| Trichosanthes kirilowii Maxim, a dioecious liana, is a member of the Cucurbitaceae. It grows in hillside, valley, brushwood, forest edge etc and can be planted all over the country. In the ancient East, it was widely used as a folk medicine because the tuber can reduce mid-term abortion and the fruit rind can help cure diseases of the heart and lungs. In 1989, McGrath purified a type 1 RIP, named trichosanthin (TCS), from the tuber of T. kirilowii. TCS displayed extreme inhibition of HIV. Hence, TCS and T. kirilowii have been studied by many researchers, and the antiviral and antitumor activities were founded. But its clinical usages were restricted by the associated fever, shock and even death. In comparison with the normal type 1 RIPs, several RIPs with a molecular mass of～10 kDa have high levels of bioactivity and exhibit low immune toxicity toward normal cells. In this study, Trichosanthrip, a novel RIP with a lower molecular mass, was purified from collected 14 populations of wild T. kirilowii, and its characterization and antitumor activity was assayed.Trichosanthrip was purified from mature seeds of 14 populations of wild T. kirilowii Maxim by ammonium sulfate fractionation precipitation, CM-52 cation-exchange chromatography and Sephadex G50 gel-filtration chromatography. About 1.0 mg Trichosanthrips with purity of 96.6% were getted from 100 g seeds kernels. Trypsin-peptides of Trichosanthrip were identified by liquid chromatography- tandem mass spectrometry, and it was a novel protein belonged to AAILTSS super family. The proteins of the family were mainly presented in seeds of a variety of plants and played a role of defence with anti-insect and anti-pathogen activities.Trichosanthrip migrated as a single band in SDS-PAGE, with an apparent molecular mass of～13 kDa. The molecular mass of Trichosanthrip was 10,964.617 Da as determined by matrix-assisted laser desorption/ionization time-of-fiight mass spectrometry. Trichosanthrip showed N-glycosidase activity on 28 S rRNA and strongly inhibited cell-free protein synthesis, with an IC50 of 1.6 ng/ml.Trichosanthrip could inhibit the growth of S180 with the IC50 of 19.91μg/ml by increased the cell of S180 in G0/G1 sterm, and meanwhile decreased the cell in S sterm.Trichosanthrip could inhibit the growth of tumor in S180 transplanted Kunming strain mice, and the inhibitory rate was 91.36% when at the dose of 1.5 mg/kg·d. Meanwhile, there were not any effect on the weight and index of thymus and spleen in S180 transplanted Kunming strain mice at this dose. And the acute toxicity of Trichosanthrip on Kunming strain mice was extreme low with LD50 was 103 mg/kg.So, Trichosanthrip has a good future in cancer care with higher antitumor activity and lower side effect. |
