induced PCOS due to its antidiabetic, anti-inflammatory, antihyperlipidemic, and antioxidant properties.


Introduction
Polycystic ovary syndrome (PCOS), a reproductive age endocrine disorder, afects 5.6-21.3% of the female population among all ethnicities [1][2][3]. PCOS is a heterogenous disorder, with a range of diferent phenotypes, which results in diferences in clinical opinions and difculties in the diagnosis and treatment [4]. Te key features of PCOS include hyperandrogenism, ovulatory dysfunction, acne, and oral contraceptives, which have the potential of developing not only obesity but also metabolic abnormalities [7,8]. Estrogen receptor modulators, i.e., clomiphene citrate and aromatase inhibitors, i.e., anastrozole, or recombinant follicle stimulating hormone (FSH) injections are given for the induction of ovulation in PCOS patients which lead to numerous pregnancies [9].
Abnormally elevated levels of androstenedione, insulin, luteinizing hormone (LH), and serum testosterone are evident in PCOS patients [10,11]. Hyperandrogenism plays a pivotal role in 90% of PCOS individuals [12]. Excessive androgen production is caused by hypothalamic-pituitaryovarian axis dysfunction, oxidative stress, insulin resistance, and steroidogenesis abnormalities, which leads to PCOSrelated symptoms [9,13]. Gonadotropin-releasing hormone originating from hypothalamus causes release of FSH and LH from anterior pituitary gland. FSH is involved in initiation and development of gametogenesis, whereas LH causes maturation of follicles and ovulation, in part by induction of various hormones involved in these developments [14,15].
Multiple studies in rodent models of PCOS demonstrated that several herbs might be benefcial for the treatment of laboratory and clinical symptoms of PCOS, such as insulin resistance, menstrual disturbances, infertility, hormonal status, and anthropometric indices. Tese herbs and their extracts can be either used alone or in combination with standard treatments [8]. Centratherum anthelminticum (CA) Kuntze (bitter cumin/black cumin) is a member of the Asteraceae family and is commonly used in Ayurvedic medicine. Over 120 chemical compounds have been discovered in diferent plant parts of CA. It is rich in phytochemicals, particularly butin, tetrahydroxy chalcone, and tetrahydroxy favone [16]. Ayurvedic medicine reveals that CA helps in healing and alleviating diabetes. Moreover, this plant is popular for being an astringent, anthelmintic, tonic, treating fever, skin problems, kidney stones, and diuretic activity. Recently, the anticancer property of the active compound from CA seeds has also been established [17].
Previous studies demonstrated that the CA had exhibited a reduction in reactive oxygen species (ROS) and lipid peroxidation and improved the antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT) to protect the damage to macromolecules such as DNA, membrane lipids, and proteins. Moreover, CA caused a signifcant decrease in both the cardiac and liver function biomarkers associated with intracellular microvesicular steatosis and lipid accumulation [18]. Multiple studies on CA showed its activity against diabetes and related complications due to antihyperlipidemic and antioxidant properties. As PCOS is associated with ROS, hyperlipidemia, and insulin resistance, therefore, CA could exhibit anti-PCOS activity in Wistar rats. Hence, the aim of this study was to explore the curative efect of CA against PCOS in Wistar rats.

Ethanol Extract Preparation.
Te plant was collected from Punjab and identifed by a taxonomist at the University of Punjab (Ref. No. LAH#61120B). Dried seeds of C. anthelminticum were ground, and the ethanolic extract was made by soaking the coarsely powdered seed in ethanol at 1 : 10. Te soaked material was kept for 14 days at room temperature in an amber-colored fask and then passed through Whatman flter paper No. 43 for fltration. Te fltrate was concentrated in a rotary vacuum evaporator at 40°C. Te brown-coloredC. anthelminticum ethanolic seed extract (CA) was kept in the refrigerator at 2-8°C till further use.

HPLC-DAD Analysis.
A solution of 100 mg extract/ 10 ml of methanol was prepared and fltered through a syringe flter into the HPLC vials (2 mL). Te injection volume was 50 μL of the sample solution was injected and analyzed by HPLC-DAD, according to the previous method [19]. Te C18 column was used for the analysis.

Experimental Animals.
Tirty-six nulliparous female Wistar rats of weight ranging from 120 to 150 g were obtained and kept at the animal house of Riphah International University. All rats were kept at 25 ± 3°C and 55-65% humidity during acclimatization and experimental study. All protocols were executed as per the guidelines of the Institutional Animal Ethical Committee (Ref. No. REC/RIPS-LHR/2022/058).

Induction of PCOS.
High-fat diet (HFD) comprising 40% fat, 20% protein, 36% carbohydrate, and 4% others (530 kcal) was given to all animals for nine weeks, except normal control (NCG) which received normal rodent chow diet [20]. Estradiol valerate (4 mg/kg) was given orally once at the start of feeding the female rats with HFD for the induction of PCOS. Te animals with a 10-20% increase in weight than the normal control group were considered obese [21,22]. To confrm the development of PCOS, vaginal smears were taken to observe estrous cycle. Moreover, three rats were randomly selected and evaluated at the 9 th week by removing their ovaries for histological examination and compared with that of NCG [21].
2.6. Study Design. Rats on a normal chow diet served as NCG. All animals except NCG received HFD and estradiol valerate and were divided into fve groups. Disease control (DCG) received distilled water, while animals in the standard therapy group (STG) orally received metformin 300 mg/kg/day [23]. Te remaining three animal groups were orally treated with CA 250, 500, or 750 mg/kg/day. Treatment with diferent therapies was started after 9 weeks of starting HFD and then continued for 28 days [24].

OGTT, Lipid, and Liver Function Parameters.
Te blood was taken from the rat tail for an oral glucose tolerance test (OGTT) on the 28 th day. For performing OGTT, 75 mg/ml glucose solution was administered via oral route to each animal and blood glucose level was checked at 0, 30, 60, 90, 120, and 180 min after glucose administration. Triglycerides and total cholesterol were estimated by using ELISA kits. Moreover, the blood sample was pulled out after 28 days of therapy by cardiac puncture under anesthesia with chloroform. Blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes. Serum was collected by centrifugation of blood at 1000 rpm and stored in a refrigerator for further use. Liver function tests i.e., glutamic-pyruvic transaminase (SGPT) and glutamic oxaloacetic transaminase (SGOT) were analyzed by standard methods [25].

Histological Examination.
For histopathological studies, the cervical dislocation technique was used to kill the anesthetized animals and ovaries were dissected out through abdominal excision and placed in 10% bufered formaldehyde. Te ovarian tissues were fxed in parafn wax and sections of 4-5 μm were prepared with a microtome. Te excised ovarian tissues were stained with hematoxylin and eosin for histological examination under a light microscope [26].

Preparation of Tissue
Homogenates. For tissue homogenate preparation, ovaries were thoroughly rinsed with ice-cold phosphate bufer saline (PBS). Te ovarian tissues were thoroughly crushed with a homogenizer. Te PBS was added to the crushed tissues to make 10% w/v homogenate. Afterward, the mixtures were centrifuged for 5 min at 1000 rpm, and supernatants were collected to determine oxidative stress parameters [27].
2.11. CAT Activity. CAT activity was estimated by a previously established method. A cuvette was flled with 1 ml supernatant which already contained 1.95 ml phosphate bufer (50 mM, pH 7.0) and 1 ml 30 mM H 2 O 2 . Absorbance change was measured for 30 S at a wavelength of 240 nm. Te CAT activity was stated in U/mg of protein. Indeed, one CAT activity unit was equal to 1 M of hydrogen peroxide decomposed per min at 25°C [28].

MDA Level.
Te MDA is a lipid peroxidation index that is determined by the double heating method. Tis method is based upon the measurement of absorbance of purple color produced by the TBA reaction. To carry out this estimation, 2.5 ml TCA was mixed with 0.5 ml supernatant. Ten, these solutions were kept in a boiling water bath for 15 min followed by cooling down to room temperature. Centrifugation was carried out at 1000 rpm for at least 10 min. Collected supernatant (2 ml) was admixed with 1 ml TBA solution (0.67%, w/v). Tese solutions were again boiled over a water bath for 15 min. Te absorbance was calculated at 532 nm after cooling [29].
2.13. SOD Activity. Free radical scavenging capacity in the tissue homogenates was measured. Tese radicals were produced by pyrogallol auto-oxidation. Te reaction mixture was comprised of 2.8 ml PBS, 100 µl pyrogallol solution (2.6 mM in 10 mM HCl), and 100 µl sample supernatant. Absorbance was recorded repeatedly at 325 nm after every 30 S. Under the assay conditions, each unit of SOD was equal to the quantity of enzyme needed to achieve 50% pyrogallol autoxidation inhibition [30].
2.14. GSH Level. For GSH estimation, a tissue homogenate supernatant of 1 ml was mixed up with an equal volume of 10% TCA. To this mixture, 1 ml phosphate solution was added along with 0.5 ml DTNB reagent was added. Absorbance was taken at 412 nm. Te values were stated as nm of GSH per mg of protein [31].

Statistical Analysis.
A one-way analysis of variance (ANOVA) test was applied to the data regarding hormonal, lipid and liver parameters, insulin, HbA1c, oxidative stress, and infammatory parameters using GraphPad Prism 5 (CA, USA, San Diego). Changes in body weight were assessed by two-way ANOVA. Moreover, the statistically signifcant data were subjected to Tukey's post hoc test. Te results were moderately signifcant when p < 0.01, while these were considered highly signifcant when p < 0.001.

Chemical Composition.
HPLC-DAD analysis revealed that kaempferol-3-pcoumaroylglucoside was present in the highest amount (146.8 ± 1.8 mg/g) of the extract followed by ferulic acid (145.8 ± 1.21 mg/g) and malvidin-3-(6-caffeoyl)-glucoside (77.1 ± 0.4 mg/g). Te detailed International Journal of Endocrinology phytochemical analysis of the CA extract is shown in Table 1, while the HPLC-DAD spectrum is presented in Figure 1.

Efect on Blood Glucose, Insulin, and Body Weight
OGTT. Blood glucose level via OGTT was recorded in all groups at the end of the treatment which revealed that the STG and all diferent doses of CA decreased the blood glucose levels 90 min after glucose administration. Te DCG demonstrated the highest rise in blood glucose. Te efect of CA 250 mg/kg on blood glucose levels was minimum among all therapeutic groups. Te efect of CA on blood glucose levels on OGTT in PCOS rats is shown in Figure 2(a).

Serum Insulin Levels.
Administration of estradiol and HFD led to a signifcant increase in insulin level (hyperinsulinemia) of PCOS animals as compared to NCG. Te PCOS rats treated with STG and diferent doses of CA demonstrated a signifcant decrease (p < 0.0001) in insulin as compared to DCG. Te efect of treatment with diferent doses of CA on the serum insulin of PCOS rats is shown in Figure 2(b).

Efect on Body Weight.
All animals had statistically indiferent body weights at the time of initiation of the experiment. With HFD for 9 weeks, there was a signifcant rise in body weight of almost 10-20 percent among all animals as compared to NCG. Tere was a continuous rise in the body weight of DCG. All treated groups demonstrated higher body weight as compared to the NCG. Te efect of treatment with diferent doses of CA on the body weight of PCOS rats is shown in Figure 2(c).

HbA1c
Level. It was found that the DCG had a signifcantly increased level of HbA1c as compared to NCG. No signifcant diference in HbA1c was observed in the diseased animals treated with metformin or CA compared with DCG. Te efect of CA groups was comparable with the DCG. Te efect of treatment with diferent doses of CA on the HbA1c of PCOS rats is shown in Figure 2(d).

Efect on Serum Lipids and Liver Enzymes
3.3.1. Serum Triglycerides. Triglyceride level was increased in the DCG as compared to NCG after administration of estradiol and HFD. Te treatment groups receiving metformin or CA 500 mg/kg demonstrated a decrease in triglycerides as compared to DCG. Te efect of treatment with diferent doses of CA on the triglyceride level of PCOS rats is shown in Figure 3(a).

Serum Cholesterol.
Hyperlipidemia is one of the main causes of PCOS. Serum cholesterol level was observed in all groups which showed that DCG had elevated levels of cholesterol as compared to NCG. Te STG and CA at all dose levels signifcantly reduced the cholesterol level as compared to DCG. Te efect of treatment with diferent doses of CA on the serum cholesterol of PCOS rats is shown in Figure 3(b).

SGOT Level.
Te level of SGOT was insignifcantly diferent among DCG as compared to NCG. However, treatment with metformin and CA 500 and 750 mg/kg signifcantly raised the level of SGOT as compared to DCG. Te treatment group CA 250 mg/kg when compared with the STG displayed a statistically comparable level of SGOT as compared to NCG and DCG. Te efect of treatment with diferent doses of CA on the SGOT level of PCOS rats is shown in Figure 3(c).

SGPT Level.
It was found that the DCG showed a statistically elevated level of SGPT as compared to NCG. However, treatment with metformin and CA did not result in any signifcant changes in SGPTas compared to DCG. Te efect of treatment with diferent doses of CA on the SGPT level of PCOS rats is shown in Figure 3(d).

FSH Level.
Te level of FSH declined in the DCG as compared to NCG. However, the treatment of PCOS rats with CA or metformin signifcantly increased the level of FSH in comparison with DCG. Te treatment with CA 750 mg/kg exhibited the maximum increase in FSH; however, the efect on FSH was lower than that of metformin. Te efect of treatment with diferent doses of CA on the FSH level of PCOS rats is shown in Figure 4(a).

LH Level.
Te LH level was increased in the PCOS rats of DCG as compared to NCG. Te STG and CA-treated groups demonstrated a signifcantly reduced level of LH as compared to DCG. However, the level of LH in STG and DCG was still elevated as compared to NCG. Te efect of treatment with diferent doses of CA on the LH level of PCOS rats is shown in Figure 4(b).

Progesterone Level.
Te progesterone level declined in the PCOS rats of DCG as compared to NCG. Administration of CA at 500 and 750 mg/kg and metformin resulted in a signifcant elevation of progesterone as compared to DCG. Te efect of CA 250 mg/kg was minimal, and the level of progesterone was insignifcant to DCG. Te efect of treatment with diferent doses of CA on progesterone level of PCOS rats is shown in Figure 4(c).

Efect on Infammatory Markers
3.5.1. IL-6 Level. It was found that the administration of estradiol and HFD resulted in a signifcant elevation of IL-6 in DCG as compared to NCG. Treatment with CA at all dose levels and STG signifcantly declined IL-6 level as compared to DCG. However, CA and STG treated rats demonstrated a higher level of IL-6 in comparison to NCG. Te efect of treatment with diferent doses of CA on the IL-6 level of PCOS rats is shown in Figure 5(a).

NF-κB Level.
Tis study showed that DCG exhibited a higher level of NF-κB level in the serum in comparison to the NCG. Treatment of PCOS rats with CA at 350, 500, and 750 mg/kg resulted in a statistically signifcant decline in NF-κB as compared to DCG. However, the treatment of PCOS rats with CA and metformin did not result in the normalization of NF-κB. Te efect of treatment with diferent doses of CA on NF-κB levels in the serum of PCOS rats is shown in Figure 5(b).

NrF2.
It was demonstrated that the administration of estradiol and HFD had resulted in a signifcant decline of NrF2 in DCG as compared to NCG. Treatment with CA at all dose levels and STG signifcantly raised the NrF2 level as compared to DCG. Te efect of treatment with diferent doses of CA on NrF2 levels in the serum of PCOS rats is shown in Figure 5(c).

Oxidative Stress in the Ovary
3.6.1. CAT Activity. It was observed that the activity of CAT in the ovary tissue homogenate of PCOS rats had reduced signifcantly as compared to NCG. In the STG, there was a signifcant increase in CAT activity (p < 0.0001) in ovaries as compared to the DCG. Although, all PCOS animals treated with CA showed a signifcant increase in CAT activity as compared to DCG; however, the CA 500 and 750 mg/kg exhibited a higher level of CAT as compared to the lowest dose of CA. Te efect of treatment with diferent doses of CA on CAT activity in the ovarian tissue of PCOS rats is shown in Figure 6(a).

Malondialdehyde Level.
Te MDA level in DCG was signifcantly higher than the NCG. Te treatment groups receiving CA at 250, 500, and 750 mg/kg or STG displayed  International Journal of Endocrinology a signifcant decrease in the MDA level as compared to DCG. Te efect of CA at 500 and 750 mg/kg or STG on MDA was comparable to that of NCG. Te efect of treatment with diferent doses of CA on MDA levels in the ovarian tissue of PCOS rats is shown in Figure 6(b).

SOD Activity.
A signifcant decline of SOD activity in the ovarian tissue of DCG was observed as compared to NCG. Administration of metformin or CA 750 mg/kg signifcantly elevated the activity of SOD in diseased rats as compared to DCG. However, CA 250 and 500 mg/kg treatments failed to bring out any signifcant increase in the SOD activity of diseased rats in comparison with the DCG.
Te efect of treatment with diferent doses of CA on SOD activity in the ovarian tissue of PCOS rats is shown in Figure 6(c).

GSH Level.
Te level of GSH was lower in the estradiol-induced PCOS rats in comparison with the NCG. Te STG and CA 250, 500, and 750 mg/kg treatments in diseased rats signifcantly increased the level of GSH as compared to DCG. However, the level of GSH exhibited by CA 250 and 500 mg/kg groups was signifcantly lower than that displayed by NCG. Te efect of treatment with diferent doses of CA on GSH levels in the ovarian tissue of PCOS rats is shown in Figure 6(d).    International Journal of Endocrinology

Histopathological Examination of Ovaries.
Histopathological slides of the ovary of all PCOS rats treated with metformin and CA extract were examined and compared with DCG and NCG for the number and size of follicles and the efect on the corpus luteum. In the NCG, the maximum number of corpus luteum and primary follicles i.e., preantral follicles (PAF) and antral follicles (AF) were observed. Changes in numbers of AF were also observed in the CA 250 mg/kg treated group, along with shrinkage of ovary tissues and decreased number of corpus luteum. Primary and secondary follicles were also seen. However, the most pronounced changes were observed in PCOS rats treated with CA 750 mg/kg group which demonstrated several follicles such as graafan follicle (GF), AF, and PAF at diferent developmental stages. Corpus luteum was also observed with decreased number of secondary follicles, thus exhibiting the restoration of fertility as shown in Figure 7.

Discussion
Like other endocrine diseases, PCOS is treated by lifestyle modifcations and pharmacological and hormonal therapy. Metformin has established its role in the management of PCOS over time, but due to its deleterious efects, pharmacological interventions with multiple herbal extracts have been assessed which showed efcacy alone or in combination with conventional therapy against PCOS [8]. In current study, the plant extract was chemically characterized with  HPLC-DAD to determine the phytochemical composition. Te efect of CA at 250, 500, and 750 mg/kg and the standard metformin therapy on PCOS rats was observed which demonstrated that these treatments caused amelioration of PCOS symptoms through efects on lipid profle, insulin levels, liver function, and serum hormones. Te histopathological observations validated the preclinical evidence.
Insulin plays a vital role in stimulating stromal androgen and ovarian thecal secretion. Insulin resistance, in the form of hyperinsulinemia, induces the overproduction of ovarian androgens. Moreover, terminal diferentiation and anovulation are induced by the impact of insulin-like growth factor (IGF)-1 on LH. Te IGF-1 enhances the production of estrogen through granulosa cells and acts in regulating aromatase concentration of granulosa cells via synergism with LH and FSH [34,35]. Insulin resistance, an important factor for the diagnostic criteria of PCOS, is usually treated with metformin. Treatment with CA improved insulin resistance as evidenced by a reduction of insulin level in PCOS rats [8]. Te phytochemicals in CA, such as quercetin glycoside, kaempferol, and ferulic acid, have antioxidant, antiinfammatory, and antidiabetic efects. Furthermore, there  International Journal of Endocrinology are multiple possible mechanisms behind the antidiabetic action of CA, such as inhibition of carbohydrate hydrolyzing enzyme activity, including α-amylase and α-glucosidase, which decrease glucose absorption in the gastrointestinal tract [17]. Steroidal hormones play a pivotal role in sexual maturation and diferentiation and are synthesized from cholesterol under the infuence of gonadotrophins. Tese steroidal hormones are produced in steroidogenic tissues involving six diferent cytochrome P450 enzymes [36]. Tese gonadotrophins are scanty in PCOS which result in reduced steroidogenesis and failure of egg production/maturation [37,38]. In PCOS women, the rate of adrenal and ovarian androgen production is signifcantly increased, while their metabolic inactivation is reduced. In addition, adipose tissues secrete adipocytokines such as leptin and chemerin, which aggravate PCOS. Te decrease in body weight reduces the secretion of adipocytokines and insulin resistance, while correction of the gonadotropin hormone release from the pituitary gland corrects the steroidal hormone homoeostasis [38].
Te present study showed that the CA had resulted in a reduction of cholesterol and triglyceride level in PCOS rats. Te efect of CA on cholesterol might be either due to the inhibition of the ß-hydroxy ß-methylglutaryl CoA reductase (HMG-CoA), the enzyme important for rate-limiting step in the biosynthesis of cholesterol, or an increase in the lowdensity lipoprotein (LDL-c) breakdown through its receptors on the liver. Te decrease in triglycerides may be related to the augmented activity of the endothelial-bound lipoprotein lipase, which leads to the hydrolysis of triglycerides into fatty acids [18]. Hyperlipidemia is directly Previously, it is demonstrated that C. anthelminticum had improved the oxidative stress and lipid profle in triton X-100-induced hyperlipidemic rabbits [18].
Te administration of HFD and estradiol infuenced the hormonal levels. Te efect of estradiol and HFD was evident on the liver as diseased rats showed a signifcant rise in levels of SGPT and SGOT. Tis study has shown the ability of CA to regulate the hormonal imbalance caused by the endocrine disorder which had caused an increase in LH and a decrease in progesterone and FSH. It demonstrated the ability of CA to prevent the progression of PCOS.
Oxidative stress induced due to hyperlipidemia is one of the associating factors that point to long-term complications of PCOS by enhancing free radical production and suppressing the antioxidant enzyme system in the body [36].
Te oxidative modifcation of LDL-c due to stress plays a major part in atherosclerotic lesions [18]. Tis study showed that CA reduced oxidative stress to prevent longterm complications of PCOS, i.e., atherosclerosis.
It is found that the reduction of SOD and CAT activities and increase in MDA level are the results of oxidative imbalance in reproductive tissues. Oxidative stress in ovarian tissues of PCOS rats is evident in previous studies. Long-term oxidative stress leads to sperm immobility and damage to DNA, enzymes, and lipid peroxidation [37][38][39].
Tere was an elevation of the SOD activity by CA 750 mg/kg in PCOS rats. Te decrease in SOD activity in untreated PCOS rats showed oxidative stress induced by HFD and estrogen. An important antioxidant having the ability to protect the cells against oxidative stress is GSH. Optimum levels in the GSH/GSSG ratio produce a reduced environment leading to protection from radical oxygen species [40]. In the present research, the GSH levels were restored in which is evident in PCOS induced by HFD and estradiol. Current study showed that CA reduced the lipid peroxidation in diseased rats at diferent dose levels.
Multiple shreds of evidence supported that high levels of proinfammatory markers TNFα, IL-6, NF-κB, and IL-1ß are associated with PCOS [41]. An elevated level of IL-6 is not termed as a prominent feature of PCOS but can be considered as a benefcial surveillance biomarker for PCOS treatment [42]. PCOS women with low-grade chronic infammation might be at risk of cardiovascular diseases and diabetes mellitus due to chronic infammation [43]. Tis study demonstrated that the treatment with CA resulted in the decline of serum levels of NF-κB, NrF2, and IL-6 in PCOS rats which resulted in a reduced risk of chronic infammation.
Histopathological substantiation in the current study showed that the ovaries of HFD and estradiol valeratetreated rats exhibited numerous cysts along with either a very lean or no granulosa layer. Moreover, diseased animals showed anovulation which was indicated by the absence of the corpus luteum. Treatment with CA led to the cyst disappearance and emergence of corpora lutea and normal follicles. Sections from CA 250 mg/kg treatment showed follicles bigger in size and lesser corpora luteum. CA at 759 mg/kg showed several corpus luteum and antral follicles with clearly diferentiate oocytes, granulosa cell layer, corona radiate, cumulus oophores, and thecal cells indicating improvement of PCOS [44].

. Conclusion
Based on the current study, it can be concluded that the extract of C. anthelminticum displayed an ameliorating efect against PCOS in rats due to anti-infammatory, antioxidant, antihyperlipidemic, and antidiabetic activities. Te plant extract was the most efcacious at 750 mg/kg. Tese in vivo activities were most probably due to the presence of favonoids and phenolic compounds which decreased the oxidative stress, insulin resistance, and HbA1c and normalized the hormonal imbalance to restore ovulation.
It is suggested that the plant extract must be evaluated for its antiobesogenic potential in diabetic rats. Furthermore, a comprehensive phytochemical analysis and bioassay-based isolation of the multiple phytochemicals responsible for these activities must be carried out to discover a safer, effcacious, and economical treatment of PCOS.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.