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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 10  |  Issue : 1  |  Page : 21-28

Ethanolic extract of Azadirachta indica ameliorates ovarian defects through phosphoinositide-3 kinase inhibition in a rat model of polycystic ovary syndrome


Department of Pharmacology, Institute of Pharmacy, Nirma University, Ahmadabad, Gujarat, 382 481, India

Date of Submission01-Jun-2020
Date of Decision28-Sep-2020
Date of Acceptance30-Oct-2020
Date of Web Publication18-Jan-2021

Correspondence Address:
Snehal S Patel
Department of Pharmacology, Institute of Pharmacy, Nirma University, Ahmadabad, Gujarat, 382 481
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2305-0500.306434

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  Abstract 

Objective: To assess the therapeutic potential of ethanolic extract of Azadirachta (A.) indica in rats with polycystic ovary syndrome (PCOS).
Methods: Thirty-five prepubertal female Sprague Dawley rats were randomly divided into five groups with 7 animals in each group. Group 1 received 0.5% carboxy methyl cellulose orally. Groups 2 to 5 received testosterone propionate (0.2 mg/kg, s.c.) dissolved in olive oil daily for 42 days to induce PCOS. In addition, group 3 was administered with A. indica extract (100 mg/kg, 0.5% carboxy methyl cellulose orally) from the 7th to 12th week, group 4 received quercetin (100 mg/kg, 0.5% carboxy methyl cellulose orally) and group 5 received wartmannin (100 mg/kg, 0.5% carboxy methyl cellulose orally). At the end of treatment, blood was collected for biochemical evaluation. Total follicular count and uterus corpus luteum count followed by PI3K gene expression in the ovary and uterus were evaluated.
Results: The ethanolic extracts of A. indica significantly reduced body weight, ovary weight and uterus weight of rats. Extracts of A. indica also significantly increased the levels of serum glucose, total cholesterol, triglyceride, low-density lipoprotein, very low-density lipoprotein, insulin, testosterone, and luteinizing hormone. Treatment also reduced lipid peroxidation and increased antioxidant parameters in the liver homogenates of PCOS-induced rats. Histological examination of the ovary and uterus confirmed PCOS occurrence and remission state in the PCOS-induced and treated groups, respectively. Moreover, A. indica and quercetin significantly downregulated PI3K gene expression. Histopathological results of the ovary and uterus also proved the protective role of A. indica.
Conclusions: A. indica leaf extract has beneficial effects in the treatment of PCOS by downregulation of PI3K gene expression.

Keywords: Azadirachta indica; PI3 kinase; Quercetin; Steroidogenesis; Testosterone propionate; Wartmannin


How to cite this article:
Patel SV, Maru H, Chavda VK, Shah JN, Patel SS. Ethanolic extract of Azadirachta indica ameliorates ovarian defects through phosphoinositide-3 kinase inhibition in a rat model of polycystic ovary syndrome. Asian Pac J Reprod 2021;10:21-8

How to cite this URL:
Patel SV, Maru H, Chavda VK, Shah JN, Patel SS. Ethanolic extract of Azadirachta indica ameliorates ovarian defects through phosphoinositide-3 kinase inhibition in a rat model of polycystic ovary syndrome. Asian Pac J Reprod [serial online] 2021 [cited 2021 Feb 28];10:21-8. Available from: http://www.apjr.net/text.asp?2021/10/1/21/306434


  1. Introduction Top


Polycystic ovary syndrome (PCOS) is a metabolic and clinically heterogeneous reproductive endocrine disease with a high prevalence in women of childbearing age characterized by polycystic ovaries, hyperandrogenism, anovulation, acne, increase in body weight, irregular menstruation, and oligo/anovulation. It is one of the main causes of infertility in females. The major clinical manifestations include irregular menstruation, amenorrhea, acne, and hursutisum[1]. The most common treatments for PCOS include hormonal contraceptives, progestin, letrozole, clomiphene or gonadotropin, and metformin. All these are short term symptomatic therapies and associated with mild to severe side effects. Taking together all these factors, there is a need for a safe, cost-effective, and long-term management strategy.

Azadirachta (A.) indica, known as neem, is a member of the Meliaceae family and has been widely used in Chinese, Ayurvedic, and Unani system of medicines in the treatment and prevention of various diseases[2]. A. indica contains various constituents including quercetin, β -sitosterol, nimbin, nimbidin, nimbolide, and limonoids and plays an important role in management of various disease like arthritis, diabetes, ulcers, cancer, bacterial and fungal infections through modulation of various pathways[3],[4],[5]. Quercetin is polyphenolic flavonoids abundantly present in fresh leaves of A. indica and is reported to have a beneficial role in treating inflammation, hypertension, mood disorders, obesity, antioxidant and gastrointestinal protective action[6]. While studying natural molecules from plant origin, we previously found that quercetin exhibited a beneficial role in the animal model of PCOS through inhibition of phosphoinositide-3 kinase (PI3K)[7]. It has been reported that PI3K in the ovary controls the androgen synthesis. Inhibition of PI3K can be a promising target in the treatment of PCOS. Hence, this study is to evaluate the therapeutic potential of A. indica rich in quercetin in rat model of testosterone propionate-induced PCOS. Gene expression is also determined to investigate the underlying mechanism.


  2. Material and methods Top


2.1. Materials

Quercetin powder was procured from Otto Chemie Pvt. Ltd, India. Testosterone propionate was procured from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. Diagnostic kits for the estimation of serum glucose, cholesterol, triglyceride, low-density lipoprotein (LDL) cholesterol, very-low-density lipoprotein (VLDL) cholesterol, and high-density lipoprotein (HDL) cholesterol, were acquired from Lab-care Diagnostics Pvt. Ltd. India. Enzyme-linked immunoabsorbent assay (ELISA) kits for the estimation of insulin, testosterone, and luteinizing hormone (LH) were obtained from Krishgen Biosystems, India. Chemicals for gene expression studies like FastRNA® Pro Green kit were obtained from MP Biomedicals. Revert Aid First Strand cDNA Synthesis Kit and polymerase chain reaction (PCR) master mix were procured from Thermo Scientific, India. GeneRulerTM 100 bp DNA Ladder from Fermentas life sciences and primers from Eurofins Genomics were procured. Other chemicals used were of analytical grade.

2.2. Sample collection and extraction

The fresh leaves of A. indica were obtained from the herbal garden of the Institute of Pharmacy Nirma University in the month of March 2015 by picking. Authentication was done by Department of Pharmacognosy, Institute of Pharmacy, Nirma University, Ahmedabad, and voucher specimen was deposited (NIP/ PCOL/2016). Leaves (1 kg) were washed with tap water followed by distilled water to remove dust particles. Leaves were then dried in hot air oven at 60 °C for 24 h. Dry leaves were then grinded to make it in a powder form using grinder. The powder was passed through sieve number 60. The sieved powder (250 g) was extracted with ethanol (500 mL, 36 h) in soxhlet apparatus. Obtained extract was subjected to rotary evaporator, subsequently concentrated under reduced pressure (invaccum at 40°C) evaporated to dryness and stored at 4 °C in air tight bottle.

2.3. Phytochemical analysis of A. indica extract and identification of the active component

Morphological and physicochemical analysis were carried out to identify and confirm the collected leaves. The extract was analyzed by the Mayer's test, cyanidin test, Borntrager test, Libermann Burchard test, foam test and ferric chloride tests for determination of the presence of alkaloids, flavonoids, glycosides, phytosterols, saponins and tannins, respectively. Quantitative estimation of quercetin in the ethanolic extract of A. indica was carried out by using thin layer chrometography method.

2.4. Animals

Thirty-five healthy prepubertal (three weeks old) Sprague Dawley female rats weighing 50-75 g were acquired from Zydus Research Centre, Ahmedabad, India and housed in a pathogen-free environment at the animal house of the Institute of Pharmacy, Nirma University. Animals were housed under well-controlled temperature (22±5) °C, humidity (55±5)%, and 12 h/12 h light-dark cycle with a well-ventilated animal house under a natural photoperiodic condition in polypropylene cages with free access to food and water ad libitum.

2.5. Treatment protocol

These 35 female rats were randomly divided into five groups (n=7 in each group). The PCOS was induced by subcutaneous injection of testosterone propionate (0.2 mg/kg) dissolved in olive oil daily for 6 weeks[8]. Group 1 was the normal control group, receiving 0.5% carboxy methyl cellulose orally. Group 2 was PCOS-induced group administered with testosterone for 6 weeks without treatment. Group 3 was PCOS-induced group and administered with A. indica extract (100 mg/kg, 0.5% carboxy methyl cellulose orally), Group 4 was PCOS-induced group and administered with quercetin (100 mg/kg 0.5% carboxy methyl cellulose orally), Group 5 was PCOS-induced group and administered with standard drug- wartmannin (100 mg/kg, 0.5% carboxy methyl cellulose orally. After 6 weeks of induction, microscopic observation of vaginal smears was carried out to confirm PCOS status of animals. Doses for A. Indica 100 mg/kg was selected based on preliminary study done taking 400 mg/kg as the highest dose, 200 mg/kg as middle dose and 100 mg/kg as the lowest dose[9]. Treatment started on the 7th week. At the end of the 12th week (on day 42 of treatment), the rats were sacrificed for morphological, biological, and histopathological evaluation.

2.6. Morphological and histopathological parameters

Body weight changes in all groups were noted daily till the end of the experiment. After 12 weeks, animals were sacrificed by using high dose of barbiturate and ovary and uterus were removed and weighed. Histopathological evaluation of ovary and uterus was carried out. The ovary and uterus were stored in 10% formalin for fixation. After fixation, the organs were trimmed and embedded in paraffin block after dehydration. Sections were trimmed by using microtome and mounted on the glass slides for staining using hematoxylin and eosin. They were observed with a light microscope at magnification of 40× (Olympus CX23, Gurgaon, India) for total follicular count (follicle, and cystic follicle) and presence of corpus luteum. The adenomyosis as well as thickness of endometrium and myometrium in myometrium was observed in uterine sections.

2.7. Serum biochemical and hormonal parameters

Glucose levels and total lipid profile like total cholesterol, triglyceride, HDL, LDL, and VLDL cholesterol in serum were evaluated by using lab-care diagnostic kits. Serum insulin levels were estimated by using Insulin ELISA kit. Serum LH and testosterone levels were measured by using ELISA kit.

2.8. Oxidative stress parameters

Liver tissues were finely sliced and homogenized in chilled Tris buffer. The homogenate were centrifuged and clear supernant was used for estimation of various antioxidant parameters like reduced glutathione (GSH), superoxide dismutase (SOD), catalase, and nitric oxide (NO) level. GSH was estimated as per prescribed protocol by Patel et al[9]. SOD was estimated as per method described by Weydert and Cullen[10]. Catalase and NO level were estimated by method by Hadwan[11] and Masic et al[12] respectively. MDA formation was determined by the method of Noeman et al[13]. Result of antioxidant activity in liver was expressed in terms of total protein content which was estimated as protocol by Demirkan et al[14].

2.9. Gene expression

The primers for PCR study were designed through Oligo Analyzer 3.1 software and nucleotide tool from NCBI database. RNA was isolated from the ovarian tissue by FastRNA® Pro Green kit. The RNAs of 1.9-2.0 ratio were taken into consideration for cDNA synthesis by Revert Aid First Strand cDNA Synthesis Kit. The forward primer: 5′-CTGCTGTAGGCCGAGTAAG-3′ and reverse primer: 5′-GTGAGACCCCAAGTCCATCG-3′ were used. Further housekeeping gene used was GAPDH. The synthesized cDNA was amplified with initial denaturation, denaturation, annealing, extension, and final extension temperatures 94°C, 95°C, 49°C, 72°C, 72 °C, respectively. Obtained PCR products were analyzed with gel electrophoresis followed by gel doc analysis.

2.10. Statistical analysis

Results were represented as mean±standard deviation (mean±SD). Statistical analysis was performed by using Graph Pad Prism 5 Statistical software. Statistical differences between the means of various groups were evaluated by using one-way analysis of variance followed by Tukey's test and data were considered to be statistically significant at P value <0.05.

2.11. Ethics statement

This study was approved by the Institutional Animal Ethics Committee of the Institute of Pharmacy, Nirma University, Ahmadabad (protocol No. IP/PCOL/MPH/17/007). The study also followed the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment, Forests and Climate Change, Government of India.


  3. Results Top


3.1. Pharmacognostic and phytochemical analysis of leaf of A. indica

The morphological characteristics like colour, odour, and taste confirmed the morphological and physical characteristics of collected leaves. In phytochemical analysis ethanolic extract of A. indica showed the presence of glycoside and flavonoids having highest concentration, while alkaloids and tannins having moderate concentration, saponins and phytosterols having low concentration. The presence of quercetin was confirmed by using thin layer chrometography with a reproducible Rf value of 0.60-0.63.

3.2. Effect of treatments on morphological parameters

Testosterone propionate significantly increased body, ovary, and uterus weight in the PCOS control group as compared with the normal control group. Treatment with A. indica, quercetin and wartmannin significantly reduced body weight as compared with the PCOS control group. In addition, treatment with A. indica and quercetin significantly decreased ovary and uterus weight as compared with the PCOS control group, while wartmannin did not significantly reduce both ovary and uterus weight [Figure 1].
Figure 1: Effect of treatments on body weight (A), ovary weight (B), and uterus weight. Values are expressed as mean±SD; n=7 in each group. # and ##: Significantly different from the normal control group, P<0.05, P<0.01, respectively. *,**, and ***: Significantly different from the PCOS control group, P<0.05, P<0.01, P<0.001, respectively

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3.3. Effect of treatments on histopathological change

In the normal control group, there were no morphological changes in ovaries and uterus, while in the PCOS control animals an increase in follicular count with large follicular cysts and absence of corpus luteum were observed in ovaries. A. indica treatment significantly reduced the number of follicles and there were fewer granulose cells, which were responsible for the cyst formation. Similar result was observed in the quercetin and wartmannin treatment groups [Figure 2]. Similarly, the PCOS control group showed a development of adenomyosis in endometrium as well as myometrium as compared with normal control group. Treatment of A. indica, quercetin, and wartmannin showed a significant reduction in adenomyosis and thickness of endometrium and myometrium [Figure 3].
Figure 2: Effect of treatments on histopathology of rat ovaries. Histological sections are stained with hematoxylin and eosin (magnification: 40×). A: The normal control group shows mature follicle with corpus luteum. B: The PCOS control group shows many follicular cysts with absence of corpus luteum. Treatment with Azadirachta indica (C), quercetin (D) and wartmannin (E) reduce the number of follicular cysts. CL: Corpus luteum; F: Follicles; FC: Cystic follicles. ##: Significantly different from the normal control group, P<0.01; *: Significantly different from the PCOS control group, P<0.05

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Figure 3: Effect of treatments on histopathology of the uterus. Histological sections are stained with hematoxylin and eosin (magnification: 40×). A: The normal control animals show normal architecture; B: The PCOS control group shows adenomyosis of the endometrium and myometrium with increased thickness; C: The Azadirachta indica treatment group shows mild adenomyosis with intact endometrium; D: The quercetin treatment group shows moderate adenomyosis with reduced thickness of the endometrium and myometrium; E: The wartmannin treatment group shows reduced adenomyosis with reduced thickness of the endometrium. MM: Myometrium; EM: Endometrium

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3.4. Effect of treatments on serum glucose and lipid profile

Serum glucose, cholesterol, triglyceride, LDL and VLDL levels were significantly increased in the PCOS control group as compared with the normal control group, while serum HDL level was decreased. Treatment with A. indica and wartmannin significantly reduced serum glucose level, while a slight reduction of serum glucose level was observed in the quercetin treated group. A. indica significantly decreased serum cholesterol, triglyceride, LDL, and VLDL levels, but there was no significant reduction in cholesterol, triglyceride, LDL or VLDL levels in the quercetin and wartmannin treatment groups. And no significant improvement in serum HDL levels was observed in any treatment group [Table 1].
Table 1: Effect of treatments on biochemical parameters.

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3.5. Effect of treatments on hormone levels

The PCOS control group showed a significant increase in serum insulin, LH, and serum testosterone levels as compared with the normal control group (P<0.001). Treatment with A. indica and quercetin significantly reduced serum insulin, LH and serum testosterone levels, while wartmannin did not produce any significant reduction in above hormonal levels as compared with the PCOS control group and other treatment groups [Figure 4].
Figure 4. Effect of treatments on hormone levels after 12th week. A: Serum insulin levels; B: Serum luteinizing hormone (LH) levels; C: Serum testosterone level. Values are expressed as mean±SD; n=7 in each group. ###: Significantly different from the normal control group, P<0.001. * and **: Significantly different from the PCOS control group, P<0.05, P<0.01, respectively.

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3.6. Effect of treatments on oxidative stress parameters and total protein

The total protein levels were normal in all groups. There was a significant reduction in GSH, catalase, SOD, and reduced NO levels in the PCOS control group. Treatment with A. indica and quercetin significantly increased GSH, catalase and SOD levels, and reduced NO levels, while no significant difference was observed between the wartmannin treatment group and the PCOS control group [Table 2].
Table 2: Changes in oxidative stress parameters.

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3.7. Effect of treatment on PI3 kinase inhibition

PCOS control animals showed a significant increase in PI3K mRNA expression in the ovarian theca cells as shown in [Figure 5]A. Treatment with A. indica, quercetin, and wartmannin significantly decreased PI3K mRNA expression as shown in [Figure 5]B.
Figure 5: Effect of treatment on phosphoinositide-3 kinase (PI3K) expression. A: Representative images; B: PI3K mRNA expression. Values are expressed as mean±SD; n=7 in each group. #: Significantly different from the normal control group, P<0.05. *,**,***: Significantly different from the PCOS control group, P<0.05, P<0.01, P<0.001, respectively

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  4. Discussion Top


PCOS is the most common endocrine disease and is more likely to develop obesity, insulin resistance and have a greater risk of hypertension, ovarian cancer, depression, and miscarriage[15],[16]. Several reports show the efficacy of various drugs used for PCOS[17]. In the present study, prepubertal androgen model is used because it produces polycystic ovaries, blocks ovulation and attenuates progesterone production. Moreover, high insulin levels in the testosterone-treated rats can lead to insulin resistance[18]. This model mimics some of the important characteristics of human PCOS.

A. indica has major chemical constituents like azadirachtin, nimbinin, nimbidin, lomonoids, margosa, isomeldenin, quercetin, nimbadiol, nimocinol, etc. Quercetin is one of the major bioactive flavonoids found in the leaves of A. indica and quantitative analysis indicates that the aqueous leaf extract contains 6 to 8 mg% (w/w) quercetin[19],[20]. Our previous studies showed that quercetin has a beneficial effect in treating PCOS by inhibiting PI3K which attributes to a decrease in the expression of CYP17A1 gene, a key player in steroidogenesis. This result led us find out whether A. indica leaf extract rich in quercetin can produce protection against PCOS.

Weight gain is a common feature of PCOS. In the present study, animal weight was increased PCOS rats and treatments with quercetin and A. indica leaf extract significantly reduce the weight gain induced by testosterone propionate. It has been reported that testosterone propionate increases body weight due to hormonal imbalance and insulin resistance in PCOS[21]. Quercetin is reported to reduce insulin resistance[22]. Therefore, quercetin reduces body weight in testosterone-induced animals via reducing insulin resistance.

Prenatal exposure to high androgen doses induces abnormal follicular growth, resulting in cyst formation and consequently the increase in ovary and uterus weight[23]. Earlier reports showed that rats treated with androgens show a significant increase in ovary and uterus weights[24]. In our study, testosterone propionate administration was found to exhibit increases in ovary and uterus weight, and treatment with A. indica, quercetin and wartmannin produced a decrease in ovary and uterus weight, which indicates beneficial effects in PCOS.

Histopathological alterations in the ovaries of prenatal androgenized female rats suggested that androgen affects the ovary development and functions[25]. Further, altered expression of caspase and matrix metalloproteinase, which are markers of apoptosis and necrosis, was reported to increase in atretic follicles in the animal model of PCOS[21]. Active constituents isolated from A. indica were reported to reduce caspase-dependent apoptosis by targeting PI3K/ Akt pathway[26]. Thus, in the present study, histopathological result shows that PCOS rats exhibited an increase in follicular and corpus luteal count in the ovary. Treatment with A. indica extract significantly improved histological changes in the ovarian cortex, which may be through PI3K/Akt signaling pathway. It suggests that A. indica extract could improve the follicular development in PCOS rats.

The leaf of A. indica has been reported to have a significant reducing effect on the blood glucose level in adrenaline-induced hyperglycaemia model[27]. Data of the present study also show a decrease in glucose level by treatment with A. indica leaf extract but quercetin did not produce changes in glucose levels, which indicates that other constituents present in A. indica leaf extract may be responsible for glucose-lowering effect.

Dyslipidaemia is very common in PCOS and characterized by elevated levels of cholesterol triglyceride, LDL and lowered levels of HDL[28]. In the present study, treatment with A. indica lowers the cholesterol and triglyceride level and also shows a decrease in LDL and VLDL, while quercetin did not produce reduction in lipid levels, which indicates that the impact of quercetin supplementation on plasma lipid levels is less as compared with the whole leaf extract.

Ovulation is strongly dependent on the stimulation of LH and its effect on the androgen production in the ovarian theca cells by activation of the PI3K/Akt pathway[29],[30]. Administration of testosterone propionate at gestational age shows a increase in serum testosterone and LH levels which were found consistent with reproductive features of clinical PCOS. Numerous reports have described that PI3K is involved in LH-induced Akt phosphorylation in theca cells which results in an increase in ovarian steroidogenesis[6]. Thus, the inhibition of PI3K leads to a decrease in the secretion of LH and testosterone hormone and this kinase might be potential target for the treatment of PCOS. The data presented here showed that the treatments with A. indica, quercetin, and wartmannin lower the testosterone and LH levels. Thus, protection produced by A. indica leaf extract may be due to inhibition of PI3K.

PCOS is characterized by hyperinsulinemia and insulin resistance. It has also been shown that PI3K inhibitors inhibited impairment of insulin secretion[31]. The present study evaluated the therapeutic effect of A. indica, quercetin, and wartmannin in PCOS induced rats. The present results showed that A. indica and quercetin have equivalent efficacy in reducing hyperinsulinemia in PCOS induced rats. The present findings provided evidence that the efficacy of A. indica and quercetin against PCOS is also through improvement in insulin resistance.

Various reports prove that testosterone stimulates the androgen to produce ovarian steroidogenic enzymes by increasing enzymatic oxidative level; this will cause inflamed ovary and uterus. According to a previous report, ethanol extract of A. indica leaves containing phenolic compounds strongly influences the activity of antioxidant enzymes and thus lead to protective effect[32]. In this study, PCOS induced rats show a high level of lipid peroxidation and decreased levels of antioxidant enzymes and the A. indica treatment group significantly reduced oxidative stress-related parameters. Thus, it seems that the alleviating effect of A. indica leaf extract against oxidative stress is ascribed to its phenolic component like quercetin.

To explore the potential mechanism of the therapeutic effect of A. indica on PCOS, gene expression of PI3K mRNA was carried out. Research studies from recent years have proven the potential relationship of PI3K signaling pathway and polycystic ovary syndrome[33],[34]. PI3K is the direct mediator of insulin-induced thecal steroidogenesis[35]. In the present study, the treatment with A. indica, quercetin, and wartmannin showed an inhibitory effect of PI3K mRNA expression. In a word, A. indica treatment demonstrated the great efficacy in ameliorating PCOS through regulating PI3K signalling pathway followed by metabolic and endocrine effect.

Present study have evaluated beneficial effect of A. indica and quercetin in animal model of androgen excess through up-regulation of PI3K signaling pathway. However, the clinical manifestations of PCOS are complex which limits translational studies using animals.

In conclusion, the ethanolic extract of A. indica and quercetin has the beneficial effect in PCOS, which is evident from the biochemical parameters and hormonal parameters, improvement in morphological and histopathological changes of ovaries. The mechanism of action of A. indica and quercetin is also confirmed by gene expression studies.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Acknowledgments

The authors are thankful to the Institute of Pharmacy, Nirma University for providing the infrastructure and facility for carrying out the research work.

Funding

The authors would like to acknowledge the funding provided by Institute of Pharmacy, Nirma University in the form of postgraduate contingency.

Authors’ contributions

Shraddha Patel contributed in terms of performing the experiment and submitting the data for further analysis. Snehal Patel designed the research work and analyzed the data with inferences. The remaining authors Harsh Maru, Vishal Chavda and Jigar Shah contributed in terms of technical support and manuscript preparation.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
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Abstract
1. Introduction
2. Material and ...
3. Results
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