Effect of hydroalcoholic extract of Berberis integerrima and resveratrol on ovarian morphology and biochemical parameters in Letrozole-induced polycystic ovary syndrome rat model: An experimental study

Abstract Background Resveratrol and Berberis integerrima (B. integerrima) are known to be natural antioxidants and regulators of human metabolism. However, the effects of resveratrol and B. integerrima on the ovarian morphology in polycystic ovary syndrome (PCOS) are not obvious. Objective This study aimed to determine the effect of the hydroalcoholic extract of B. integerrima in combination with resveratrol on some biochemical parameters and ovarian morphology in the letrozole-induced PCOS rat. Materials and Methods Seventy adult female Sprague-Dawley rats aged 10-12 weeks weighing 200 ± 20 gr were randomly divided into seven groups (n = 10/each). Group I): normal; Group II): vehicle; Group III): letrozole-induced PCOS 1 mg/kg letrozole orally, rats receiving 1 cc normal saline orally; Group IV): PCOS + receiving 150 mg/kg metformin orally; Group V): PCOS + receiving 20 mg/kg resveratrol orally; Group VI): PCOS + 3 gr/kg barberry orally; and Group VII): PCOS + receiving 3 gr/kg barberry and 20 mg/kg resveratrol orally. All animals were followed-up for 63 days. The biochemical parameters and histological assessments of ovaries were performed. Results Resveratrol alone and/or in combination with B. integerrima treatment in rats led to a significant decrease in low-density lipoprotein, triglyceride, malondialdehyde, and tumor necrosis factor-alpha concentrations (p = 0.02). The groups IV, V, VI, and VII showed a decrease in insulin resistance and an increase in the superoxide dismutase, total antioxidant capacity, and high-density lipoprotein (p = 0.01). No significant difference was observed between the level of serum glucose in the treatment groups. Number of cystic follicles had a significant decrease in barberry, resveratrol, and their combination groups (p < 0.001). Conclusion Resveratrol, B. integerrima, and their combination as natural products with fewer side effects might be effective as an alternative medicine in treatment of PCOS.


Introduction
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders of fertility in women that affects 6-14% of them during the reproductive years. In the Iranian population, the prevalence of PCOS has been reported to be 7.1%, 11.7%, and 14.6% according to the National Institute of Health, Androgen Excess Society criteria, and the Rotterdam consensus, respectively (1,2). This syndrome is associated with a range of reproductive, endocrine, and metabolic specifications, including obesity, being overweight, hyperandrogenism, anovulation, and infertility that results in hyperinsulinism and type II diabetes.
There are numerous researches about inflammation in chronic diseases such as PCOS, and the inflammatory markers include C-reactive (CRP) protein, interleukin (IL) 6, and tumor necrosis factor alpha (TNF-α). As a result of inflammation, the risk of developing type 2 diabetes, cardiovascular disease, ovarian dysfunction, and hyperandrogenism increases because of the free radicals. Furthermore, oxidative damage is aggravated by the decrease in the body's antioxidant defense mechanisms such as superoxide dismutase (SOD) and catalase (CAT) activities that act as free radicals' scavengers. Total antioxidant capacity (TAC) is sensitive to changes in plasma antioxidant levels and degree of insulin resistance. Therefore, a wide range of oxidative stress biomarkers, including malondialdehyde (MDA), protein carbonyl, TAC, SOD, glutathione peroxidase (GPx), and glutathione (GSH) have been tested in the patients (2)(3)(4).
Due to the low efficacy and safety of the synthetic drugs and the increased risk of insulin resistance and hyperlipidemia, there is a growing tendency toward the consumption of medicinal plants instead of synthetic compounds (5).
B. integerrima is a regular ingredient of Iranian cuisine (6). The high antioxidant capacity of this plant is because of the presence of natural flavonoids and phenolic compounds such as anthocyanins and carotenoids in Berberis which are known to be antidiabetic, anticancer, and antimicrobial natural agents. Research on the cytotoxicity of B. integerrima extract demonstrated no cellular toxicity (7)(8)(9).
Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is commonly found in grapes, peanuts, and some types of berries as a natural polyphenolic compound. It is recognized as a powerful antioxidant with antiplatelet, anticancer, antiinflammation, and antiaging effects. Several studies have demonstrated direct antioxidant effects of resveratrol, and its protective effects may be due to the enhancement of antioxidant capacity in both humans and rodents (10,11). As a result, antioxidants are extremely important because they inactivate the reactive species and increase the protection of biological sites (9).
In this study, the effectiveness of resveratrol, B.
integerrima, and their combination were evaluated in the PCOS rat models for the first time. Moreover, the biochemical parameters, including oxidative stress, inflammatory and lipid profiles, as well as ovarian pathology were determined.

Induction of PCOS
Seventy adult female Sprague-Dawley rats aged 10-12 wk weighing 200 ± 20 gr were used in this experimental study. The rats were kept ad libitum in steel cages with controlled temperature and humidity, 22 ± 2-C and 55 ± 5%, respectively, at 12 hr light/dark cycle. Letrozole orally induced PCOS in rats; this model has histological and biochemical similarities with PCOS in humans. This was done by inducing 1 mg/kg letrozole (Aburaihan Pharma.co., Tehran, Iran)dissolved in 0.9% NaCl solution (Razi Pharma.co., Tehran, Iran) administered orally for 21 days (12). The flowchart of the study is shown in Figure 1.
All animals were weighed on day 1, after PCOS induction (day 21), and at the end of the study (day 63). After 63 days, all study groups were made to fast overnight and were anesthetized via ketamine (Alfasan.co., Holland) (100 mg/kg) and xylazine (Alfasan.co., Holland) (10 mg/kg). Blood samples were collected by cardiocentesis, and their sera were separated to be used for the assessment of biochemical parameters. Afterward, the animals were sacrificed and their ovaries were harvested, cleaned of fat, weighed, and fixed in 10% formalin.

Barberry extract preparation
To prepare the extract, powdered fruit of B.
integerrima (herbarium number 2518) was mixed with 70% ethanol (Razi Pharma.co., Tehran, Iran), stirred for 72 hr, and placed in a percolator. The remainders were placed in rotary devices and dried in a vacuum in color glass until usage.

Antioxidant activity
2, 2-diphenyl-1-picrylhy-drazyl (DPPH) assay is a powerful technique to determine the scavenging potential of antioxidant. DPPH is characterized as a stable radical containing the delocalization electron with deep violet color (15). The odd electron of DPPH absorbed a hydrogen atom from antioxidants and the reduction of violet color took place. The antioxidant capacity was calculated by measuring the reduction in the absorbance at 517 nm. The modified Brand-Williams method has been used in this study (16). Plants extract was diluted with methanol at various concentrations. Quercetin as a positive control was used. A stock solution of DPPH (110 μM) was prepared freshly by dissolving the appropriate amount in methanol. A volume of 20 μl of different concentrations of the sample extract or standard liquid and 180 μl of methanol (with or without DPPH) were mixed in 96-well plate at 25°C temperature for 30 min (17).
The DPPH scavenging effect was calculated as follows: Where A 0 is the absorbance of the control, and A 1 is the absorbance in the presence of

Determination of total phenolic content (TPC) by Folin-Ciocalteu assay
Based on the Folin-Ciocalteu method, 10 μL of Folin-Ciocalteu reagent and 5 μL of the tested sample or gallic acid as standard were added to 96-well plate and diluted with 160 μL distilled water. The contents were then mixed thoroughly for around 10 min. After 10 min of incubation, 30 μL of 25% Na 2 CO 3 was added and the mixture was further incubated for 90 min at room temperature in the dark. Finally, the absorbance was measured at 725 nm (18) and the concentration of the total phenolic was calculated as milligrams of gallic acid equivalents per gram of dry extract.

Biochemical markers
Fasting blood glucose (FBG) was measured by an enzymatic colorimetric method using a biochemical Auto-analyzer (BT-1500, Italy) and a commercial diagnostic kit (Pars Azmoon Co., Iran).
SOD and TAC were determined via the enzymatic colorimetric method with the microplate reader using enzymatic kits (ZV-TAC-A96) purchased from Zellbio Co., Germany. Additionally, MDA activity was determined via spectrophotometric analysis using thiobarbituric acid reactive substances (TBARS) method (19).
The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula (20): Fasting plasma insulin ( ) × fasting plasma glucose (mM)

Ovarian histopathology
Consecutive serial sections with 5-micron thickness from formalin-fix ovary were prepared, and then from each 10 sections one slide and from each ovarian sample 3 sections were stained with hematoxylin and eosin (22). The thin sections were dehydrated, cleared, and eventually mounted within Entellan® (Merck Co., Germany) and cover slipped. The prepared slides were evaluated using a light microscope (Olympus CX31, Japan).

Ethical consideration
The study was conducted in accordance with the recommendations of the European Council Directive (86/609/EEC) on November 24, 1986, regarding the protection of animals used for experimental purposes (http://data.europa.eu/eli/ dir/1986/609/oj). The animal protocols were also approved by the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (Code: IR. SUMS. REC.1394.S1208).

Statistical analysis
The results have been reported as mean ± standard deviation. Additionally, data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test. P < 0.05 was considered to be statistically significant. All analyses were done using the SPSS statistical software, version 22 (SPSS Inc., Chicago, IL, USA).

Biochemical assessment
According to Table I,

Insulin resistance
According to the results presented in Table II,

Histomorphological changes
In this study, a significant decrease in the  Figure   4F).

Determination of TPC and antioxidant activity
The

Discussion
Generally, the animal models are used to gain clinical insight and high degree of evolutionary conservation into human reproductive disorders, especially PCOS (23). PCOS induced by letrozole is histologically and biochemically similar to PCOS in humans that increases testosterone but decreases progesterone and estrogen levels, eventually leading to the appearance of cysts that inhibit the aromatase enzyme (24). Based on Webber and colleagues study in 2003, PCOS could result in an increase in the number of antral follicles, ovarian stroma, hyperplasia theca cells, and ovarian cortical thickness (25). Moreover, it can be concluded that the number of primordial and primary forms of prenatal follicles is increased in PCOS compared with normal ovaries. Indeed, the number of primary follicles (early growing) is increased significantly in both ovulatory and anovulatory PCOS, with a reciprocal decrease in the proportion of primordial follicles compared to normal ovaries. These results are in line with those of the present investigation.
The histomorphological slides in the resveratrol group showed fewer cysts and the presence of corpus luteum in the ovaries, indicating follicle maturation and ovulation. This was in the same line with the results of a previous study by Ergenoglu and co-workers In that study, the treatment of PCOS-induced rats with 10 mg/kg resveratrol led to the development of antral follicles to the normal state (11). These favorable changes indicate that barberry, resveratrol, and their combination may have positive effects on PCOS symptoms. Interestingly, the histomorphological changes in the barberry, resveratrol, and their combination groups were totally comparable to those in the metformintreated group.
In this study, it was found that B. integerrima is a predominant source of antioxidant (DPPH, assay, IC 50 = 33.1 + 1.07 µg/mL). Our data are supported by a few investigations on antioxidant activity and phenolic content amounts of Berberis species (26)(27)(28). High amounts of antioxidant and polyphenol present in Berberis act as free radical scavengers, making it a necessary aid to human health.
Our results revealed a significant increase in body weight among the rats suffering from letrozole-induced PCOS in comparison with the control and vehicle groups, which was consistent with the results obtained by Maharjan (29). On the other hand, our findings confirmed that there is a significant decrease in body weight in the groups treated with barberry, metformin, and resveratrol. These effects are consistent with the previously published data.
Evidence indicate that there is an excessive occurrence of alterations in lipid profiles and lipedema in women with PCOS (30). The current results demonstrated that barberry extract and resveratrol could reduce TC, LDL, and TG levels following an increase in HDL concentrations. Besides, the simultaneous use of barberry and resveratrol provides better results in comparison with metformin in terms of HDL and TG concentration.
Recent studies have shown that Peroxisome proliferation-activated receptor-α (PPAR-α) is predominantly expressed in tissues that have fatty acids metabolizing function such as liver, heart, kidney, and muscles. Activation of PPARα could reduce serum TG and also raise HDL level. Barberry is able to activate PPAR-α and AMP-activated protein kinase (AMPK) which is accompanied by reduction of lipid accumulation in adipocytes (31). Similarly, Derosa and colleagues examined the potential application of Berberine in the regulation of plasma lipids in patients with cardiovascular diseases and found that Berberine present in barberry could decrease TC, LDL, and TG levels following an increase in HDL concentration (32).
Another study found that Berberine might improve the liver function and secretion of bile acids. It seems that the high levels of polyphenolic compounds might inhibit intestinal cholesterol absorption, inactivate 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoA) enzyme, and reduce the intestinal production of chylomicrons in berberine which in turn results in constructive effects of this substance on lipid profiles (33).
The hypocholesterolemia-inducing effect of resveratrol may be explained by its phenolic hydroxyls compounds that result in oxidation of unsaturated fatty acids, as well as a decrease in circulating cholesterol, downregulation of HMG-CoA reductase, and an increase in the expression of the LDL receptors (LDL-R) in hepatocytes. In addition, it is reported that the alternations in medium and long-chain triglyceride concentrations, decreased apolipoprotein C-III, reduced activity of hepatic acyl-CoA cholesterol acyltransferase, and the upregulation of genes involved in lipid metabolism might be linked to the hypotriglyceridemic effect of resveratrol which is in line with our findings (34)(35)(36).
A clinical trial demonstrated that 250-1000 mg daily consumption of resveratrol declined LDL in patients with type II diabetes. In the same way, treatment with resveratrol (150 mg daily) lowered plasma TG in healthy obese men (37).
It was previously reported by Victor and colleagues that changes in the mitochondrial membrane potentially resulted in the activation of kappa B nuclear factor ( a pro-inflammatory transcription factor) that induces reactive oxygen species (ROS) , TNF-α, and endothelial dysfunction in patients with PCOS (38). Although, TNFα could increase the inflammatory factors such as IL-1, IL-6, and inducible nitric oxide synthase, resveratrol might play an essential role in inhibiting the expression of inflammatory factors (39). A number of studies showed that resveratrol could decrease TNF-α concentration and improve SOD and glutathione peroxidase 1 levels, in addition to disabling superoxide and hydrogen peroxide which is in correlation with the findings of the present study (40,41). A number of studies suggested that Berberine exerts its antiinflammatory effects by inhibiting cyclooxygenase-2, which is an enzyme that is responsible for inflammation, as well as nuclear factor kappalight-chain-enhancer of activated B cells. In this way, it directly scavenges superoxide-free radicals in the system, increases the expression of sirtuin 1, and decreases oxidative stress by reducing expression of nicotinamide and adenine dinucleotide phosphate oxidase which are critical sources for ROS production in cells. In line with the present research, the review study performed by Imenshahidi and co-workers showed that barberry and resveratrol might suppress TNF-α, IL-1, nitric oxide (NO), and MDA through the aforementioned mechanism (9,19,42,43). According to this mechanism, resveratrol and barberry increase SOD and TAC levels. As a result, the simultaneous consumption of barberry and resveratrol increased the antioxidant activity and significantly reduced the inflammation in the PCOS group compared with the metformin group at TAC level. In other words, the simultaneous consumption of barberry and resveratrol led to more desirable results possibly due to their synergistic effects.
The high prevalence of abnormal blood sugar levels in PCOS patients indicates the presence of deficiencies in insulin secretion as well as its function. One study found that androgens could cause insulin resistance and change insulin action in the target tissues in PCOS patients, which may eventually increase visceral adiposity and reduce the secretion of adiponectin, which is the major insulin-sensitizing adipokine (44).
According to the studies, resveratrol and barberry might reduce insulin resistance by regulating insulin signaling pathway through increasing protein kinase B (PKB) expression. This is following the increase in the activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and expression of glucose transporter type 4, sirtuin 1, and enhancing glucose uptake in the absence of insulin. In addition, the inhibition of insulin secretion from new island cells, reducing intestinal glucose absorption by inhibiting αglucosidase activity, and stimulating healthy pancreatic beta cells are other results of this pathway. A study has discovered that berberine reduces insulin resistance in metabolic syndrome (45)(46)(47).
The results of this study revealed that the administration of barberry extract and resveratrol in adult female rats with PCOs can lead to significant changes in their insulin resistance.
However, there were no significant changes in the serum glucose levels. This might be a result of low antioxidant dosages and short study duration.

Conclusion
In conclusion, it can be stated that the combination of Resveratrol and B. integerrima (as natural products) can decrease biochemical factors related to the pathogenesis of PCOS. They have the promising antioxidant capacity and anti-inflammatory activities that might ameliorate the complications, and they might be able to regenerate the ovarian morphology to the normal state. Future studies in larger groups, as well as randomized clinical trials on resveratrol and barberry, will help researchers in introducing new treatments for PCOS patients.