Study setting
This study was carried out in the research laboratory of the Department of Anatomy, Faculty of Basic Medical Sciences, Nnamdi Azikiwe University, College of Health Science, Nnewi Campus, Anambra State and lasted about 5 months as part of a thesis in the Department of Anatomy of Nnamdi Azikiwe University.
Plant collection, identification, and extraction
The aerial parts of the plant PO was obtained from marshy areas at Awka, Anambra State, Nigeria. The botanical identification (ID/155A/P. oleracea) and authentication were done in the herbarium of the Department of Botany, Edo State University, Ekpoma, Edo State. A large amount of the harvested plant was washed free of soil, and roots separated from the aerial part. It was air-dried for 2 weeks then oven-dried at 40 °C. The dried plant was ground giving a yield of about 500 g. The sample was macerated and extracted with 70% methanol (1:2 wt/vol.) for 72 h at room temperature (26–28 °C). The resulting solution was filtered, sieved, and 70% methanol was evaporated at a temperature of 40 °C to give a percentage yield of 10.2% of the starting material.
Sample size determination
The sample size was determined using the “resource equation” method for animal studies which is based on the degree of freedom of analysis of variance (ANOVA). However, this study considered an attrition rate of 10% for each group, thus giving a total sample size of fifteen (15) animals (five animals per group) [18, 19].
Animal source, care, and handling
The animals were procured from the animal house of the Faculty of basic medical sciences, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, Nnewi Nigeria. The Wistar rats were acclimatized for 2 weeks after which the animals were housed in standard cages, five per cage in a controlled temperature room (25 °C), with a 12 h light:12 h dark cycle; lights on at 6:00 a.m. Standard laboratory rat chow and tap water were made available throughout the study period. Fifteen (15) normal cyclic female Wistar rats weighing between 150 and 200 g were used for the experiment. The experimental procedures complied with ARRIVE guidelines [20] and the National Institutes of Health guide for the care and use of laboratory animals (2011). Animal health status was monitored throughout the experiment according to the federation of European Laboratory Animal Science Associations (FELASA) guidelines [21].
Vaginal smear cytology and estrous cycle evaluation
The estrous cycle of the animals was observed by vaginal smear cytology before grouping and was repeated at the end of MEPO administration as only the normal cyclic rats were included in this study. The vagina smear was carried out consistently in the morning between 20:00 and 21:00 h throughout the smear duration. Before vaginal smear, each animal is held in position by grasping each rat on the suspended tail while fixing the animal on a stable flat surface with a mild clampdown of the hind using the dorsum of the hand. Vaginal secretion was collected by inserting a plastic pipette filled with 10 μl of normal saline (NaCl 0.9%) into the tip of the vagina for a suction aspiration of the cells and vaginal mucus. The secretion collected was then placed on the slide and viewed under a light microscope with × 10 objective lenses. The proportion among the three types of cells (the round and nucleated ones—epithelial cells; the irregular anucleated ones—cornified cells; and the little round ones—leukocytes) were used for the determination of the estrous cycle phase of the animals. The smear cytology assessment was done by two individuals to avoid bias. Each animal was examined for two 4-day cycles to establish the regularity of the estrous cycle before inclusion in the study. In the end, 15 normal cyclic female Wistar rats were selected for this study. The cytology protocol followed in this study was as documented by Long and Evans [22], Mandl [23], and McLean et al. [24].
Experimental procedure
Female Wistar rats of proven fertility (normal cyclic) were divided into three (3) groups of five (5) animals each, based on their body weight. Group A served as the control group and they received only distilled water. Doses of 400 mg kg−1 day−1 and 800 mg kg−1 day−1 were administered to groups B and C orally for 14 days. One gram of the methanolic extract of PO was diluted with 20 ml of distilled water to constitute the administration stock. The administration was based on the bodyweight of each rat in each group and remnant of the extract was discarded after each use. All administration was done orally using the oral cannula. The dosage, duration of administration, and experimental design were based on our previous study [17].
Animal sacrifice and blood sample collection
After the last day of administration (14th day), the animals were fasted overnight and sacrificed on the next day by cervical dislocation. Blood samples were collected by orbital puncture into sample collection tubes with the appropriate labels after which the animals were dissected and the ovary and uterine tissues were harvested and weighed. The average weight of the left and right ovaries and uterine horns were recorded for each animal and used to determine the organ weights.
Hormonal analysis
The collected blood was allowed to stand for 15 min in the specimen container under room temperature before centrifugation at 1000 g for 5 min in a refrigerated centrifuge and the serum was extracted. Analysis for follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone, and estradiol were carried out using AccuBind enzyme-linked immunosorbent assay (ELISA) microwells for FSH, LH, progesterone, and estradiol respectively purchased from Monobind Inc. Lake Forest CA USA with the respective product codes—4925-300, 4825-300, 425-300, and 625-300 respectively. AccuBind procedure was used for the assessment of FSH, LH, Estradiol, and progesterone [25]. The laboratory technician was unaware of the treatment allocation.
Histopathological studies
Ovarian and uterine tissues were fixed in Bouin’s fluid immediately after excising them and were passed through ascending grades of alcohol before clearing in xylene and embedding in paraffin. Tissues were sectioned at 5 m and stained with hematoxylin and eosin to give contrasting colors to different elements of the cells or tissue thus making them conspicuous and easy to study. The tissues were viewed under the light microscope and the micrographs were taken using the digital micrography system. The histopathological assessment and procedure used in this study were based on the study by Rowley et al. [26].
Data analysis
The data analysis was done using the IBM statistical package for social science (SPSS) version 23.0. The data was cleaned and tested for normality using the Kolmogorov-Smirnov test. The animal body weights were analyzed using the dependent t test while the relative organ weights and serum hormonal levels were analyzed using the one-way analysis of variance (ANOVA) and post-hoc LSD. Tables were used for result presentation, and values were considered significant at p < 0.05. All the data generated for each animal were used for the analysis and each animal study group was considered as a single experimental unit.
