Chemical and Drug Preparation
Aluminum chloride (CAS Number: 7446-70-0) was obtained from Sigma-Aldrich. Aluminum chloride stock solution was prepared by dissolving 1g of Aluminum chloride in 10mls of distilled water. Capsules of vitamin E (Gujarat liquid pharmacaps Pvt) were obtained from a reputable drug store in Zaria, Kaduna, Nigeria. The stock solution was prepared using Tween 80, containing 60 mg of vitamin E in 0.2 ml of the suspension. The stock solution containing vitamin E was then shielded from direct light to avoid photodegradation.
Plant Material Acquisition and Extraction
Tamarindus indica leaves were collected from the Botanical garden of Ahmadu Bello University, Zaria. The leaves were authenticated in the Herbarium unit of the Department of Botany and assigned a verification number of 2417. The extraction of the leaves was carried out by maceration, followed by subsequent fractionation (16).
Phytochemical Screening
Phytochemical screening of the crude extract and the fractions was carried out using standard methods (17), to reveal the presence of chemical constituents. Gas chromatography-mass spectrometry (GC-MS) analysis on the chemical composition of ethyl acetate leaf fraction of Tamarindusindica was done using GCMS-QP2010 PLUS SHIMADZU, Japan) (see additional file 1).
Experimental Animals
Apparently healthy (20) adult non-pregnant females and 10 adult male Wistar rats were acclimatized for two weeks in the Animal house of Human Anatomy Department, Ahmadu Bello University, after which, the vaginal smear was taken from all the female rats and examined under a light microscope for the staging of their estrous cycle. The female rats in the proestrus phase were caged overnight with the mature male in the ratio of 2:1 (female: male); the presence of vaginal plugs the following morning indicated mating and assumed to be day zero of pregnancy (18–20). The dams and their pups were allowed free access to feed and water before and during the experiment, under similar ventilated and spacious housing condition. A total of 40 male pups were used in the study.
Dosage Determination
A dosage of 200 mg/kg bw was adopted for AlCl3 based on previous studies in Wistar rats (21,22). The adopted dosage for Ethyl acetate leaf fraction of Tamarindus indica (EATI) were 400 and 800 mg/kg bw (low and high dose respectively). A dosage of 300 mg/kg bw was adopted for vitamin E based on previous studies using the Wistar rat model (23).
Experimental Design
Gestational rats on day 7 day (day 0 of the experiment) were administered the extracts for two weeks i.e. day 21 of gestation. To reduce biases, animals were assigned random numbers and independently assigned to groups (n=4) in line with the ARRIVE guidelines on experimental animals.
Group 1: 2 ml/kg bw of distilled water, p.o (negative control)
Group 2: 200mg/kg bw of AlCl3, p.o (positive control).
Group 3: 200mg/kg bw of AlCl3 and 400 mg/kg bw EATI, p.o
Group 4: 200mg/kg bw of AlCl3 and 800 mg/kg bw EATI , p.o
Group 5: 200mg/kg bw of AlCl3 and 300 mg/kg bw of Vitamin E, p.o (comparative control).
Neonatal rats on post gestation days 7 and 21 were euthanized using thiopental sodium since was ethically acceptable in experimental animals (24,25), by intraperitoneal injection of 5mg/kg thiopental sodium. The skull was dissected and the brain tissues were harvested from neonates for quantification of brain total Cu, Zn, Fe, and Ca.
Sample Preparation
Two grams of the harvested brain tissue were weighed and transferred to a beaker, then 10 ml of solvent (NHO3+HCl) was added. The mixture was then heated for 45 minutes to 1 hour at 100 0C, to allow complete digestion. The digested mixture was then allowed to cool for 15-20 minutes. The mixture was then filtered and readied for brain metal quantification using Atomic Absorption Spectrophotometer (AAS) (Perkin-Elmer Corp., Norwalk, Conn. 06856) and the concentrations were recorded in ppm.
Statistical analysis
Data obtained were analyzed using the GRAPH PAD prism. The results were expressed as mean ± SEM; differences in means were compared using one-way analysis of variance (ANOVA) and considered significantly different at p ≤ 0.05.