Isolation,Purification,Structural Identification And Activity Analysis Of Compounds In Tachypleus Tridentatus

Today,the marine environment is likely to be a prolific source of new natural antitumor products.For the past several years,many antineoplastic materials with original structures have been identified from marine organisms.And this presents a marvelous prospect in drug discovery area.Horseshoe crabs have been known as “living fossils” because they lived on earth 400 million years in the Paleozoic.There are four species(one American and three Asian species)in the world: Limulus polyphemus from North America to Mexico,Carcinoscorpius rotundicauda from India to Philippines,Tachypleus gigas from India to Malaysia,and Tachypleus tridentatus from China.To be honest,rarely has a country like China who take more than 98 percent in the source of Horseshoe crabs.So,antineoplastic materials was isolated and identificated from Tachypleus tridentatus by using modern scientific methods.Main results for the study are as follows:(1)The Tachypleus tridentatus was dismembered into shell,leg,entrail and tail parts.Analyzed the methyl alcohol extract of different parts of Tachypleus tridentatus by High performance liquid chromatography and evaluated the similarity by Traditional Chinese Medicine(TCM)chromatographic fingerprint similarity evaluation system version A.By constantly optimized,the HPLC conditions were as follows: mobile phase of acetonitrile-0.1% formic acid solution with gradient elution at flow rate of 0.2 mL/min,the detection wavelength of 220 nm and the sample concentration of 1 mg/m L.The result of similarity analysis showed that the chemical composition similarity is under 60%.Under the condition of 60% acetonitrile and water solution,there were the similar characteristics between tail and other parts.while the mobile phase was 100% acetonitrile solution,the result was quite different.So,the extract of Tachypleus tridentatus tail were firstly separated and purified,which existed large difference relatively compared with the compositions of other parts.(2)The extract of Tachypleus tridentatus tail were separated and purified by Sephadex LH-20 resin,positive phase silica gel column chromatography and reversed phase C18(RP-C18)silica gel column chromatography.The structures were identified combined with thin layer chromatography(TLC),UV detection,electrospray ionization mass(ESI-MS),nuclear magnetic resonance(1H-NMR,13C-NMR,1H-1H COSY,DEPT135,HMBC and HSQC).Eleven compounds,kynurenic acid,linoleic acid,cholesterol,methyl linoleate,2-methyldecane,bis(2-ethylhexyl)phthalate,pentadecan-2-one,cholesterol,ethy carbonate,dodecyl 2-methylacrylate and N-Cbzglycine ethyl ester were identified from the separation and purification of the methyl alcohol extract of Tachypleus tridentatus tail through chromatography and spectroscopic techniques.(3)MTT methods were used to measure the activity of inhibiting liver carcinoma cells in vitro of 11 kinds of compounds.The MTT test indicated that only kynurenic acid,linoleic acid,pentadecan-2-one and Dodecyl 2-methylacrylate had significant inhibitory effect on Hep G2 within 24 hours.The result of cellular toxicity test indicated that kynurenic acid,linoleic acid and pentadecan-2-one were nontoxic to the LO2.The MTT test indicated that the IC50 of kynurenic acid,pentadecan-2-one and cisplatin were 75.50 ?g/mL,48.65 ?g/m L and 19.39 ?g/m L within 72 hours.The results showed that only kynurenic acid and pentadecan-2-one had significant inhibitory effects on HepG2 cells,and nearly no efficacy on LO2 cells.Meanwhile,The Dodecyl 2-methylacrylate had cytotoxic activity on LO2 cells.