Rhamnocitrin ameliorates ovarian brosis via PPARγ/TGF-β1/Smad2/3 pathway to repair ovarian function in polycystic ovary syndrome rats

Background: Polycystic ovary syndrome (PCOS) is one of the major endocrine disorders in women, characterized by androgen excess, chronic anovulation and ovarian brosis. Rhamnocitrin is an herbal bioactive avonoid that has anti-inammation and antioxidant effects. We intended to investigate the impacts of Rhamnocitrin on PCOS-induced ovarian brosis and its underlying mechanisms. Dehydroepiandrosterone (DHEA) induced-PCOS rats were treated with Rhamnocitrin. HE staining was performed to detect ovarian histological features. Ovarian brosis was evaluated by Sirius Red and Masson staining. Vaginal smear was examined to exhibit estrus cycle stage and vaginal cornication. The serum hormone levels of FSH, LH, E2 and T were measured with ELISA. The related mRNAs and proteins of brosis factors and PPARγ/TGF-β1/Smad2/3 signaling were detected by RT-qPCR and western blot. The weights of rat bodies and ovaries were recorded. PPARγ inhibitor T0070907 and its agonist GW1929 were employed for the mechanistic investigation.


Introduction
Polycystic ovary syndrome (PCOS), a common disorder of anovulatory infertility, is one of the major endocrine-metabolic disorder in reproductive age women [1]. Pathophysiology of PCOS is a complex interplay among numerous factors containing androgen excess, disordered gonadotropin secretion, insulin resistance, follicular arrest and ovarian dysfunction [2]. PCOS is characterized by hormonal imbalance, including the leutinizing/follicle-stimulating hormone (LH/FSH) ratio, gonadotrophin-releasing hormone (GnRH), insulin, parathyroid hormone (PTH), estrogens, androgens and cortisol [3]. Signi cantly, no general treatment is available for PCOS, and therapeutic approaches for PCOS treatment mainly target hyperandrogenism, ovarian dysfunction and metabolic disorders [4].
Flavonoids are well known to be bioactive polyphenols that associated with anti-in ammation and antioxidant effects due to their highly reactive oxygen radicals [5]. Flavonoids are traditionally utilized to prevent or treat various diseases containing PCOS and reproductive system dysfunction [6]. For instance, supplementation of quercetin down-regulates the level of resistin in plasma and the concentrations of LH and testosterone in overweight PCOS patients [7]. Troxerutin, a natural avonoid, protects against dihydrotestosterone (DHT)-induced PCOS in rats via modulating neurotransmitter release [8]. Treatment of rutin apparently ameliorates ovarian malfunction and systemic insulin resistance in PCOS rats [9]. Literatures have reported that Rhamnocitrin has the e cacy of anti-in ammation and antioxidant activity [10,11]. However, limited data are available assessing the effects of Rhamnocitrin on ovarian dysfunction in PCOS.
Polycystic ovarian morphology in PCOS is featured with a thickening of ovarian capsule and stroma induced by up-regulation of collagen deposition and brous tissue [12]. As nuclear receptors, peroxisome proliferator-activated receptors (PPARs) are involved in modulating brosis [13]. Enormous researches have con rmed that PPARγ exerts a protective function in tissue from brosis and have explored natural products that have latent PPARγ-activation e ciency [14,15]. As one of the multifunctional cytokine families, transforming growth factor-β (TGF-β) functions in numerous physiological and pathological processes, such as tissue brosis and wound healing [16]. Studies present that TGF-β1 plays a critical function in extracellular matrix remodeling in ovary [17]. TGF-β1 acts as a vital regulator in tissue brosis largely via activating its downstream small mother against decapentaplegic (Smad) signaling, including Smad2 and Smad3 [18]. Activation of TGF-β1/Smad3 signaling pathway suppresses development of ovarian follicle through facilitating granulosa cell apoptosis in PCOS [19]. PCOS rodent model induced by dehydroepiandrosterone (DHEA) displays critical characteristics similar to PCOS women, including a high androgen state, abnormal secretion of serum hormones, blocked follicular development, de cient corpora lutea and presence of cystic follicles and so on [20]. Furthermore, DHEAinduced PCOS rats are validated to present ovarian collagen deposition and hyper brosis, which compromises ovarian functions [12, 21,22]. Here, DHEA induced-PCOS rats were applied to explore the role of Rhamnocitrin in PCOS, including ovarian histological features, estrus cycle stage, serum hormone levels, ovarian brosis and brosis factors expressions. Besides, we then validated whether PPARγ/TGF-β1/Smad2/3 pathway was implicated in effects of Rhamnocitrin on ovarian brosis to modulate ovarian function. This study aims to provide a potential effective drug for patients with PCOS.

Animals
Sprague-Dawley (SD) rats (female, 21 days old, 50-60 g) were obtained from Experimental Animal Center of Guangxi Medical University. Rats were housed in SPF environment at 24 °C with 12:12 h light-dark cycle. They were provided with free access to water and food. This research was performed according with the guidelines for the use of laboratory animals and approved by the institutional research animal committee of Guilin Medical College.Approval Number: GLMC201803009.

Experiment design
The rats were randomly divided into 7 groups (n=6). For PCOS induction, injectable sesame oil was used to dissolve DHEA. Then rats received a daily hypodermic injection of DHEA solution with a dose of 0.2 ml (6 mg/100 g) at nape of the neck for 21 consecutive days, fed with high fat diet. Naïve rats were adopted as controls. Additionally, some rats also orally treated with three levels of Rhamnocitrin (5mg/kg/day;10mg/kg/day;20mg/kg/day,p.o;Carbosynth,UK) daily during treatment period. For PPARγ inhibitor treatment, rats were injected intraperitoneally with T0070907 (10 mg/kg, Sigma-Aldrich, St Louis, MO, USA) once daily. For PPARγ agonist treatment, rats were treated with GW1929 (15 mg/kg, Sigma-Aldrich) by oral gavage once daily. Estrous cycle and body weight of rats were recorded, during process and treatment. At day 21, rats were killed, and the blood and ovaries of rats were collected. After that, fat around the ovaries was removed and the ovaries were weighed.

Serum hormone measurement
The rat orbital veins blood was received administration. After separated from blood samples, serum was stored under −80°C. The Follicle-Stimulating Hormone (FSH), luteinizing hormone (LH), estradiol (E2) and total testosterone (T) in serum were evaluated with ELISA following the guideline. Testing kits for FSH, LH, E2 and T were obtained from Elabscience (Elabscience, USA). Experiments were carried out in triplicate and repeated three times.

Hematoxylin and eosin (H&E) staining
After xed with 10% neutral-buffered formalin, ovaries were embedded in para n. After that, they were sectioned into 3 μm thick slices. Hematoxylin and eosin were utilized to stain para n slices and optical microscope (Leica Microsystems, Wetzlar, Germany) was applied to con rm pathological structural variations of rat ovaries.

Sirius Red and Masson staining
After xed with 10% neutral-buffered formalin, ovaries were embedded in para n. Subsequently, they were sectioned into slices (3 μm). They were then stained with Sirius red or Masson staining to reveal ovarian brosis and con rm inhibitory impact of Rhamnocitrin on brosis. For Masson staining, tissue slides were then incubated at 37°C for 2 h with Bouin solution, which contained acetic acid (5 mL), 10% formalin solution (25 mL) and saturated picric acid (75 mL). After that, according to the manufacturer's direction, slides were stained using Masson Trichrome Staining kit (Sigma-Aldrich) and collagen bers were stained in blue. For Sirius red staining, tissue sections were administered for 20 min with Picro-Sirius red stain solution kit (Abcam, Cambridge, MA, USA) and washed for 1 min with tap water in accordance with the manufacturer's guideline. The muscle, blood vessels and epithelium appeared yellowish while collagen bers were stained in red. These stained specimens were visualized under light microscope (Leica Microsystems).

Determination of the Estrus Cycle Stage
Toluidine blue stain was utilized to determine variations of vaginal exfoliative cytology in rats. After was soaked in physiological saline, aseptic cotton swab was put on vaginal wall of rats and smeared clockwise. Cotton swab was taken out and smeared on slide in same direction. Afterwards, cells on slide were xed for 15 min with 4% paraformaldehyde. Toluidine blue (Servicebio, MA) was applied to stain vaginal smear following instruction. Microscope imaging system (Nikon, Japan) was used to visualize the images.

Western blot
Ovarian tissues were milled separately with RIPA lysis buffer. After protein collection from each specimen, protein concentration detection was carried out with BCA protein assay kit (Biovision, Milpitas, CA, USA) according with the instruction. The electrophoretic separation is run using SDS-PAGE gels (TakaRa Biotechnology Co., Ltd., Dalian, China). Protein (50 μg) were separated with 10% SDS-PAGE prior to transfer onto PVDF membranes. After that, they were blocked at room temperature in 5% bovine serum albumin for 2 h. Subsequently, they were incubated with primary (12h at 4°C) before treatment with Immobilon Western Chemiluminescent HRP Substrate (Abcam) was used to develop the blots. GAPDH was adopted as internal control. Protein expression quantitative analysis was performed with ImageJ software 1.8.0.
Quantitative real-time PCR RNA was collected with Trizol Reagent (Takara) according with the guideline. cDNA was generated with RNA (1 μg) with Prime Script TM RT Reagent kit (Takara) followed by ampli ed by RT-PCR with the following cycling conditions: 15 minutes for reverse transcription at 37°C, 5 seconds for inactivating reverse transcriptase at 85°C, followed by cold-storage at 4°C. cDNA was ampli ed by Brilliant II Fast SYBR green QPCR master mix (Sigma-Aldrich, MO, USA) with ABI 7500 Fast Real-Time PCR System (Applied Biosystems) using the following cycle: 30 seconds for pre-denaturation at 95°C, 5 seconds for PCR ampli cation at 95°C (45 cycles), and 30 seconds for primer annealing at 60°C. Primer was produced from Macrogen Inc. (Seoul, Korea). GAPDH expression was applied as control. The relative level analysis was achieved by the comparative CT method (2 -ΔΔCT ). The primers were showed in Table 1.

Statistical analysis.
Statistical analyses were evaluated with SPSS 21.0 software. All data are shown as means ± SEM. Difference betweeen two goups was compared using Student's t test. Results for multiple comparisons was implemented by ANOVA followed by Tukey's post hoc test. The criterion of statistical signi cance was set as P value less than 0.05.

Rhamnocitrin recovered ovarian function in PCOS rats
Morphological analysis was performed on ovarian tissues of rats. In model group, the disordered ovary structure was observed, corpus luteum and follicles were noticeably decreased, whereas antral follicles were remarkably increased in comparison by control group (Fig. 1A). Additionally, compared to control group, granular cells were arranged loosely and layer of cells developed thinner (Fig. 1A). Compared to PCOS group, corpus luteum was increased after Rhamnocitrin administration (Fig. 1A). Furthermore, antral follicles were evidently reduced and granular cell layer thickness was increased after Rhamnocitrin administration (Fig. 1A). Results of vaginal smears presented that estrous cycle was regular, with corni ed squamous epithelial cells in estrus of control group (Fig. 1B), however, PCOS rats exhibited the irregular estrous cycle, with numerous leucocytes and in continuous diestrus phase (Fig. 1B). After Rhamnocitrin treatment, estrous cycle returned to regular and normal gradually (Fig. 1B). These data revealed that Rhamnocitrin recovered the abnormal ovarian morphology and estrous cycle in PCOS rats.
Among these groups, no obvious difference was exhibited in body weight prior to any intervention, (Fig. 1C). After treated orally by DHEA for 21 days with high-fat diet, we observed signi cantly increased body weight of rats in comparison with control group (Fig. 1C). Rat weight in rhamnocitr group was apparently decreased compared to PCOS group, especially in high dose Rhamnocitrin rats (Fig. 1C).
Ovary weight were also recorded, and the result revealed that in comparison to control group, ovary weight in PCOS rats was effectively increased (Fig. 1D). Whereas the ovary weight was reduced in Rhamnocitrin treated rats, in comparison with that of PCOS rats (Fig. 1D). Besides, the serum hormone levels of FSH, LH, E2 and T were analyzed. The serum levels of LH, E2 and T were obviously up-regulated in PCOS group compared to control group, and Rhamnocitrin administration apparently down-regulated serum LH, E2 and T level when compared to PCOS group (Fig. 1E, F, G). Additionally, Rhamnocitrin treatment signi cantly elevated the decreased FSH level in PCOS group (Fig. 1H).
3.2 Anti brotic effects and regulating PPARγ/ TGF-β1/Smad2/3 signaling of Rhamnocitrin on ovaries of PCOS rats Sirius Red and Masson staining were used to measure the role of Rhamnocitrin in ovarian brosis. Our data demonstrated that in comparison to control group, ovarian interstitial brosis was elevated in PCOS rats ( Fig. 2A), whereas ovarian interstitial brosis was suppressed after Rhamnocitrin treatment ( Fig. 2A), unveiled by Matson staining. In addition, Sirius Red also showed the similar results (Fig. 2B). These data suggested that ovarian brosis was inhibited by treatment with Rhamnocitrin.
In rat ovarian tissues, mRNA expressions of brosis factors, including α-SMA, Collagen1, MMP-2, MMP-9, TIMP1 were determined by qRT-PCR (Fig. 2C). Data presented that brosis factors mRNA expressions in PCOS group were remarkably elevated in control group (Fig. 2C). In ovarian tissues, mRNA expressions of brosis factors were decreased considerably in presence of Rhamnocitrin as comparing to PCOS group (Fig. 2C). The protein expression of brosis factors exhibited the similar results with qRT-PCR (Fig. 2D). Besides, it was presented that in ovarian tissues from PCOS group, PPARγ was obviously decreased, while TGF-β1 and p-Smad2/3 levels were evidently increased in comparison to control group (Fig. 2D). There was a signi cant increase in expression of PPARγ, while decrease in expression of TGF-β1 and p-Smad2/3 after Rhamnocitrin treatment (Fig. 2D). In addition, no signi cant difference was detected in protein expression of Smad2/3 among these groups (Fig. 2D), suggesting that Rhamnocitrin exerted a role of inhibiting Smad2/3 phosphorylation.

Rhamnocitrin restored ovarian function by activating PPARγ in PCOS rats
PPARγ agonist GW1929 and PPARγ inhibitor T0070907 were administrated to explore whether PPARγ signaling pathway was involved in ovarian function recovery of Rhamnocitrin in PCOS rats. We found that in comparison with the PCOS group, High-Rhamnocitrin supplementation showed elevated thickness of granular cell layer, reduced antral follicles and increased corpus luteum (Fig. 3A). Whereas T0070907 reversed the alteration of ovarian tissues induced by Rhamnocitrin in PCOS rats (Fig. 3A). Additionally, GW1929 treatment in PCOS ratsexhibited the similar histological characteristics of ovaries with that in High-Rhamnocitrin treated model rats (Fig. 3A). In addition, we found that Rhamnocitrin rescued the irregular estrous cycle induced by PCOS (Fig. 3B). In PCOS rats treated by GW1929, the estrous cycle also returned to regular and became normal, which was similar with that in model rats treated by Rhamnocitrin (Fig. 3B). However, the effect of high-Rhamnocitrin on estrous cycle was antagonized by T0070907 in PCOS rats (Fig. 3B).
In comparism with PCOS group, Rhamnocitrin treatment reduced the weight of PCOS rats, which was similar with GW1929 addition in PCOS rats (Fig. 3C). The reduction of body weight induced by Rhamnocitrin could be reversed after T0070907 treatment (Fig. 3C). Ovary weight were also recorded, and the result was in agreement with body weight (Fig. 3D). We then measured the serum hormone levels of FSH, LH, E2 and T. The data showed that the serum LH, E2 and T levels were obviously increased in PCOS group, compared to that in control group (Fig. 3E, F, G), and both Rhamnocitrin and GW1929 treatment could reduce the elevated LH, E2 and T level of PCOS rats (Fig. 3E, F, G). Compared to control group, FSH level in PCOS group was decreased (Fig. 3H), which could be signi cantly elevated after Rhamnocitrin or GW1929 treatment (Fig. 3H). On the contrary, Rhamnocitrin induced-alterations of serum hormone levels were antagonized by T0070907 supplementation (Fig. 3E, F, G, H).
3.4 Rhamnocitrin exerted anti brotic effects on ovaries through PPARγ/TGF-β1/Smad2/3 signaling in PCOS rats Next, we investigated whether PPARγ signaling pathway was implicated in anti brotic effect of Rhamnocitrin on ovaries of PCOS rats. Sirius Red and Masson staining were used to evaluate ovarian brosis. The data from Matson staining presented that compared with the control group, ovarian interstitial brosis was elevated in PCOS rats (Fig. 4A). However, ovarian brosis of PCOS rats could be suppressed by administrated with Rhamnocitrin (Fig. 4A), whereas this alteration was reversed by T0070907. GW1929 mimicked the function of Rhamnocitrin on ovarian brosis in PCOS rats (Fig. 4A). In addition, the similar results were showed by Sirius Red staining (Fig. 4B). These data suggested that GW1929 or Rhamnocitrin treatment inhibited ovarian brosis of PCOS rats, while T0070907 could antagonize anti brotic effects of Rhamnocitrin on ovaries in PCOS rats.
The qRT-PCR data illustrated that mRNA expressions of brosis factors including α-SMA, Collagen1, MMP-2, MMP-9, TIMP1 in PCOS group were remarkably up-regulated, in comparison by control group (Fig. 4C). After Rhamnocitrin or GW1929 treatment, mRNA levels of these brosis factors in ovarian tissues were apparently down-regulated (Fig. 4C). Effect of Rhamnocitrin on mRNA levels of brosis factors were reversed by T0070907 application (Fig. 4C). The effects of GW1929 and T0070907 on protein expressions of α-SMA, Collagen1, MMP-2, MMP-9, TIMP1 in ovaries detected by western blot showed the similar results with mRNA expressions measured with qRT-PCR (Fig. 4D). Besides, protein expressions of PPARγ, TGF-β1 and p-Smad2/3 were also determined. Treatment with Rhamnocitrin or GW1929 in PCOS rats could reverse the down-regulated PPARγ and the up-regulated TGF-β1 and p-Smad2/3 in the ovarian tissues, compared to PCOS group (Fig. 4D). Conversely, T0070907 antagonized the effects of Rhamnocitrin on the expressions of PPARγ, TGF-β1 and p-Smad2/3 in PCOS rats (Fig. 4D). In addition, there was no signi cant difference in Smad2/3 protein expression among these groups ( Fig. 4D).

Discussion
The judicious application of appropriate therapeutic approaches for treatment of PCOS are required, mainly addressing hyperandrogenism, ovarian dysfunction and associated metabolic disorders [4]. Ovarian brosis in PCOS is characterized by increased interstitial brosis and collagen deposition, leading to up-regulated ovarian capsule and stroma [12]. Currently, the usage of natural products such as avonoids in the treatment of different diseases has attracted great attention [6]. In the present work, which is the rst of its kind to the best of our knowledge, we evaluated that Rhamnocitrin supplementation had bene cial effects on ovarian morphology and estrous cycle disorders, body and ovary weights, hormonal status, ovarian brosis in rats with PCOS. Besides, Rhamnocitrin ameliorates ovarian brosis through PPARγ/TGF-β1/Smad2/3 pathway in PCOS rats. The present study provides a potentially effective therapeutic candidate of Rhamnocitrin for treating PCOS, the hallmark of anovulatory infertility and endocrine-metabolic disorders in reproductive age women.
Studies have shown that Rhamnocitrin, an herbal bioactive avonoid, has been reported to exert various pharmacological effects. Rhamnocitrin extracted from Nervilia fordii inhibits vascular endothelial activation [10]. Rhamnocitrin isolated from Prunus padus var. seoulensis is validated to be a reversible human monoamine oxidase (hMAO) inhibitor [23]. Rhamnocitrin 3-O-b-isorhamninoside induces human lymphoblastoid cell apoptosis via the extrinsic apoptosis pathway [24]. Obtained from Rhamnus alaternus L. (Rhamnaceae), Rhamnocitrin 3-O-b-isorhamninoside is evaluated to have antioxidant and antigenotoxic activities [11]. However, as far as we know, whether Rhamnocitrin can restore the ovarian damage in PCOS remains unclear. Due to ethical issues, studies on humans have limitations, hence animal models of PCOS aids in studying various aspects beginning from etiology to the treatment [20]. In current study, we rst investigate impacts of Rhamnocitrin on DHEA -induced PCOS rats. Our study demonstrated that a number of indicators of ovarian dysfunction, including corpus luteum, antral follicles, the thickness of the granular cell layer and estrous cycle, were restored by Rhamnocitrin supplementation in DHEA treated rats. In addition, the observations that Rhamnocitrin inhibited DHEAinduced up-regulated LH, E2, and T serum levels while promoted the down-regulated FSH serum level, suggest its bene cial role in hormonal secretion and release. Previous study reveals that total avonoids of dodder can decrease ovary index, affect serum hormone secretion and improve endometrial hyperplasia, achieving a protective effect on PCOS rats [25]. Rutin, a avonoid, ameliorates hyperandrogenism, acyclicity and infertility to improve PCOS phenotypes [9]. Flavonoids from Nervilia Fordii can down-regulate FSH serum level, up-regulate of LH and T serum levels and recover estrous cycle, exerting a therapeutic e ciency for PCOS treatment [26]. Our results are in line with these previous ndings that herbal bioactive avonoids can exert a protective effect on PCOS. Taken together, these observations support the conclusions that Rhamnocitrin has restored ovarian function successfully in PCOS rats induced by DHEA, might probably be considered as one candidate components for PCOS treatment.
Study demonstrates that PCOS rats exhibit ovarian brosis which results in function disorder of ovary [22]. As far as we know, in recent studies, the effect of Rhamnocitrin on tissue brosis is still unclear. In this study, we discovered Rhamnocitrin was able to repress ovarian interstitial brosis, indicated by Matson and Sirius Red staining. The inhibited mRNA and protein levels of brotic factors, including α-SMA, Collagen1, MMP-2, MMP-9 and TIMP1, in Rhamnocitrin administration rats may contribute to explain inhibitory mechanism on ovarian brosis. Recent researches present that PPARγ/TGF-β1/Smad signaling pathway participated in tissue brosis development. For example, capsaicin inhibits hepatic brosis via activating PPARγ to suppress TGF-β1/Smad Pathway [27]. Dual PPAR-α/γ agonist saroglitazar attenuates renal brosis via inhibiting TGF-β/Smad signaling pathway [28]. PPAR-γ agonist triterpenoid alleviates brogenesis via TGF-β/Smad and Akt pathway [29]. TGF-β1 from endometriomas modulates TGF-β1/Smad signaling pathway to promote brosis and adhesion to ovary [30]. TGF-β1/Smad3 pathway participates in ovarian brosis inhibition and results in ovarian function restoration in primary ovarian insu ciency (POI) rats after human umbilical cord mesenchymal stem cell (hUMSC) transplantation [31]. Whereas, whether PPARγ/TGF-β1/Smad signaling is implicated in ovarian brosis and dysfunction of PCOS is still unclear. Here, we demonstrated that PPARγ was decreased while TGF-β1 and p-Smad2/3 was elevated in ovarian tissues of PCOS rats, Rhamnocitrin could restore abnormal expressions of PPARγ, TGF-β1 and p-Smad2/3. In subsequent study, we demonstrated that PPARγ inhibitor T0070907 antagonized those bene cial effects of Rhamnocitrin on PCOS rats, including ovarian morphology and estrous cycle disorders, body and ovary weights, hormonal status, ovarian brosis and the related brosis factors expressions, whereas PPARγ agonist GW1929 markedly mimiced the functions of Rhamnocitrin. These data further prove that Rhamnocitrin plays its therapeutic e ciency via regulating PPARγ/TGF-β1/Smad2/3 pathway.
In addition to the implication into the development of tissue brosis, PPARγ, TGF-β1 and Smad signaling also participates in other important pathological processes of ovarian dysfunction. Previous research shows that upregulation of PPAR-γ and aromatase Cyp19a1 in ovarian steroidogenic pathway can be a potential cure for PCOS [32]. The decreased bioavailability of TGF-β1 is correlated with an improvement in some abnormal clinical parameters of PCOS [33]. Enhanced Smad2 level by plasmid can reduce apoptosis and improve cell viability of rat ovarian granulosa cells [34]. TGF-β1/Smad3 pathway regulates apoptosis of granulosa cells, through inhibiting ovarian follicle development of PCOS [35]. Therefore, what needs to be emphasized is that other potential mechanisms may also exist in the function of Rhamnocitrin on PCOS, which need to be further investigated in the future study.
In conclusion, we demonstrate here that Rhamnocitrin ameliorates ovarian brosis in PCOS rats through regulation PPARγ/TGF-β1/Smad2/3 pathway, which signi cantly improves PCOS induced dysfunctions. To our best knowledge, this is the rst report to identify Rhamnocitrin, an herbal bioactive avonoid, as potential novel therapeutic option for PCOS pharmaceutical treatment.

Declarations Data Availability
The data obtained in this research are available from the corresponding author on reasonable request.