| Due to the aggravation of environmental pollution, nutrition disequilibrium and pressure from life, cancer has been the leading cause of deaths worldwide. The search of active fraction or compound from traditional Chinese medicine is one of the effective ways in the development of drugs that have antitumor activities. Poria cocos is the dry sclerotium of Poria cocos (Schw). Wolf which contains lanostane triterpenes, and these compounds were reported to have antitumor activities, generally.The kinds of basic skeketon of these compounds were very few and there are usually many isomers of these compounds. To purify these compounds is very difficult and this does not benefit the research of these comounds on their antitumor activity. However during haverst, the epidermis of poria cocos was usually discarded for its dosage is very low in clinic. Researches showed that the content of lanostane triterpenes in the epidermis of poria cocos was ten times higher than in the scleritum. Moreover, the varieties of lanostane triterpenes were very rich. Overall, to separate triterpene acids from the epidermis of poria cocos and investigate its antitumor activity will be a good and practicable idea.In present research, the lanostane triterpene extract of the epidermis of poria cocos has been prepared and its antitumor activity in vitro and vivo were investigated, and the lanostane tirterpene extract was further separated by conventional column, high-speed counter-current chromatography and preparative supercritical fluid chromatography, the obtained triterpene acids were subjected to antitumor acitivity in vitro, and the structure-activity relationship of antitumor bioactivity was also discussed. All contents were described as follows:1. Qualitative and quantitative analysis of the triterpene extract from the epidermis of Poria cocosLC-MS was used for the identification of ethyl acetate extract from the epidermis of Poria cocos, a total of 13 triterpene acids were identified. The total content of triterpene acids of the ethyl extract was analysed by Ultraviolet spectrophotometer, and its content was more than 50%. The results showed that the ethyl acetate extract can be used as triterpene acid extract for further research of chemical constituents and bioacitivities.2. Investigation of antitumor activity of the triterpene extractThe bioactivities of triterpene acids from poria cocos were predicted by network pharmacology, and these triterpene acids are considered to be related with breast cancer, cervical cancer, lung cancer, et al. The antitumor activity of triterpene acids extract on Lewis lung cancer bear mice was investigated; the results showed that the inhibitory rates were 41.7%,58.87% at 65 mg/kg and 90 mg/kg, respectively. Further research of the antitumor activity of the triterpene extract with different polorities in vitro showed that the triterpene acids have the tendencies of structure-activity relationships.3 Chemical constituens of triterpene extract from the epidermis of Poria cocos3.1 Separation of compounds from triterpene extract by conventional column chromatographyFirstly, the triterpene extract was separated by silica gel column, and the obtained fractions were further separated by ODS medium-pressure chromatography and preparative HPLC, repeatedly. As a result, a total of 26 triterpene acids were obtained, and they were identified as dehydrotrametenolic acid (1), trametenolic acid (2), dehydroeburicoic acid (3), eburicoic acid (4), pachymic acid (5),3-O-acetyl-16a-hydroxytrametenolic acid (6), poricoic acid AE (7), dehypachymic acid (8), poricoic acid GE (9), poricoic acid AM (10), poricoic acid BE (11), poricoic acid GM (12), 3-O-acetyl-16α-hydroxydehytrametenolic acid (13), (3β,16α)-3-acetyloxy-16a-hydroxy-24-methylenelanost-5,7 (9), 11-tetraene-21-oic acid (14), polyporenic acid C (15),16a-hydroxyeburiconic acid (16),3β,15α-dihydroxylanosta-7,9 (11),24-triene-21-oic acid (17),3β,16α-dihydroxylanosta-7,9 (11),24-trien-21-oic acid (18), dehydrosulphurenic acid (19),16a-hydroxytrametenolic acid (20), dehydrotumulosic acid (21), tumulosic acid (22), poricoic acid A (23),16α-hydroxy-3,4-secolanosta-4 (28),7,9 (11),24 (31),25 (27)-pentaene-3,21-dioic acid (24), poricoic acid B (25) and poricoic acid G (26). Compound 17 and 19 were firstly separated from Poria cocos, compound 9,11,14 and 24 were all new compounds.3.2 Preparative separation of compounds from triterpene extract by high-speed counter-current chromatographyAn efficient method for the preparative separation of triterpene acids from the triterpene extract was established and involves the combination of pH-zone-refining CCC and conventional HSCCC. pH-Zone-refining CCC was used to concentrate the triterpene acids using a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (3:7:5:5, v/v), trifluoroacetic acid (10 mM) was added to the upper phase as a retainer and ammonia (10 mM) was added to the lower phase as an eluter. As a result,200 mg concentrate of triterpene acids was obtained from 1.0 g of crude extract. The concentrate was further separated by conventional high-speed counter-current chromatography using a solvent system composed of petroleum ether-ethyl acetate-methanol-water (0.8:1.2:1.2:0.9, v/v), yielding 50 mg of poricoic acid A and 5 mg of poricoic acid B from 120 mg concentrate, respectively.3.3 Preparative separation of constituents from the sclerotium of poria cocos by high-speed counter-current chromatographyA method for the acid-alkali extraction and preparative separation of triterpene acids from poria was established. The triterpene acids were enriched and separated into two fractions after extraction at the optimized pH value. The two fractions were subjected to high-speed counter-current chromatography for the preparative separation of triterpene acids, separately. As a result, dehydropachymic acid, pachymic acid,3-epi-dehydropachymic acid, poricoic acid B, dehydrotumulosic acid, and 3-epi/-dehydrotumulosic acid were obtained with purities of 94.1%,96.2%,93.5%,85.9%,80.1%, and 93.1%, respectively. The structures were identified by ESI-MS,’H-NMR, and 13C-NMR.3.4 Preparative separation of constituents from triterpene extract by preparative supercritical fluid chromatographyTo separate lanostane-type triterpene acids with bad resolutions in reverse C18 column from the epidermis of poria cocos. The ethyl acetate extract was enriched by silica gel and reverse C18 column at middle pressure system. The enriched sample was subjected to semi-preparative supercritical fluid chromatography with the solvent system composed of methanol-CO2 (15%-85%) in isocratic elution mode. As a result, three triterpene acids were obtained and identified as polyporenic acid C, poricoic acid AM, and 3-Oacetyl-16a-hydroxydehytrametenolic acid by NMR and MS, the purities of these compounds were 93%,80% and 99%, separately.4 The antitumor activity of triterpene acids in vitroThe antitumor activity of two compounds including poricoic acid AM and 3-O-acetyl-16-hydroxytrametenolic acid with different substituents in chemical structure was investigated in vitro on 8 kinds of cancer cell lines. The results showed that these all two compounds showed cytoxity in the 8 kinds of cancer cells. The results were consistent with that of the prediction of bioactivities of triterpene acids from Poria cocos by net work pharmcology and the anti-lung cancer bioactivities in vitro and vivo on Lewis bear mice.For further research of triterpene acids, the antitumor acitivities of a 24 triterpene acids were test in vitro on MGC-803 and HepG2 cells, and the inhibitory activities of these compounds on these two cells were the same. The exsistence of hydroxyl in position 16 or 15, the double bond in B and C cycle of the structure will increase the acitivity. In 24 (25)-ene triterpene, if the hydroxyl in position 3 was acetylated, the inhibitory activity will increase, while in 24 (31)-ene triterpene, the inhibitory activity will decrease.In conclusion, the bioactivity of triterpene from poria cocos was predicted using network pharmacology method, and the triterpene extract of the epidermis of Poria cocos was prepared and found to inhibit the growth of tumor on Lewis lung cancer bear mice in vivo. Further investigation of antitumor experiment in vitro showed that the triterpene extract have antitumor activity. Based on the bioactivity, the extract was further separated by conventional column, high-speed counter-current chromatography and preparative supercritical fluid chromatography, a total of 26 triterpenes acids was obtained and four were new compounds, two were firstly separated from Poria cocos. Experiments of the antitumor activity in vivo of these compounds showed structure-activity relationships. This study provide theory base for the application and development of triterpenes from poria cocos as antitumor constituents. |
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