Collection, preparation of plant materials and crude extract
Waltheria indica Linn. plants were obtained from Ibadan, Oyo State, Nigeria, and the voucher number (UIH-22371) for the plant was obtained from a university herbarium. The details of the collection and preparation of crude extract of Waltheria indica Linn. root was reported by [6].
Sequential extraction of crude ethanol extract of Waltheria indica Linn. root
The crude ethanol extract of Waltheria indica Linn. root (100 g) was sequentially extracted with hexane, dichloromethane, and ethyl acetate in order of increasing polarity.
Column chromatographic separation of dichloromethane (DCM) fraction
The glass column was packed with silica gel using n-hexane. The Waltheria indica Linn. root extract absorbed with silica gel was packed into the column layer, de-aerated, and then allowed to settle. Filter paper discs were used to separate the layers [7].
The mobile phase consisted of 3 solvents: hexane (nonpolar), ethyl acetate (mid-polar), and methanol (polar). The solvents were mixed in various proportions to achieve a “gentle gradient” in terms of separation. The various proportions of solvents were pushed through the bed. The fractions obtained were pooled together using thin-layer chromatography (TLC).
Separation of column fractions by TLC
A line of about 1.5 cm from the bottom of the silica-coated plate was drawn. The samples obtained from the column chromatographic separations were spotted (using a capillary tube filled with the fraction) on this line, equidistant from each other. The plates were placed in a TLC chamber saturated with ethyl acetate: methanol as mobile phase. The plate was thereafter removed from the chamber when the solvent had risen towards the end of the plate. The position of the solvent front was marked.
The TLC plate was thereafter examined under ultraviolet light. Fractions with similar retardation factor (Rf) were pooled together.
Experimental animals and dosing protocol
Ninety healthy adult male Wistar rats were used in this experiment. The rats were obtained from the experimental animal house of the Faculty of Veterinary Medicine, Nigeria. The rats were fed with pelletized grower poultry feeds (Vital feeds®, Grand cereals Limited, Jos, Nigeria) and water was provided ad libitum. All experimental protocols were carried out according to internationally approved principles for the handling of experimental animal, use, and care. The ethical approval was obtained from University of Ibadan Animal Care and Use Research Ethics Committee with reference number UI-ACUREC/APP/2016/002. Thirty rats were randomly divided into ten groups (n = 5). Three doses (200, 500, and 1000 μg/Kg BW) each of the solvent fractions (hexane, dichloromethane, and ethyl acetate) were administered to each group, while the control was administered with distilled water. Thereafter, the chromatographic fractions (DF1–DF7) were also administered to rats (n = 5) at 1000 μg/Kg BW, while the control (n = 5) was given distilled water. The administration was done using oral gavage for 15 consecutive days. The dosage was chosen based on the LD50 of Waltheria indica Linn. root [5] and the trypanocidal dose of Waltheria indica Linn. root [8].
Determination of sperm parameters and serum levels of testosterone, follicle stimulating hormone, luteinizing hormone, and prolactin
Sperm collection
Rats were anesthetized with combination of xylazine (10 mg/kg) and ketamine (90 mg/kg). The rats were thereafter sacrificed by cervical dislocation and the epididymis excised. Sperms were squeezed from the caudal epididymis of anesthetized adult male Wistar rats.
Sperm parameters
Epididymal sperm concentration, motility, and live–dead ratio were determined according to methods of [8,9,10], respectively, while the sperm morphology was determined as described by [11]. The detailed procedure was reported by [5].
Hormonal assay
Serum levels of testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin were assayed using ELISA kits (Calbiotech Inc., USA) following the kit manual.
Preparation of the testicular homogenates and assay of testicular protein and cholesterol
The testes were homogenized in cold 0.25 M sucrose solution (1:5 w/v) using a tissue homogenizer (Bio-Gen PRO200®, Oxford, USA). The homogenates were spun at 5000 rpm for 30 min using a centrifuge (Axiom Medical Ltd, UK). The supernatants obtained were then used for the determination of the protein and cholesterol using Randox assay kits (Randox Diagnostics, Crumlin, UK).
Histopathology of the testis
Briefly, fixed testes in 10% buffered formaldehyde were dehydrated through ascending concentrations of ethanol (70, 90, and 95%). They were cleared in xylene, impregnated, and embedded in molten paraffin wax (melting point 56 °C). The embedded tissue in paraffin wax was sectioned using a semi-automatic microtome (Kedee®, China) at a preset thickness of 4 μm. The satisfactory sections were picked up with microscope glass slides that had been coated on one side with glycerin egg albumin (to prevent detachment from slides during staining procedure). The slides carrying the sections were then labeled with a diamond pencil. They were then arranged in a slide carrier and then put in an oven (DHG-9023A oven, Lab science, England) to dry. After staining with hematoxylin and eosin (H&E), the slides were passed through ascending concentration of alcohol (20–100%) for dehydration and then cleaned with xylene. A thin glass-covered slip was placed on the covering–mounting medium, and underlying tissue sections were allowed to dry. The slides were then examined under the microscope (Olympus®, Germany) for histopathological changes and photographed with camera (AmScope®, Japan) mounted on the microscope [12].
Statistical analysis
The data obtained were expressed as mean ± standard deviation (Mean ± SD). The data were subjected to one-way analysis of variance (ANOVA), and differences between the control and treatment groups were determined by Dunnett’s multiple comparison test using GraphPad Prism® (Version 5.0, San Diego, CA). P values ≤ 5% were regarded as significant.
