2.1. Preparation of AR extract
Black cohosh roots and rhizomes were obtained from medicinal plant store (Haraz) in Cairo, Egypt and were authenticated at the herbarium of Botany Department, Faculty of Science, Cairo University, Giza, Egypt, using American Herbal Pharmacopoeia (38). Plant sample (0.5 kg) was initially washed with running tap water followed by deionized distilled water, sun dried and grinded, then was extracted by 70% ethanol (2 × 1.5 L) at 25°C during 48 h. Solvents were evaporated under vacuum, in a evaporator (Büchi, Switzerland), at 45–55 °C and 60 rpm (39). The obtained viscous extract was dried at 37°C for 24 h, then weighed and percentage yields were calculated (7.6 % w/w). The extract was stored at −20°C until use, where it was freshly dissolved in 0.5% carboxy methyl cellulose (CMC) for the preparation of doses.
2.2. UPLC-MS fingerprinting of the AR extract
The AR extract was dissolved in acetonitrile (2 mg/ml) and centrifuged at 13,000 g for 15 min at 4 °C. The supernatant was filtered through a 0.2 μm PTFE filter while the first 2 ml were discarded. The metabolomic analysis was performed in ACQUITY UPLC (Waters, USA) coupled with SYNAPT G2-S high definition mass spectrometry (Waters Corp, Manchester, England). The sample injection volume was 5 µL, and the flow rate was set at 400 µL/min. Chromatographic separation was achieved using ACQUITY UPLC HSS T3 Column (1.8 μm, 2.1 X 150 mm, Waters Corporation, Milford, USA). The column temperature was kept at 45 °C and gradient elution was implemented utilizing water with 0.1% formic acid (A), and acetonitrile with 0.1 % formic acid (B). Initially, 1 % of the mobile phase B was used for 2 mins, and linearly inclined as the following gradient: 35-60 % B (2-4 min), 60-80 % B (4-8 min), and 99 % B (8-8.5 min) and finally declined to 10 % B till 11.5 min.
G2-S high definition mass spectrometer (HDMS) (Waters Corp, Manchester, England) equipped with Z-spray source controlled by MassLynx v4.1 was used for mass spectrometry analysis in both positive and negative ESI ionization modes using HDMS mode of operation. The scanning mode parameters were: source temperature; 120 °C, desolvation temperature; 500 °C, cone gas flow; 50 L/h, desolvation gas flow; 1000 L/h, collision energy ramp; 20-50 eV, capillary voltage; 2.5 kV, and acquisition mass range; 50-1200 m/z (40). Data acquired in a profile mode and corrected with separate lock mass spray switching between the injected samples and external reference permitting the MassLynx to continuously ensure mass analysis accuracy. Leucine enkephaline (1 ng/μL) was used as an external reference in 1:1 acetonitrile-water containing 0.1 % formic acid at a flow rate 5 ul/min via a lock-Spray interface, generating a reference ion for positive ion mode [M-H]+ and negative ion mode [M-H]− of 556.2771 and 554.261 m/z, respectively (41).
Three technical replicates were implemented in a randomized batch sequence. To enable proper column equilibration and conditioning, the mobile phase was run for 1.5 h, followed by six quality control (QC) samples before each batch analysis. In line with the published guidelines and to overcome the UPLC-MS analytical drifts, QC samples were injected at regular intervals during the experimental sequence (42). Features were considered reproducible if their coefficient of variation (CV) among the sample replicates were < 25%, and the fold change (FC) > 2, ANOVA p-value and Q value < 0.01 against blank samples Q value is an adjusted p-value, calculated using an optimized False Discovery Rate (FDR) approach.
Progenesis QI software (Waters Corp., USA) was used for data processing and putative identification of metabolites of interest by comparison with literature and available databases such as metabolomic profiling CCS library (Waters Corp., USA), LipidBlast and Progenesis MetaScope imported databases including HMDB, MONA, and GNPS and Chemspider imported data sources such as NIST and PlantCys.
2.3. In vivo experiments
The experimental protocol was approved by the Institutional Animal Care and Use Committee of Cairo University (CU-IACUC; VetCU11112018017).
Virgin, cyclic, adult female Wistar Albino rats (160–200 g) were used in this study. Animals were acquired from Laboratory Animal Colony, Helwan, Egypt and housed in the animal house of Faculty of Veterinary Medicine, Cairo University, Egypt, and allowed to acclimatize for two weeks. During the study, all animals were caged in standard polypropylene cages and maintained in a controlled environment of (22 ± 3) °C temperature, (55 ± 5) % humidity and a 12 h light/ dark cycle. The rats were provided with a standard diet and water ad libitum.
2.4. Experimental protocol
Forty-eight female rats were randomly allocated into six groups of eight each. Animals of group one served as negative control and received a daily oral dose of (1 ml) of vehicle (0.5% CMC) using gavage for 49 days. The induction of PCOS was done guided by an established rat model that previously described (43). Animals of groups two to six received letrozole (LTZ) (Natco Pharma Limited Hyderabad) at a dose of 1 mg/kg dissolved in 0.5% CMC once daily for 21 days for induction of PCOS. Different samples (vehicle, standard and extracts) were then received orally for 28 days. Group 2 (positive control) received only the vehicle and Group 3 received clomiphene citrate in 0.5% of CMC at a dosage of 1 mg/kg (Fertyl-Super® tablets were obtained from Ar-Ex Laboratories Private Limited, Goregaon (E), Mumbai) as a standard ovulation induction drug (44). Groups (4-6) were treated for 28 days with AR extract; 7.14 mg/kg, vitamin C; 500 mg/kg and AR extract with vitamin C, respectively. From day 6 of treatment, daily vaginal smears of all rats were obtained for ovulation monitoring, where PCOS was suggested by an indiscriminate estrous cycle with prolonged diestrus phase (45, 46). On day 50 of the study, all the rats were anesthetized with ketamine 91 mg/kg, i.p. Duplicate blood samples were collected in sodium heparin tubes for separation of plasma and in gel separator tubes for collection of serum samples. The serum was separated by blood centrifugation at 3000 g at 4º C for 10 min and used for different biochemical assays. The animals were then sacrificed, ovaries and uteruses excised, cleaned of fat, weighed and divided into triplicates; two sets; stored at -80º C to be used for real-time reverse transcriptase-PCR and antioxidant assays. Other set of the ovaries in addition to liver tissue were fixed in 10% neutral buffered formalin for histopathological examination. The relative weight of the ovaries was calculated as the ratio of the ovary (wet weight in mg) to the body weight (g) for each animal at the day of scarification.
2.5. Hormonal profile
Serum total testosterone was measured using a commercial ELISA kit (chemux bioscience Inc, USA) and the beta subunit chain of luteinizing hormone (LH) level was measured using rat lutropin subunit beta ELISA kit (EIAab, China) following the instructions of the manufacturers.
2.6. Malondialdehyde (MDA) and antioxidants biomarkers determinations
Ovarian and liver tissues were separately homogenized in 10 ml cold buffer per gram tissue using a glass homogenizer then tissue homogenate was centrifuged at 5000 rpm for 15 min at +4 °C. This buffer consists of 50 mM potassium phosphate with a pH 7.5 for MDA and 50 mM potassium phosphate with a pH 7.5 and 1 mM ethylenediaminetetraacetic acid (EDTA) for reduced glutathione (GSH). The supernatant was used to measure GSH and MDA concentrations according to the standard protocols (47, 48).
2.7. Biochemical parameters
Serum glucose level, lipid profile (cholesterol, triglycerides, and high-density lipoproteins (HDL)) and liver enzymes (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) were measured using commercial kits (spectrum, Egypt). The very low-density lipoprotein (VLDL) and Low-density lipoprotein (LDL) concentration was calculated as following:
(VLDL) = triglycerides/5
(LDL) = total cholesterol – (HDL + VLDL)
2.8. Histopathological examination
Ovaries and liver from different groups were collected and fixed in 10% neutral-buffered formalin and processed to obtain 3-4 µm paraffin-embedded sections. The sections were stained with hematoxylin and eosin (H&E) and the morphometric analysis of the ovaries was performed (49, 50). The number of follicular cysts, its average and diameter alongside with the thickness of granulosa cell layer and the thickness of theca cell layers were measured.
2.9. Immunohistochemical analysis
The tissue sections were deparaffinized, rehydrated and pre-treated with 10 mM citrate buffer for antigenic retrieval. Sections were incubated overnight at 4° C in a humidified chamber with the following primary antibody: rabbit monoclonal anti-Ki 67 antibody (Dako, Denmark) in 1:25 dilution. The tissue sections were incubated with a biotinylated goat anti-rabbit and mouse antibody (Thermo Scientific, USA), streptavidin peroxidase (Thermo scientific, USA) and 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma). The slides were counterstained with Mayer’s hematoxylin then dehydrated and mounted. The primary antibody was replaced by PBS for negative controls. The stained sections were analyzed by Leica Qwin 500 Image Analyzer (Leica, Cambridge, England). In each field, the immunolabeled dark brown areas were recorded. The percentage of positively stained area in the granulosa cell layer, theca cell layers and interstitial stromal cell layer was calculated (51).
2.10. Quantitative real-time PCR for aromatase (Cyp19α1) gene
Total RNA was extracted from female rat ovarian tissue samples using RNeasy mini kit (Qiagen, Hilden, Germany). The quantity and purity of the total RNA was measured by the NanoDrop spectrophotometer. The cDNA synthesis was carried out using reverse transcriptase (Invitrogen, California, USA) and oligo-dT following the manufacturer protocol. The cDNA samples were then submitted to qPCR using the following forward (5’-CTGCTGATCATGGGCCTCCT-3’) and reverse (5′-CTCCACAGGCTCGGGTTGTT-3′) primer pairs for Cyp19α1 gene. The cDNA was amplified by 40 cycles of denaturation at 95° C for 45 s, annealing at 59° C for 45 s and extension at 72° C for 45 s. The 95° C step was extended to 4 min during the first cycle. The amplicon size was confirmed by 2% agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Invitrogen). The β-actin gene was furtherly amplified in the same reaction to serve as the reference gene (52). Each measurement was repeated three times, and the values were used to calculate the gene/β-actin ratio, with a value of 1.0 used as the control (calibrator) and the normalized expression ratio was calculated (53).
2.11. Statistical analysis
The different analytical measurements in the biological samples were carried out in triplicate and results are expressed as the mean ± SD, a, b, c, d superscripts indicate statistical significance compared to the negative control group at P < 0.05 (n=5). Data for multiple variable comparisons were analyzed by one-way analysis of variance (ANOVA) test to analyze the significant differences (P < 0.05) between groups using SPSS version 24 package for Windows.