Screening Of The Chemical Compositions Isolated From The Holotrichia Diomphalia Bates And Their Antitumor Activities

Objective:Holotrichia diomphalia Bates has a long history of the application in Chinesemedicine (TCM). Holotrichia diomphalia Bates used alone treat tetanus and eye diseases,the components used in the treat stroke, pediatric asthma, ulcer and schistosomiasiscirrhosis. In recent years, as people in-depth study of the Holotrichia diomphalia Bates,the Holotrichia diomphalia Bates extract has anti-tumor, treatment of aphthous, eyediseases and other bioactive. The Holotrichia diomphalia Bates is of great value anddevelopment in animal drugs. But there is few explicit reported about anti-tumor activityof chemical compositions isolated from the Holotrichia diomphalia Bates. The chemicalcompounds of alkali chemical constituents extracted from the Holotrichia diomphaliaBates showed significant anti-tumor activities. We isolated the chemical compounds fromalkali chemical constituents, and evaluated their antitumor activity in order to givesupports to the exploitation of Holotrichia diomphalia Bates resources for the foundation.Methods:Single factor analysis and L9(34) orthogonal test were used to get the best extractionmethod of alkali chemical constituents from Holotrichia diomphalia Bates. The anti-tumoractivities of alkali chemical constituents in vitro were evaluated in human cancer cells by MTT assay. S180sarcoma mice were used for the anti-tumor test in vivo. The alkalichemical constituent was analysed by GC-MS. The alkali chemical constituents extractedby the best extraction method were isolated by Sephadex LH-20, silica gel and C18column,etc.The chemical compounds were identified through the methods of physical inspection,chemical inspection and spectral techniques, such as1H-NMR,13C-NMR and MS. Theanti-tumor activities of Monomer compounds in vitro were evaluated by MTT assay inB16and HepG2cancer cells. B16sarcoma mice were used for the anti-tumor test in vivo.The effects of two monomers compounds to immune system were observed by detectingthe spleen index and thymus index. Apoptosis morphological changes were observed byHE staining. Cell apoptosis was detected by TUNEL staining.Results:The optimal condition was dipping the coarse powder of Holotrichia diomphaliaBates in95%alcohol extraction for24h, condensing, acidating and extracting with EtOAc,then alkaline was extracted with chloroform. The alkali chemical constituents inhibitedhuman cancer cells and development of S-180transplanted tumor in mice in a dosedependent manner. The chemical constituents were analysed by GC-MS， then69components were identified and9nitrogen-containing compounds were found by analysisof GC-MS. There are14compounds were isolated from the alkali chemical constituents.They were identified as Cholesterol, Cis-15-tetracosenoic acid, Elaidic acid, Oleic acid,9H-pyrido(3,4-b)indole,1,2–Cyclohexanediol, Hexadecenoic acide, Oil methyl ester,5,10-Diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a:1’,2’-d]pyrazine,2-piperidone, Indol-3-aldehyde, Pyridine-3-carboxamide,4-methyl-5-(β-hydroxyethyl)thiazole. Oxindole,Cis-15-tetracosenoic acid and Pyridine-3-carboxamide monomer compounds significantlyinhibited B16cells in vivo and in vitro. Cis-15-tetracosenoic acid and Pyridine-3-carboxamide monomer compounds promote cells apoptosis in vivo.Conclusion:14compounds were isolate from the Holotrichia diomphalia Bates, the oleic acidconstituents were isolated5, the alkali chemical constituents were isolated7, the steroidalconstituents were isolated1, the alcohol constituents were isolated1. The compounds5,10-Diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a:1’,2’-d]pyrazine9H-pyrido(3,4-b)indole, Cis-15-tetracosenoic acid,4-methyl-5-(β-hydroxyethyl)thiazole Oil methyl ester were isolated from the species for the first time from the Holotrichia diomphalia Bates. Inthe active evaluation, cis-15-tetracosenoic acid and Pyridine-3-carboxamide monomercompounds were significantly inhibited in vivo and in vitro.