Studies On The Chemical Constituents And Antitumor Activities Of Hemsleya Pengxianensis Var. Jinfushanensis

Hemsleya pengxianensis var.jinfushanensis belongs to Cucurbitaceae Hemsleya and mainly distributed in Yunnan,Sichuan,Guizhou and Chongqing provinces of China.It is rich in cucurbitane triterpenes and glycosides,lignans,organic acids and other constitutes.Modern pharmacology research revealed that H.pengxianensis var.jinfushanensis exhibited the anti-tumor,antibacterial,anti-inflammatory,anti-HIV activity,liver protection,gastric ulcer treatment and other activities.This paper continued the depth study of H.pengxianensis var.jinfushanensis following abundant previous research.Objective:To study the chemical constituents of H.pengxianensis var.jinfushanensis,and their cytotoxic activity.To study the anti-tumor activity focusing on the mechanism of strong cytotoxic activity compounds to provide promising target for the development of new anti-tumor drugs.Methods:The ethyl acetate extract was isolated and purified by Silica gel column,ODS column,prep-HPLC and re-crystallization methods.Their structures were identified on the basis of IR,MS,and NMR spectra.The S180 tumor-burdened mouse models were established,and tumors were excised after administration to observe the inhibitory effect of the ethyl acetate extract.The cytotoxic activities of 30 compounds against HT29 cells?human colon cancer?were evaluated by MTT method.PI staining and Annexin V-FITC/PI double staining were employed to examine the effect of 16,25-O-diacetyl-cucurbitane F and 25-O-acetyl-23,2-4-dihydrocucurbitacin F on cell cycle and apoptosis.The tubulin-tracker red staining,the actin-tracker green staining and Hoechst 33342 staining were used to examine the influence of the cell cytoskeleton.Results:Thirty compounds were obtained from the ethyl acetate fraction of H.pengxianensis var.jinfushanensis and identified as hemslepenside J?1?,hemslepenside K?2?,hemslepenside L?3?,hemslepenside M?4?,hemslepenside N?5?,hemslepenside O?6?,hemslepenside P?7?,hemslepenside Q?8?,hemslepenside R?9?,hemslepenside S?10?,scandenoside R8?11?,3-O-?-D-glucopyranosyl-3?,27-dihydroxycucurbita-5,24?Z?-diene-11-o-ne-27-O-?-D-glucopyranoside?12?,delavanoside A?13?,3?,11,26-trihydroxycucurbita-5,24?E?-diene-26-O-?-D-glucopyranosyl?1?6?-?-D-glucopyranoside?14?,carnosifloside I?15?,hemslepenside I?16?,jinfushanoside C?17?,jinfushanoside D?18?,hemslepenside G?19?,hemslepenside H?20?,scandenoside R3?21?,scandenoside R4?22?,jinfushanoside J?23?,carnosifloside??24?,carnosifloside??25?,scandenoside R6?26?,jinfushanoside K?27?,amabiose?28?,16,25-O-diacetyl-cucurbitane F?29?,25-O-acetyl-23,24-dihydrocucurbitacin F?30?.Tumor growth inhibition ratios of the ethyl acetate extract of H.pengxianensis var.jinfushanensis at the dose of 300 mg/kg,200 mg/kg and 100 mg/kg were 47.83%,37.27%,30.43%,respectively.Thirty compounds were subjected to cytotoxic test and the results showed that compounds 29 and 30 showed strong cytotoxic activities.The cell cycle experiments showed that compounds 29 and 30 could significantly induce the cell cycle arrest of colon cancer HT29 cells in G₂/M phase in a dose-dependent manner compared with negative control?P<0.05?.The apoptosis assay showed that compounds 29 and 30 could significantly increase the percentage of apoptotic and necrotic cells in a dose-dependent manner compared with negative control?P<0.05?.The immunofluorescence staining showed that compounds 29 and 30 could induce the F-actin aggregation and severe destruction of microfilament cytoskeleton structure.Conclusion:Thirty compounds were isolated from the ethyl acetate extract of H.pengxianensis var.jinfushanensis,among which 1¹0 were new compounds.The ethyl acetate extract of H.pengxianensis var.jinfushanensis could significantly inhibit the tumor growthofS180tumor-bearingmice.16,25-O-diacetyl-cucurbitaneFand25-O-acetyl-23,24-dihydrocucurbitacin F showed strong cytotoxic activities against various cancer cells and they might be potential suppressor of HT29 cells through its effect on F-actin,cell cycle,and apoptosis.