Materials
Procurement, identification and authentication of plant material
Ripe seeds of Datura stramonium L. (Solanaceae) (Fig. 1), were collected from the plant habitat in Anyigba, Dekina Local Government Area, of Kogi State, North Central Nigeria, in July 2018. The plant sample was identified by Prof. M. Adukwu, of the Herbarium of the Department of Botany, Faculty of Agriculture, Kogi State University, Anyigba, Kogi State, Nigeria. The identity and authenticity of the plant was also confirmed with the exact sample deposited in online databases of http://www.theplantlist.org/ and http://www.ipni.org/.
Preparation and processing
The plant seeds were carefully separated from the whole plant, freed from sand and debris, and air-dried to a constant weight. The dried seeds were pulverized into powdered form, stored in airtight bags prior to aqueous crude extraction.
Study rodents
A total of twenty (20) adult Swiss mice (24-29 g), and Thirty (30) healthy adult Wistar rats of both sexes (180-200 g), were procured from the Animal House of the Faculty of Basic Medical Sciences, College of Health Sciences, Kogi State University, Anyigba, and were be kept in well ventilated laboratory cages. They were acclimatized to the laboratory environment for a period of seven days under standard environmental conditions, with a 12 h light/dark cycle. The animals were fed with standard feed pellets and drinking water ad libitum, and were accorded humane care throughout the period of the experiment, in line with the regulations and ethical approval of the Ethics and Biosafety Committee of the Faculty of Biological Sciences, University of Nigeria, with Reference No. UNN/FBS/EC/1046, and in accordance with the International ethical guidelines for care and use of laboratory animals [42].
Drugs, chemicals and reagents
All chemicals and reagents used for the conduct of this study were of analytical grade. Drugs used were purchased from reputable pharmaceutical outlets in Lokoja, Kogi State, Nigeria. Freshly prepared phosphate buffer at pH 7.4 and normal saline were used for the study. Distilled and deionized water were obtained from the National Centre for Energy Research and Development (NCERD), University of Nigeria, Nsukka. Hydrogen peroxide (Interstate Chemical Co. Hermitage, PA), Bovine serum albumin (BioClot GmbH), Phosphotungstic acid (CDH Fine Chemicals, India). Orthophosphoric acid (Prime Chemicals, Gujarat, India). Ascorbic acid, oxalic acid, batophenanthroline, sodium hydroxide, iron (III) chloride, petroleum ether and ethyl acetate (Sigma Aldrich, St. Louis, MO, U.S.A.). Chloroform, methanol, ethanol, sulfuric acid, and hydrochloric acid (British Drug House, England). Fehling’s solutions A and B (Sisco Research Lab., India). Xylene (Shantou, Guangdong, China). Cyclophosphamide was purchased from Cadila Healthcare Limited, Marketed by Zydus Oncosciences, Baxter Oncology, Frankfurt Germany. Levamisole (47.3 mg) was purchased from Ecomed Pharma Limited, Ogun State Nigeria. Glutathione peroxidase (GPX) activity assay kit was obtained ‘Ready to use’ from Cayman Chemical, Ann Arbor, Michigan, USA. The kits for High-density lipoprotein (HDL) and Low-density lipoprotein (LDL) were products of Prestige diagnostics, Geigorim Co Antrim, United Kingdom. Catalase (CAT) and superoxide dismutase (SOD) assay reagent kits were procured from Randox Laboratories Ltd., United Kingdom. Immunoglobulins A, G, and M ‘Ready to use’ assay kits were procured from Weiner’s Laboratory, California, U.S.A.
Methods
Crude plant extraction
Crude plant extraction was carried out according to the method of Sofowara [43] with slight modifications. A known weight, 257.6 g of the powdered seeds were soaked in 2567 mL liters of distilled water and allowed to stand for 48 h at room temperature. The mixture was then filtered using a vacuum pump, and the filtrate was lyophilized to yield 20.35 g of the aqueous seed extract of D. Stramonium L. (ASEDS). The percentage yield of ASEDS was determined using the formula given below.
Phytochemical analyses
The phytochemical screening of ASEDS was carried out using the methods of Harborne [44], and Pearson [45].
Determination of LD50 of ASEDS
The acute toxicity was conducted in accordance to the method of Lorke [46]. The study was conducted in two phases using a total of twenty (20) male Swiss mice of (24-29 g). In the first phase, 9 mice are divided into three groups of 3 mice each. Groups 1, 2 and 3 animals were orally administered 10, 100 and 1000 mg/kg body weight (b.w.) of ASEDS respectively; while a mouse used as the control was as only administered physiological saline. In the second phase, 1600, 2900 and 5000 mg/kg b.w. of ASEDS were administered orally to the remaining nine mice respectively. A mouse served as the control. The treated mice were observed for 24 h for signs of acute intoxication as well as mortality.
Induction of immunosuppression
Immunosuppression in Wistar rats was induced according to the protocol of Joshua et al. [28]. Accordingly, 10 mg/kg b.w. of Cyclophosphamide was administered orally for 27 days to induce immunosuppression in designated rat groups.
Experimental design
The experimental design of Kyakulaga et al. [47] as modified, was adopted. Thirty (30) rats were divided into six groups of 5 rats each. Group 1 served as normal control and received 1 mL/kg b.w. distilled water throughout the duration of the experiment. Group 2 served as negative control and was pretreated with 10 mg/kg b.w. Cyclophosphamide orally for 27 days followed by 1 mL/kg b.w. distilled water given orally for 28 days’ post treatment. Group 3 served as standard control, and were pretreated with 10 mg/kg b.w. Cyclophosphamide followed by 5 mg/kg b.w. of Levamisole [32]. Groups 4, 5 and 6 made up the ASEDS test groups. Rats in these groups were orally pre-treated with 10 mg/kg b.w. Cyclophosphamide for 27 days, followed by 60, 90, and 120 mg/kg b.w. of ASEDS respectively, administered orally for 28 days. Thereafter, the rats were fasted overnight, and euthanized by cervical dislocation prior to sacrifice on day 29 [48]. Fresh blood samples were collected via cardiac puncture and emptied into neatly labeled plain tubes. Serum was obtained by centrifugation at 3000 rpm for 10 minutes, and stored in the refrigerator for subsequent biochemical analyses.
Determination of biochemical indices
Total leucocytes and differential cell counts were determined using an automated hematology analyzer (URIT-330) based on Coulter’s method as described by Robinson [49] Serum catalase activity was assayed according to the method of Sinha [50]. Superoxide dismutase activity was assayed by the inhibiting of auto-oxidation of epinephrine, using the protocol of Pajovic et al. [51]. Glutathione peroxidase activity was assayed according to the method of Paglia and Valentine [52] as described by Ekpo et al. [48]. The concentration of serum Vitamin A was determined by the method of Rutkowski et al. [53]. Serum vitamin C concentration was determined by the method of Rutkowski et al. [54], while serum vitamin E concentration was determined by the method of Rutkowski et al. [55]. High density lipoprotein concentration in serum was determined according to the method described by Kameswara et al. [56], while Low-density lipoprotein concentration was determined by the method of Arsman et al. [57], using an Auto-analyzer (URIT-810).
Determination of serum immunoglobulin A, G and M concentrations
Serum immunoglobulin A, G, and M concentrations were determined according to the method of Pressac et al. [58]. The method is based on the principle that immunoglobulin reacts with specific antigen to generate insoluble immune complexes. The turbidity of the complexes is directly proportional to the immunoglobulin concentration in the sample which can be measured using a spectrophotometer. Briefly, a calibration curve was drawn from serial dilutions of the calibrator protein in saline solution at 1:10, 1:20, 1:40, 1:80, and 1:160 using saline solution as zero point. 40 µL of diluted calibrator protein was mixed with 900 µL of Reagent A (buffered saline solution). The mixture was homogenized and the absorbance of the dilutions were measured at 340 nm as OD1, after calibrating the instrument to zero with distilled water as blank. An aliquot of 160 µL of Reagent B (antibody monospecific anti-IgA, IgG, or IgM) was then added to the mixture and incubated at room temperature for 30 min. The absorbance of the reacting mixture (OD2) was measured against the blank. The difference in absorbance (OD2 - OD1) for each calibrator protein dilution, including the zero point was calculated. A plot of calibrator protein concentrations in mg/dl against the differences in absorbance was plotted. For the sample, an aliquot of 10 µL was mixed with 100 µL of saline solution to achieve a 1:10 dilution. Thereafter, 40 µL of the diluted sample was mixed with 900 µL of Reagent A. The mixture was homogenized and the absorbance of the dilution was measured at 340 nm as OD1 using distilled water as blank. Thereafter, 160 µL of Reagent B was then added to the mixture and incubated at room temperature for 30 min. The absorbance of the reaction mixture (OD2) was measured against the blank. The differences in absorbance was determined, and the concentration of immunoglobulin A, G, and M respectively, in the serum (mg/dl) was determined from the standard calibration plot.
Statistical analyses
The data obtained from the study were analyzed using IBM Statistical Product and Service Solutions (SPSS) version 21.0 (Chicago, IL), and GraphPad Prism version 7.0. Significant differences in the means were established by the one-way analysis of variance (ANOVA), post hoc multiple comparison, Duncan homogenous subset, and Turkey multiple comparison test. Student’s t-test was used to compare mean body weights in the acute toxicity study. The results were presented as mean ± standard deviation of replicate measurements. Mean values with p ≤ 0.05 were significantly different.