Purification, Detection Of Curcin From The Seeds Of Jropha Curcas And The Primary Investigation Of Its Antitumor Activities

Jatropha curcas L., a member of the family Euphorbiaceae, is widespread in tropical and subtropical countries. A type I RIP(Ribosome inactivating Protein),designate as curcin,was isolated and purified from J.curcas seeds by a improved method of precipitation with 60%-80% saturation of (NH₄)₂SO₄ followed by chromatography on SP-Sepharose FastFlow. The production rate of curcin is about l%‰.A sensitive nonradioactive method to determine the activity of ribosome-inactivating proteins (RIPs) based on rabbit reticulocyte lysate translation in vitro system and luciferase assay system was established. Using this assay we investigated the RIP activity of Curcin samplel (purified by ion exchange chromatography) and Curcin sample2 (purified by molecular sieve chromatography). Curcin samplel IC₅₀ = 0.045±0.009nM; Curcin sample2 IC₅₀= 0.217±0.026nM. Because of the avoidance of radioactive amino acid,the process of this assay is convenient,rapid,safe and also accurate.At the meantime,we probed into and optimized the terms of the assay,including reaction time,temperature, pH value and concentration of buffer.MTT assay and the method according to NCI were performed to determine the inhibitory efficiently of curcin on HepG2, SP2/0, NCL-H446, S-180, SGC7901,but curcin is nontoxic to them. In another side, after treated with curcin,apparent apoptotic cells of S-180 were observed under fluorescencemicroscope; Meanwhile, an apoptotic peak of sub-diploid was also observed by FAM.Site-directed polyethyleneglycol(PEG) modification (PEGylation) was proceeded by couple PEG to a pre-selected Cys site, the PEG-curcin conjugate was homogeneous and showed a single band on SDS-PAGE.