Caffeine is the most frequently ingested pharmacologically active substance in the World. It is found in common coffee, tea, soft drinks, in products containing cocoa or chocolate, and in medications among others. Because of its wide consumption at different levels by most of the people, the scientific community as well as the general public have expressed interest in the potential for caffeine to produce adverse effects on human health. This study was carried out to isolate and characterize caffeine from root bark extract of Piliostigma thonningii and to study the toxicity profile of the isolated isolate. The ethyl acetate fraction was gradiently eluted in a silica gel column to afford an isolate coded EA, in which its purity was confirmed by preparative thin layer chromatography (TLC). Structure of the isolate was elucidated using spectroscopic techniques such as 1H NMR, 13C {H}NMR and Infrared spectroscopy (IR). and literature comparison suggested the isolated compound EA as an alkaloid namely 1,3,7-Trimethylpurine-2,6 dione. Drosophila melanogaster oral toxicity of the EA against 30 adults fruit flies (15 males and 15 females) was carried out using standard procedure. results showed mortality rate of 0 % at 14 mg/10g diet for 7 days. The Locomotor assay was done using negative geo-axis assay, results showed 70 % of flies reached the top of the glass bottle (10 cm height) at concentration of 5 mg/10g diet. The isolate EA from P. thonningii root bark is safe for oral consumption up to a concentration of 14 mg/10gdiet.
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