Chemicals and reagents
Folin-Ciocalteu reagent was procured from Sisco Research Laboratory (SRL), Mumbai, India. LC/MS grade solvents and analytical standards such as gallic acid and quercetin were procured from Sigma Chemicals Co. (Bangalore, India). All other chemicals employed were of standard analytical grade from Merck India.
Collection of plant materials
Plants such as S. asoca, P. longifolia and T. orientalis were collected from Herb garden of the Centre. The bark of S. robusta was collected from Chhattisgarh. All the materials were authenticated by Plant Systematics and Genetic Resources division of the Centre, and herbarium specimens were deposited in CMPR Herbarium (CMPR 11252, CMPR 11253, CMPR 11251, and CMPR 11255)
Extraction of materials
The plant materials were shade dried and pulverized. Five grams each of the sample was successively extracted with various solvents like n-hexane, chloroform, and methanol by refluxing for 6 h. Crude extracts were prepared separately with methanol, water, and hydroalcohol (ethanol:water 50:50) using reflux extraction method. The process was repeated in triplicates. The final extracts were pooled and concentrated at 40 °C using a Rotary evaporator (Heidolph, Germany) and it was made up to 100 ml with respective solvents in standard flasks. The extracts were kept under refrigeration for various phytochemical analyses.
Estimation of total polyphenolics
The total phenolic content (TPC) was determined using Folin-Ciocalteu reagent [6, 7]. TPC was expressed as gallic acid equivalents (GAE) in mg/g of the sample.
Estimation of total catechins
Catechins and other flavonoids were isolated by overnight shaking at 70 °C in incubator shaker. Twenty grams of powdered materials was macerated with 200 ml of methanol and kept for overnight shaking at 250 rpm. The residue was concentrated to dryness under reduced pressure in rotary evaporator (Heidolph, Germany) and it was redissolved in LC/MS grade methanol.
Estimation of total catechins was carried out using diazotized sulfanilamide as reported previously [8]. The absorbance of the samples and standards was measured at 425 nm using spectrophotometer (Shimadzu, Japan) against reagent blank. The concentration of catechins in the sample extract was estimated from standard curve of catechin.
High-performance thin-layer chromatographic (HPTLC) analysis
HPTLC analysis was performed using CAMAG HPTLC system (Switzerland). Samples were applied using automatic sample applicator, CAMAG ATS-4 on aluminum-backed pre-coated silica gel 60F254 TLC plate (Merck, India). Mobile phase was standardized as toluene, ethyl acetate in the ratio of 8:2. The chromatogram was developed in a saturated Twin Trough chromatographic chamber (Camag, Switzerland). The developed plate was visualized under UV at 254 nm and 366 nm and in visible light after derivatizing with anisaldehyde-sulphuric acid reagent followed by heating at 105 °C for 5 min.
High-performance liquid chromatography (HPLC) analysis
Comparative HPLC profiling was done using Shimadzu High-Performance Liquid Chromatographic system equipped with LC-10ATVP pump, SPD M10AVP Photo Diode Array Detector in combination with CLASS-VP 6.12 SP5 integration software. The mobile phase used for the separation was HPLC grade methanol (A) and 0.1% formic acid in water (B) in a gradient elution, with % B at 0.01–65, 5–50, 8–40, 12–30, 15–20, 18–10, 23–30, 25–40, 28–50, and 30–60. The column used was Merck HIBAR Lichrospher RP 18e (5 μm) with a Phenomenex guard column (4 mm × 2 mm i.d, 5 μm). The samples were injected using a 20-μl loop (Rheodyne Rohnet Park, CA, USA). The flow rate was maintained at 0.8 ml/min. The PDA signal was recorded at 360 nm.
LC/MS characterization of catechin fractions
LC-ESI/MS analysis of catechin fraction was conducted on Agilent 6520 accurate mass Q-TOF LC/MS coupled with Agilent LC 1200 equipped with Extend-C18 column of 1.8 μm, 2.1 × 50 mm. Gradient elution was performed with LC/MS grade acetonitrile (A) and 0.1% acetic acid in methanol (B) at a constant flow rate of 0.7 ml/min, with an increase in the volume of B %: 5–20%, 12–30%, 19–40%, 26–50%, and 30–40%. The MS analysis was performed using ESI in negative mode with the following conditions: drying gas (nitrogen) flow 5 L/min, nebulizer pressure 40 psig, drying gas temperature 325 °C, capillary voltage + 3000 V, fragmentor volt 125 V, and Oct RF Vpp 750 V. The mass fragmentation was performed with varying collision energy 4 V/100 DA with an offset of 8 V.
Acute oral toxicity studies of plant extracts
Two hundred and fifty grams of various samples such as S. asoca bark, S. asoca stem, P. longifolia bark, S. robusta bark, and T. orientalis bark were extracted with water using soxhlet extraction method for 72 h. The final extracts were concentrated to dryness under reduced pressure using rotary evaporator (Heidolph, Germany).
The experiment was conducted on Wistar rats (females) weighing 147 to 204 g and aged 8 to 9 weeks obtained from the Animal House, J.S.S. College of Pharmacy, Ootacamund, Tamil Nadu, India. The rats were distributed into 5 groups with 6 animals in each group. The experimental procedures relating to the animals were authorized by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (approval no. JSSCP/IAEC/OT/Ph.D/Ph.Cology/06/2017-18) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care [9].
The extracts were prepared from a plant material having a high safety margin, and hence it was decided to use 2000 mg/kg (limit test) for this study. The test item was prepared immediately prior to administration on respective treatment days. A quantity of 2 g of the test item was dissolved in distilled water and the volume was made up to 10 ml to get a test item concentration of 200 mg/ml. Homogeneity of the test item in the vehicle was maintained during treatment by constant stirring and mixing. The test substance was administered soon after preparation.
The prepared test item solutions were administered once orally as gavage to the fasted (16-18 h) rats at the dose volume of 10 ml/kg b.wt. to deliver a dose of 2000 mg/kg b.wt. Food was offered about 3–4 h after dosing. Water was not withheld.
The treated rats were observed five times during day 1 (day of administration) i.e., at 30 min and four times hourly (post-administration) intervals and once daily, and thereafter for a total of 14 days. The clinical signs were recorded on all working days. The body weights of rats were recorded on test day 1 (pre-administration), day 8 (7 days post-administration), and day 15 (14 days post-administration). The rats were euthanized by using diethyl ether anesthesia and necropsied.
Evaluation of estrogenic activity
The samples, Saraca asoca bark (AB), Saraca asoca stem (AS), Polyalthia longifolia bark (PB), Shorea robusta bark (SB), and Trema orientalis bark (TB), were studied for their estrogenic activity in ovariectomized Wistar rats [10].
The experiment was conducted on 72 numbers of Wistar rats (females) weighing 64 to 99 g and aged 4 weeks obtained from the Animal House, J.S.S. College of Pharmacy, Ootacamund, Tamil Nadu, India . The rats were distributed into 5 groups with 6 animals in each group. The experimental procedures relating to the animals were authorized by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (JSSCP/IAEC/OT/Ph.D/Ph.Cology/05/2017-18) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care.
Animals were divided into 12 groups of 6 each.
- I.
Groups 1 and 2 served as sham and control, respectively, and received distilled water (10 ml/kg., p.o.).
- II.
Groups 3 and 4 received AB extract at a dose of 200 and 400 mg/kg, p.o., respectively.
- III.
Groups 5 and 6 received AS extract at a dose of 200 and 400 mg/kg., p.o., respectively.
- IV.
Groups 7 and 8 received PB extract at a dose of 200 and 400 mg/kg., p.o., respectively.
- V.
Groups 9 and 10 received SB extract at a dose of 200 and 400 mg/kg., p.o., respectively.
- VI.
Groups 11 and 12 received TB extract at a dose of 200 and 400 mg/kg., p.o., respectively.
Bilateral ovariectomy was performed in all the groups, except sham group via dorsolateral approach under general anesthesia. In sham group animals, only placebo surgery was performed. After 1 week of recovery, animals were given respective treatments for a period of 8 weeks. The estrogenic activity of the test items was assessed by analyzing the estrus cycle, uterine weight, uterine glycogen content, serum lipid profile, and histopathology of the uterus.
Estimation of uterine glycogen content
Uterine tissue (15 mg) was homogenized in 20 ml 30% KOH, and heated at 100 °C for 30 min, to inactivate enzymes and destroy free glucose. To isolate glycogen, samples was diluted with 1.2 vol 95% ethanol, frozen at − 80 °C for 60 min, then thawed and centrifuged at 9600×g for 10 min. The supernatant was discarded and the pellets dried overnight. To breakdown glycogen to glucose, 100 μl 1.0 N HCl was added to each tube and heated at 90–100 °C for 2.5 h. Glucose concentrations was measured by using Glucose Assay Kit (120235, M/s. Erba Mannheim, Transasia Biomedicals Ltd. Solan, HP, India). The percentage of glycogen is calculated using the standard formula.
Estimation of lipid profile
The blood sample collected was allowed to clot for 45 min at room temperature. The serum was separated by centrifugation at 3000 rpm at 30 °C for 15 min and used for assaying total cholesterol, triglycerides, HDL, and total protein using Precision Biomed assay kits.
Histopathological studies
The uterus tissue was fixed in the 10% formalin, dehydrated in gradual ethanol (50–100%), cleared in xylene, and embedded in paraffin wax. The sections, with 5–6 μm thickness, were then prepared using rotary microtome and stained with hematoxylin and eosin dye for microscopic observation of histopathological changes in the uterus.
Statistical analysis
For determination of significant intergroup differences of each parameter, one-way analysis of variance (ANOVA) was carried out. Dunnett’s test was used for individual comparisons after significant ANOVA results. The differences with p < 0.05 were considered statistically significant. Graphpad Prism-6 software (Graphpad Software Inc., USA) was used for the statistical analysis.