PHYTOCHEMICAL ANALYSIS OF ACTIVE CONSTITUENTS OF ALSTONIA SCHOLARIS AND THEIR CYTOTOXICITY IN VITRO

The non-infected bark of Alstonia scholaris R. Br. was carefully peeled off, shade dried, and coarsely powdered with the help of a hand club. Powdered dried bark was extracted in 95% ethanol at room temperature and evaporated in vacuo (40°C). The ethanolic extract was suspended in distilled water and subsequently extracted in hexane, chloroform, ethyl acetate and n-butanol. The solvent of each fraction was removed in a rotary vacuum evaporator under reduced pressure. The crude chloroform extract was mixed with 3% HCl and ethanol. The aqueous layer was collected and the pH was adjusted to 10 by adding NaOH, thereafter it was further extracted with chloroform to get a crude alkaloidal fraction. The alkaloidal fraction was subjected to column chromatography over silica gel and eluted with chloroform: methanol (50:50). The elutes between fractions 40-100 were collected and subjected to preparative HPTLC in the solvent system of ethyl acetate: benzene (1:1). The HPTLC showed the presence of two alkaloids, which were evaluated for their cytotoxic effects in neoplastic cell lines by MTT assay. The compound which showed greater cytotoxicity was subjected to UV, NMR, Mass, and IR spectra and identified as echitamidine-N-oxide-19-o-β-D-glucopyranoside (EOG). The presence of EOG in the chloroform fraction was further confirmed by semipreparative RPHPLC (H₂O/CH₃CN-0%-80%; CH₃CN in 80 min, flow rate 3 mL/min, UV detector – l 250 nm) and was also identified based on the literature