Toxicological evaluation of methanolic extract and alkaloidal fraction of crotalaria retusa linn
The study was designed to investigate the toxic potentials of methanolic extract and alkaloidal fraction of leaf and stem of Crotalaria retusa. This was with a view to understanding its mechanism of toxicity to animals and plants. Fresh leaves and stems of C. retusa were collected, cut into bits, washed, drained and air dried in the laboratory over three (3) weeks and pulverized into powder. Methanolic extract (ME) of powdered material was prepared by soaking 350 g in 800 ml of 70% (v/v) methanol for 48 h, filtered and concentrated to dryness at 35oC on a rotary evaporator. Crude alkaloidal fraction was prepared from ME by a procedure that consisted of acid dissolution with 10% (v/v) hydrochloric acid, basification to pH 9 with 1M sodium hydroxide solution and extraction with chloroform. Rat diets containing 0-5% (w/w) of ME were prepared and administered to fifteen (15) albino rats randomly divided into three (3) groups of five (5) rats each over a period of eighteen (18) days. Brown bean (Phaseolus vulgaris) seeds were grown with varying concentrations (0-100 µg/ml) of crude alkaloid fraction with strychnine (10 µg/ml) as reference alkaloid. Leaves and stems were collected from beans seedlings for biochemical analyses. On day 19, rats were sacrificed, dissected and blood was collected by cardiac puncture into an anticoagulant (3.8% w/v trisodium citrate) for plasma preparation and livers were collected asceptically for preparation of tissue homogenates. Plasma and liver homogenates were used for biochemical analyses such as assay of enzyme activities and evaluation of concentrations of metabolites. Analyses of bean seedlings involved determination of percentage germination and evaluation of biochemical parameters in the leaves and stems. Phytochemical screening of Crotalaria retusa methanolic extract revealed the presence of alkaloids, flavonoids, tannins, cardiac glycosides, triterpenoids, steroids and saponins. Administration of C. retusa methanolic extract supplimented-diets for 18 days caused increase in the activities of plasma L-aspartate and L-alanine aminotransferases, acid and alkaline phosphatases activities while there were decrease in the activities of the enzymes in the liver homogenates of the treated animals. Moreover, activities of plasma α-mannosidase activities decreased (p<0.05) significantly, concentrations of creatinine, bilirubin, urea, albumin and sugar also increased significantly in the treated albino rats. Also percentage germination of bean seeds reduced with increasing concentrations of alkaloid fraction. The activities of antioxidant enzymes (superoxide dismutase and catalase) increased with alkaloid concentration in the leaves and stems of bean seedlings. The levels of metabolites (proline, reduced glutathione and ascorbic acid) increased significantly (p<0.05) in the stems and leaves of treated bean seedlings. However, there was reduction in the total protein and sugar contents of the leaves and stems of bean seedlings which implied stress. The results of this study revealed that the mechanisms of action of both the extract and its alkaloid fraction involved the induction of oxidative stress that resulted in the generation of reactive oxygen species (ROS). The study concluded that both the methanolic and chloroform extracts of C. retusa were toxic to both plants and animals and that induction of oxidative stress was the underlying mechanism of toxicity.