All chemicals used in this study were of analytical grade. They were products of Sigma Aldrich, Germany.
Fresh fruits of garden egg (Solanum aethiopicum) were obtained from the Agric Farm of the Faculty of Agricultural Sciences of the University of Nigeria, Nsukka and were identified by Mr. Ugwuozor, a taxonomist of Botany Department, University of Nigeria, Nsukka. A voucher specimen was deposited in the herbarium unit of the Department of Botany, University of Nigeria, Nsukka. The plant was chopped into tiny bits, air-dried for 2 weeks and milled with a mechanical grinder. The ground plant (500 g) was macerated in methanol for 24 hrs, filtered with a white cloth and the filtrate concentrated using a rotary evaporator (IKA, Germany) at an optimum temperature of 40–50°C.
Swiss albino mice (22–28 g) and adult Wistar rats (120–200 g) of both sexes obtained from the animal house of the Faculty of Biological Sciences, University of Nigeria, Nsukka were used. They were housed in metal steel cages and acclimatised in the laboratory for seven days before the experiments. They were given free access to water and fed with growers mash (Niger Feeds, Nigeria) bought from the local market. The research was conducted in accordance with the ethical rules and recommendations of the University of Nigeria committee on the care and use of laboratory animals and the revised National Institute of Health Guide for Care and Use of Laboratory Animal (Pub No.85-23, revised 1985).
Fresh whole blood (3 ml) was collected intravenously from healthy human volunteers into heparinised tubes to prevent coagulation.
Acute toxicity study
The acute toxicity test of the garden egg extract was carried out by the method of Lorke with some modifications to define the range of lethal dose and safe dose for the extract. Eighteen Swiss albino mice starved of food for 18 hr but allowed access to water were used for the study. They were grouped into six (6) groups of three mice each and treated intraperitoneally (i.p.) with the plant extract at varied dose levels (100, 1000, 1500, 2000, 3000, and 5000 mg/kg). The animals were then observed for nervousness, dullness, in-coordination and or mortality for 24 h. Based on the results of the preliminary toxicity testing, the doses of the extract for further studies were decided to be 100, 200 and 400 mg/kg bodyweight of the rats.
The phytochemical analysis of the extract was carried out based on procedures outlined by Trease and Evans. The phytochemicals assayed include alkaloid, glycosides, steroid, terpenoids, flavonoids, tannins, resins and saponinis.
Leukocyte mobilization test in rats
The effect of the methanol extract of Solanum aethiopicum on in vivo leukocyte mobilization induced by an inflammatory stimulus was evaluated in albino rats using the method of Rebeiro et al.,. Twenty five (25) adult Wistar rats of either sex (120 g – 200 g) divided into five groups of five rats each were used for the test. Groups 2, 3 and 4 were administered varied doses of the extract (100, 200 and 400 mg/kg), while groups 1 (vehicle control) and 5 (treatment control) received normal saline and indomethacin (50 mg/kg) respectively. Three hours after oral administration of the extracts, normal saline or reference drug, each animal in the respective groups (n=5) received intraperitoneal injection (i. p) of 0.5 ml of 3% w/v agar suspension in normal saline. Four hours later, the animals were sacrificed and the peritoneal cavities washed with 5 ml of a 5% solution of EDTA in phosphate buffered saline (PBS). The peritoneal fluid was recovered and both total and differential leukocyte counts (TLC and DLC) were performed on the perfusates using a manual cell counter after staining with Wright’s stain. The percent inhibition of leukoctye migration was calculated using the formula:
Where T and C represents the leukocyte count of the treated and control groups respectively.
Vascular permeability test in rats
The effect of the extract on acetic acid induced vascular permeability was assessed by a modified method of Whittles. Twenty five (25) adult Wistar rats of either sex (120 g – 200 g) divided into five groups of five rats each were used. The animals were fasted for 10 hours prior to the experiment and were then administered with varied doses of the extract and drug as stated above.
Three hours later, each animal was given a 0.5 ml intravenous injection of 1% Evans blue solution. Vascular permeability was induced thirty minutes afterwards, by (i. p) injection of 1 ml of 0.6% acetic acid. The animals were sacrificed 20 minutes later, and their peritoneum washed with 10 ml of normal saline. The recovered peritoneal fluid was centrifuged and the absorbance of the supernatant measured at 610 nm using a spectrophotometer.
Preparation of erythrocyte suspension
Fresh whole blood (3 ml) collected from healthy volunteers into heparinised tubes was centrifuged at 3000 rpm for 10 min. A volume of normal saline equivalent to that of the supernatant was used to dissolve the red blood pellets. The volume of the dissolved red blood pellets obtained was measured and reconstituted as a 40% v/v suspension with isotonic buffer solution (10 mM sodium phosphate buffer, pH 7.4). The buffer solution contained 0.2 g of NaH2PO4, 1.15 g of Na2HPO4 and 9 g of NaCl in 1 litre of distilled water. The reconstituted red blood cells (resuspended supernatant) were used as such.
Assay of membrane stabilisation
The effects of the garden egg extract on haemolysis of HRBC induced by heat and distilled water was evaluated using the method of Shinde et al. with some modifications.
Heat induced haemolysis
Samples of the extract used were dissolved in isotonic phosphate buffer solution. A set of 5 centrifuge tubes containing respectively, 5 ml graded doses of the extracts (100, 200, 400, 600 and 800 μg/ml) were arranged in quadruplicate sets (4 sets per dose). Two sets of control tubes contained 5 ml of the vehicle and 5 ml of 200 μg/ml of indomethacin respectively. HRBC suspension (0.1 ml) was added to each of the tubes and mixed gently. A pair of the tubes was incubated at 54°C for 20 minutes in a regulated water bath. The other pair was maintained at −10°C in a freezer for 20 minutes. Afterwards, the tubes were centrifuged at 1300 g for 3 min and the haemoglobin content of the supernatant was estimated using Spectronic 21D (Milton Roy) Spectrophotometer at 540 nm. The percent inhibition of haemolysis by the extract was calculated thus:
Where OD1 = absorbance of test sample unheated
OD2 = absorbance of test sample heated
OD3 = absorbance of control sample heated.
Hypotonicity induced haemolysis
Samples of the extract used in this test were dissolved in distilled water (hypotonic solution). The hypotonic solution (5 ml) containing graded doses of the extracts (100, 200, 400, 600 and 800 μg/ml) were put into duplicate pairs (per dose) of the centrifuge tubes. Isotonic solution (5 ml) containing graded doses of the extracts (100 – 800 μg/ml) were also put into duplicate pairs (per dose) of the centrifuge tubes. Control tubes contained 5 ml of the vehicle (distilled water) and 5 ml of 200 μg/ml of indomethacin respectively. Erythrocyte suspension (0.1 ml) was added to each of the tubes and mixed gently. The mixtures were incubated for 1 hr at room temperature (37°C), and afterwards, centrifuged for 3 min at 1300 g. Absorbance (OD) of the haemoglobin content of the supernatant was estimated at 540 nm using Spectronic 21D (Milton Roy) spectrophotometer. The percentage heamolysis was calculated by assuming the heamolysis produced in the presence of distilled water as 100%. The percent inhibition of haemolysis by the extract was calculated thus:
Where OD1 = absorbance of test sample in isotonic solution
OD2 = absorbance of test sample in hypotonic solution
OD3 = absorbance of control sample in hypotonic solution
Data obtained were analyzed using SPSS version 15.0 (SPSS Inc. Chicago, IL. USA). All values are expressed as mean ± SD. Data were analysed by one-way ANOVA and difference between means was assessed by Duncan’s new multiple range. P<0.05 was considered statistically significant.