| Dichrocephala integrifolia, as the plant of Dichrocephala genus (Compositae), distributed in tropical Asia, Africa and Oceania with a very high medicinal value. It included Dichrocephala integrifolia, Dichrocephala benthamii, Dichrocephala chrysanthemifolia, Dichrocephala bicolor and Dichrocephala latifolia. The top three plants can be found in China according to "the Flora of China Account". As recorded in "Diannanbencao", the herb of Dichrocephala benthami and Dichrocephala chrysanthemifolia is called Dichrocephala integrifolia.This medicine tastes bitter and it’s cold. It is effective in malaria, dysentery, diarrhea, mouth sores, ulcers, women leucorrhea, hepatitis, etc. All of above is also recorded in "Chinese Herbal Medicine in Yunnan","the Catalog of Guizhou Medicine Plant","Yunnan Traditional Chinese Medicine Prescriptions" and other medical books.At present, some terpenoids, volatile oil, flavonoids and steroids have been found in this plant, their pharmacological activities have been further studied. In order to promote the comprehensive development and utilization of this genus plants and looking for more active ingredients, we studied the aerial part of Dichrocephala integrifolia and separated the petroleum ether, dichloromethane, ethyl acetate and n-butanol extraction part systematically. We confirmed the structures of the compounds that we obtained and screened their pharmacological activities.Objective:The aims of this task is study the chemical constituents of the aerial part of Dichrocephala integrifolia systematically by using silica gel column chromatography preparation, preparative thin layer chromatography, high performance liquid chromatography and recrystallization purification technology. So as to establish effective methods of isolation and purification of pure compounds and find new chemical composition. Using UV, IR, MS and ID-and2D-NMR (1H-NMR,13C-NMR,1H COSY, HMQC, HMBC, NOESY) to complete the structure identification of these compounds. At the same time, we can also use the activity screening to get some active ingredients for further development and utilization.Methods:The natural drying acrial parts of Dichrocephala integrifolia (6kg) were extracted three times with95%ethanol under reflux, The extraction successively partitioned with petroleum ether, dichloromethane, ethyl acetate and n-butanol in order, and get enriched every part. After silica gel column chromatography, PTLC, HPLC, glucan, macroporous adsorption resin column, recrystallization and other purification methods were used in the process of separation and purification. By using these methods, compounds were getted. The plane and stereochemical structures of these compounds were confirmed by the analysis of spectroscopic data including UV, IR, MS and HRMS-ESI, ID-and2D-NMR and comparised with the references data. Finally some compounds activities against three humen liver cancer cell lines (Bel-7402, SMMC-7402, Hep G2) were tested by MTT colorimetric method.In experiment, all of the silica gels were GF254, all of the adhesive is CMC-Na which concentration is0.5%-0.7%and all of the chromatosheets were made through artificial. Common analysis methods are observation under254nm,365nm UV analyzer and coloration by10%sulfuric acid in alcohol, vanillin, phosphomolybdic acid, iodine vapor observation.A preliminary analysis of purity methods are:a single peak by HPLC; a single spot in TLC when spread liquid are three system at least.Results:Through the systematically separation to the acrial part of Dichrocephala integrifolia,37compounds were obtained and their struct-ures were identificated. Only26structures were confirmed because of t-he limitation of time. Types of the compounds are terpenoids, fatty aci-ds, sterols and its glycosides, alkaloids, phenylpropanoids and volatile o-il.26compounds are stigmasta-7,22-dien-3-ol(1), n-tetradecoic acid(2), dichrocephol C(3), dichrocephol B(4),3-O-[6’-O-palmitoyl-β-D-glucos y１] spinasta7,22-dine(8), ergosta-6,22-diene-3,5,8-triol(12),3’-O-meth yle-upatorin(13), methyl pheophorbide b(14), N-benzoylphenylalaninyl-N-benzoylphenylala-ninate(15), aurantiamide acetate(16), isobutyl phthala te(17), n-butyl phthalate(18), methyl phaeophorbide a(20),16-hydroxycl eroda-3,13-dien-15,16-olide-18-oicacid(22), eudesm-11-ene-4α,7p,9p-tr iol(23),stigmasta-7,22-dien-3β-O-β-D-glucoside(24),1,8-terpenediol(26),quercetin-3-O-glucoside(27),(1R,3S,4R,5S)-3,4-bis[[(2E)-3-(3,4-dihydroxyphe-nyl)-1-oxo-2-propenyl] oxy]-1,5-dihydroxy-cyclohexanecarboxyli c acid(28),(1R,3R,4S,5R)-3,4-bis[[(2E)-3-(3,4-dihydroxyphenyl)-1-oxo-2-propenyl] oxy]-1,5-dihy droxy-cyclohexanecarboxyli c acid(29),quercetin-3-O-rutinoside(31), kae mpferol3-β-D-glucoside(32), kaempferol-3-O-rutinoside(33),3-(β-D-glucopyranosyloxy)-2-hydroxy-olean-12-ene-23,28-dioicacid,28-(glu copyranosyl-(lâ†'2)-xylopyranosyl-(lâ†'4)-grucopyranosyl) ester(35),2-hyd roxy-olean-12-ene-23,28-dioicacid-3-()-β-D-glucopyranoside(36),3-(β-D-glu copyranosyloxy)-16-methanoyl-olean-12-ene-28-oicacid,28-(glucopyran-osy1-(1â†'4)-2-ethanoyl-xylopyranosyl-(1â†'4)-glucopyranosyl) ester (37).Activity screening results showed that compounds15,16and23have antiproliferative activity against three human hepatoma carcinoma cells (Bel-7402, SMMC-7721, Hep G2).Conclusions:In this paper, we had a systematically study to the aerial part of Dichrocephala integrifolia. The experimental results show that the solvent and the methods using in experiment are feasible. Liquid-liquid extraction, silica gel column chromatography, PTLC, HPLC, glucan, macroporous adsorption resin column, recrystallization and other purification methods were used in the process of separation and purification,37compounds were isolated.26strucures were confirmed by a variety of spectroscopic methods. Among of them, compounds23,35and37were new, compounds2,8,12-16,22,24,26,36were isolated from this genus for the first time, compounds firstly isolated from Dichrocephala integrifolia were compounds3-4,18,28-29,31and33, compound22might be a new natural product after comfired their stereochemical structure.N-benzoylphenylalaninyl-N-benzoylphenylalaninate (15) firstly exhibited antiproliferative activity against SMMC-7721and Hep G2human hepatoma carcinoma cells; aurantiamide acetate (16) and eudesm-11-ene-4,7,9-triol (23) were both firstly shown antiproliferative activity against Bel-7402, SMMC-7721, Hep G2cells. |
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