The Effect Of Gynostemma Damulin A And Damulin B On HepG2 Cells In Vitro

ObjectiveGypenosides?Gyps?are the major effective components in Gynostemma pentaphyllum?Thunb.?Makino.This paper reported the processe of separation and identification of Gyps from heat-processed G.pentaphyllum.The mechanism of damulin B inhibiting HepG2 cells had been discussed through selectivity,cell cycle and apoptosis.The purpose was to discover potential drug candidates for the treatment of liver cancer,and provide theoretical basis for rational administration in clinic.Methods1.Liquid-liquid extraction,silica gel column chromatography and semi-preparative HPLC were used to isolate and purify the total extract from heat-processed G.pentaphyllum.The chemical structures were identified with UV,IR,ESI-MS and NMR data.2.The activity of damulin B against HepG2 cells was investigated using CCK-8 assay.Median inhibitory concentration(IC₅₀)was used to measure the cytotoxicity.3.Set different gradient of concentration-time to test the relationship between inhibition activity and function of time and dose effect detection of damulin B on the proliferation of HepG2 cells which was be tested through CCK-8 method.The inhibitory effect of damulin B was better than the positive control drug ginsenoside Rg3 on the proliferation of HepG2 cells.4.Colony formation assay was used to detect the anti-colony activity of damulin B.Flow cytometry was employed to test the effect of damulin B on HepG2 cell cycle using PI/Rnase staining.5.Tunel assay was used to test the effect of damulin B on HepG2 cell apoptosis.The qualitative fluorescence images and quantitative flow cytometry analysis had been studied from apoptosis ratio,mitochondrial membrane potential,the intracellular Ca2+ concentration and the production of the intracellular reactive oxygen species?ROS?.Results1.Two gypenosides from the total extract of heat-processed G.pentaphyllum,were isolated and identified as damulin A and damulin B.2.Through CCK-8 assay,Damulin A was found to have no obvious inhibitory effect on proliferation of HepG2 cells.3.Damulin B significantly inhibited the proliferation of HepG2 cells,and the inhibition of proliferation was dose and time dependent.When the concentration was 25?g/ml,the inhibition rate was more effective than that of the positive control drug ginsenoside Rg3?S?at the same concentration.4.When treated with damulin B,the apoptotic cells obviously increased;the apoptotic cell ratio increased;Green/Red fluorescence ratio decreased by using JC-1 assay and this indicated that mitochondrial membrane potential depolarization was obvious.The intracellular Ca2+concentration increased obviously and the mean fluorescence intensity tested by FCM increased.The production of intracellular reactive oxygen species obviously increased and the mean fluorescence intensity increased.5.The migration ability of HepG2 cells after damulin B treatment was significantly reduced,and the drug concentration was adjusted to 25?g/ml and then treated for 6 hours,12 hours,and 24 hours.Conclusion1.damulin A has no obcious inhibitory effect on proliferation,which may be related to the position of double bonds in the structure.2.The inhibitory effect of damulin B on HepG2 cells has the following characteristics:the inhibitory effect of damulin B on HepG2 cells was significantly stronger than that of ginsenoside Rg3 in the effective inhibition concentration range.3.The possible mechanism of damulin B inhibiting HepG2 cells were as follows:? damulin B could arrest HepG2 cells at G0/G1 phase.?damulin B induced the depolarization of the mitochondrial membrane potential,increased intracellular Ca2+ concentration,promoted the production of reactive oxygen species.