Toxicological implications of the therapeutic use of Acalypha wilkesiana leaves in traditional medicine

One hundred gram of the powdered leaves was soaked in 400 ml of absolute ethanol (95%) for 72 h. The mixture was occasionally stirred using a magnetic stirrer, to ensure proper mixture of the vessel content. After 72 h, the mixture was filtered using a sintered funnel and the extract (filtrate) concentrated using a rotary evaporator. The concentrate of the extract was weighed and used for the study [10].

One hundred gram of the powdered leaves was soaked in 400 ml of distilled water for 72 h, and treated as described above for ethanol extract [10].

Eighteen adult male experimental rabbits of the New Zealand strain, weighing between 0.9–1.5 kg were used for the study. The rabbits were housed in clean disinfected cages in the animal house of the Department of Biochemistry, University of Benin and maintained on a 12-h light and dark cycle. They were allowed to acclimatize to the new environment for a period of three weeks, with free access to feed (standard pelletized growers feed from UAC- Vital Feed, Jos, Plateau State) and water. The experimental procedures performed on the animals were approved by the Animal Ethics Committee of the Faculty of Life Sciences, University of Benin, Nigeria. The use of rabbits for the study was also according to the Ethical Guidelines Involving Whole Animal Testing of the Animal Ethics Committee, Faculty of Life Sciences, University of Benin. The experimental animals were then randomized into three groups (groups A, B and C) of six rabbits each [11].

Groups A, B and C animals were treated as follows;

Five grams of the concentrated extracts were suspended in distilled water and measured amounts (dose) administered to the experimental animals with respect to their body weight. The extracts (aqueous or ethanol) were administered orally at a dose of 300 mg/kg body weight to the experimental rabbits for a period of twenty-one (21) days [11].

The dose “300 mg/kg body weight” was selected after a preliminary study (though unpublished) was done with graded doses (i.e. 200, 250, 300, 350 and 400 mg/kg body weight) of the plant extracts administered to salt loaded rabbits. The 300 mg/kg body weight dose was found to be more effective.

Prior to administration (Basal/day 0) with the extract, blood samples were collected from the veins located on the dorsal side of the ear lobes of the experimental rabbits, using sterilized hypodermic needles. At days 7, 14 and 21 after administration of the extracts, blood samples were also collected. Blood samples were collected in plane (universal) bottles immersed in ice. Immediately after collection of blood, the tubes were centrifuged at 3500 rpm for 10 min and clear serum obtained which were used for further analysis [11].

Alanine aminotransferase (ALT) assay was done by the method of Reitman and Frankel (1957) [12]. ALT was measured by monitoring the concentration of pyruvate hydrazone formed with 2, 4 – dinitrophenyl hydrazine at 546 nm. Aspartate aminotransferase (AST) assay was also done by the method of Reitman and Frankel (1957) [12]. AST was measured by monitoring the concentration of oxaloacetate formed with 2, 4- dimtrophenyl hydrazine at 546 nm. Gamma Glutamyl Transferase (GGT) was determined by the method of Szasz (1969) [13]. The substrate L-γ-glutamyl-3-carboxy-4-nitroanilide, in the presence of glycylglycine was converted by γ-GT in the sample to 5-amino-2-nitrobenzoate which was measured at 405 nm, to determine the increase in absorbance. Alkaline Phosphatase (ALP) was determined by the method of Rec. GSCC (1972) [14], which measured the intensity of the yellow colour PNP formed from PNPP by ALP action at 405 nm. Lactate Dehydrogenase (LDH) was done by the method of Weissharr et al., (1975) [15]. The reduction of pyruvate to lactate involved the concomitant oxidation of NADH to NAD⁺ which was monitored at 340 nm. Creatinine Kinase was by the method of Bauer (1976) [16]. The creatine that was liberated reacted with diacetyl and α-naphtol to form a colored product which was read at 520 nm. Bilirubin was determined by the method of Jendrassik and Grof, (1938) [17]. The principle involves the reaction of direct (conjugated) bilirubin with diazotized sulphanilic acid in alkaline medium to form a blue colored complex. Total bilirubin was determined in the presence of caffeine, which released albumin bound bilirubin, by the reaction with diazotized sulphanilic acid and absorbance was read at 578 nm.

Data are represented as Mean ± S.E.M (n = 6). Significance of Difference was tested by Student T-Test, ANOVA and Turkey-Kramer test, using the GraphPad Instat Version 3 (GraphPad Software Inc. San Diego, California U.S.A.). Statistical Significance was set at P < 0.05.