Extraction Of Resveratrol From Polygonum Cuspidatum And Its And Its Effect On Human HEPG-2Cell Apoptosis

Objective:1.To optimize extraction process for extracting Resveratrol fromPolygonum Cuspidatum by microwave assisted and do preliminaryidentification.2.To observe the inhibitory effect of Resveratrol standard sample onproliferation in cell line HepG-2, dose-effect relationship is determined, choosethe half of inhibition concentration as reference of the investigation of theanti-hepatoma effects of Resveratrol.3.Provide a new experimental evidence for development and utilization ofCuspidatum.4.To study the anti-tumor effect and its mechanism of Resveratrol andprovide theoretical basis for its clinical application.Method:1.The Resveratrol was extracted by microwave assistant. During this course,standards sample was used as index to control the abstraction. All theexperiments were determined with UV. The extraction condition was optimizedthrough L9（34）orthogonal experiments.2.HepG-2cells were treated with Resveratrol at different concentrations,the changes of cell morphology were studied through inverted microscope. 3.The changes of cell morphology were observed by fluorescence stainingwith Hoechst33342staining after different concentrations of Resveratrol（100μmo/L,50μmol/L,25μmol/L,12.5μmol/L）affecting HepG-2cells48h.4.The inhibitory effect of Resveratrol by different concentrations ofResveratrol were valuated by MTT （methyl thiazolyl tetrazolium） after24h,48h,72h on HepG-2cells.5.Cell cycle was detected by flow cytometry analysis after differentconcentrations of Resveratrol affecting HepG-2cells48h.Results：1.The optimal extraction condition was100W of microwave power,30minof microwave time,25℃of extraction temperature, and1:20of solid-liquidratio. The extraction rate of Resveratrol was91%at the optimum parameters,which was better than water bath extraction.2.Under an inverted microscope could be seen in the negative group inhuman HepG-2cells were fusiform, plump shape, into a piece arrangement,abundant cytoplasm and uniform distribution of large nuclei; in the positivegroup(DDP5μg/L), Cell structure is unclear, nucleus fragmentation, dissolving,pyknosis. A large number of dead cells floated in the cell suspension. Eachexperimental group cells after Resveratrol treatment, cell derangement, irregularcontour, membrane shrinkage, change the park, off the uneven distribution ofcytoplasm within the cell, the cell size becomes smaller, fewer number of cells.3.Fluorescence staining in control hoechest33342group HepG-2cellmembrane integrity, full of nuclear morphology, nuclear quality stained uniformcoloring lighter; HepG-2cells affected by Resveratrol were observed under thecell membrane occurred shrinkage the nucleus is relatively small, thefluorescence enhancement is obviously the nucleus chromatin concentration and fragmentation was round cell debris, namely apoptotic bodies, showing thetypical apoptotic morphological features.4.MTT Results showed that Resveratrol showing time-dependent anddose-dependent inhibition of the growth of human HepG-2cells. Experimentalgroup after24hours, inhibition of human HepG-2cells was not obvious (P>0.05) at low concentrations (12.5μmol/l),25μmol/l,50μmol/l,100μmol/l groupsinhibition with the control group compared to the difference of statisticalsignificance (P<0.05). The inhibition ratio were48.68%,32.56%,11.17%,9.80%respectively. The25μmol/l,50μmol/l,100μmol/l in human HepG-2cellsinhibited with increasing concentrations after48hours and72hours, and had ahigh degree of statistical significance (P<0.05).48hours of concentration toinhibit the rate were48.68%,32.56%,11.17%,9.80%respectively.72hours ofconcentration to inhibit the rate were58.75%，35.73%，16.46%，14.68%respectively.5.Flow cytometry analysis detected HepG-2cells after Resveratrolaffecting48h. The result showed that the cells in the negative group were totallyin G1phase. As increasing of Resveratrol concentration， G0/G1phase wasdecreasing gradually and S phase was increasing. S phase ratio of100μmol/l，50μmol/l groups were77.50%，65.21%respectively，which were significantlyabove contrast group and had a high degree of statistical significance (P<0.01).S phase ratio of25μmol/l,12.5μmol/l groups were44.82%，54.00%respectively， which were significantly above contrast group and had a highdegree of statistical significance (P <0.05).Conclusions:1.Microwave-assisted extraction method was an efficient, energy-efficientextraction method，which provided a new way to the separation and purification of the active ingredients of natural products and provided the technical supportsfor mass production of Resveratrol.2.The cell morphology was changed after different concentrations ofResveratrol affecting HepG-2cells.3.Resveratrol inhibit the growth of human HepG-2cells and inhibition wastime dependent and dose-dependent manner.4.Resveratrol can induce HepG-2cell apoptosis. As increasing ofResveratrol concentration， the number of apoptosis cells was increasinggradually.5.Cell growth arrest at S phase after Resveratrol inhibit HepG-2cells.