Effect Of Scutellaria Baicalensis Formula Granule On Proliferation And Apoptosis Of HepG2 Cells

Objective: Scutellaria baicalensis formula granule(SBFG)is a traditional Chinese medicine formula,which is made from Chinese herbal medicine Scutellaria baicalensis by extracting,concentrating,drying and pelleting with dextrin.As a new form of modern chinese medicine,the quality standard and the pharmacological action need to be studied and improved urgently.This research is done to establish the quality inspection method of Scutellaria baicalensis formula granule(SBFG)and control the product quality;to observe the effect of SBFG on proliferation and apoptosis of HepG2 cells and preliminary discuss its mechanism,to provide the basis and reference to the further development and clinical application of SBFG.Methods: 1 The research of quality standardTake 3 batches of pilot products as the research object,researched from the aspects of thin layer identification and content determination;determine the quality of SBFG,establish quality standard.1.1 Thin layer identification methodRespectively studied on the preparation method of test sample,developing solvent system,thin layer plate etc by using thin layer chromatography.1.2 Content determination methodTake the baicalin content as the index,adopt the HPLC method to determine the content of baicalin in SBFG,and study on methodology,ultimately formulate SBFG content determination method and limit,as the important basis to evaluate the quality of SBFG.2 Effect of SBFG on proliferation and apoptosis of HepG2 cellsThe effect of SBFG on HepG2 cells proliferation was analyzed by MTT method.Apoptosis was observed by Hoechst 33342 staining method.The mitochondria membrane potential(MMP),cytochrome C,membrane permeability,nuclear size,nuclear DNA content,and changes of cell count was tested by high content screening assays(HCS).Results: 1 The results of quality study 1.1 Qualitative identification resultsSet up the thin layer identification method of SBFG.1.2 High performance liquid chromatography(HPLC)method for determining the content of BaicalinUsing HPLC method to establish the determination method of the content of baicalin in SBFG.Methodology test showed that baicalin sample quantity within the scope of 0.0930 to 0.4650 μg,had good linear relationship,the regression equation is:Y=3.5326 ×106 X+2.9300 ×10,r=0.9999;baicalin average sample recovery rate was 102.19%(n=6).The RSD was 1.71%,less than 2.0%,which show that the method is accurate and reliable,good repeatability,which can realize to control the content of baicalin in SBFG.2 The influence of SBGF on HepG2 cell proliferationSBFG obviously inhibited the proliferation of HepG2(P<0.01),inhibition rate was concentration dependent,IC50 value was(0.383 + 0.016)g/L.Morphological changes of apoptosis after Hoechst 33342 staining,the nuclei of HepG2 cells became chromatoid,condensed or fragmented.HCS indicated that MMP and cytochrome C release were significantly increased at the concentration of 0.1 g/L of SBFG(P<0.01),0.4 g/L of SBFG led to a lower MMP and higher cytochrome C release,meanwhile,the membrane permeability was significantly increased at this concentration(P<0.01),nuclear size was decreased significantly and the nuclear DNA content was obviously increased(P<0.01),reduction of the cell number was observed in the concentration of 1.6 g/L of SBFG(P<0.01).Conclusion: This study established a TLC identification method for SBGF and the content of baicalin in HPLC method;and found SBGF can obviously inhibit human hepatocellular carcinoma HepG2 cells proliferation,induce apoptosis,significant antitumor activity,the mechanism related to decrease MMP,accelerate cytochrome C release and increase membrane permeability;the related research results provides the experimental basis for the quality control and clinical application of SBGF.