Toxicity Activity-Guided Characterization of Toxic Constituents in Azadirachta indica Seed

Background: Azadiradita indica seed has been used in traditional system of medicine. It is known to be natural medicine with many benefits. Toxicological studies have reported that toxic effects may be related to the complex mixtures of active constituents and other chemicals which increase the risk of adverse reactions. Objective: The purpose of the study was designed to isolate and characterize the toxic constituents with a view of recommending for clinical trials. Materials and Methods: The study was conducted at the Department of Biochemistry, University of Nigeria, Nsukka. The seeds were collected, identified and extracted with conventional Soxhlet extraction technique. Chromatographic techniques were used for fractionation and isolation of the toxic constituents. A total of eighty- four (84) adult Albino rats of both sexes were randomly assigned into fourteen (14) groups containing 6 rats in each group. Each group I-VI received one of the 100, 500, or 1000 mg/kg of Methanol Extract (ME) or Hexane Extract (HE), respectively. Group VII was the control and received 0.5 ml/kg of the vehicle, 3% v/v Tween 80. Group VIII-XIV received 100 mg/kg of Pet-ether-ethylaceate fraction (PEF), Methanol fraction (MF), Methylene Chloride /Acetone fractions 9:1, 8:2 and 7:3 (MAC-1, MAC-2, MAC-3), Isolate 1 (TN-1), and Isolate 2 (TN-2), respectively. Extracts and fractions were administered orally once daily for 30days for animals in group I-XII. Groups XIII-XIV were treated for only 10 days. On days 10, 20, 30, 3 ml of blood was withdrawn from each rat by an ocular puncture for liver function test (ALT and AST). Body/ organ weights were equally used as a toxicity guide in the separation of toxic constituents. Results: The ME and various fractions caused significant (P<0.05) increase in the activities of ALT and AST. The isolated compounds TN1 and TN2 also caused a significant effect on AST and ALT. The toxic effect of TN-2 was higher than that exhibited by TN-1. The methanol and fractions caused significant (P<0.05) dose related increase in the body weight of treated animals. TN-1 and TN-2 caused significant (P<0.05) reduction in the organ weight of the treated animals. Conclusion: Although there was no evidence of lethality for acute toxicity of the extract, chronic oral administration of the extract and solvent fractions caused hepatotoxicity. Structure elucidation revealed TN-1 and TN-2 to be 6- deacetylnimbin and Nimbolide, respectively.