Study On The Effect Of The Extraction And Purification Technology Of "Qian Jin Wen Wu Tang" And The Activity Of High Glucose-induced Hk-2 Cell

Objective:Guided by Korean medicine theory,the extraction and purification technology of traditional Korean medicine "QIAN JIN WEN WU TANG"(QJWWT)was optimized,the effect of different isolated components on the oxidative stress and the expression of alpha smooth muscle actin(α-SMA)in normal human proximal tubular epithelial cells(HK-2)cultured in vitro was studied,in order to provide the experiment basis for the development of this prescription into multi-component Chinese Medicine treating diabetic nephropathy.Method:With the extraction of total polysaccharides,total flavonoids and total saponins as index,optimize the extraction process parameters through the design of orthogonal text,with clarity,stability and the retention of total flavonoids,total polysaccharides,and total saponins as index,refine it with the adsorption clarification refining and determine the refining process parameters;after separating the effective part of "QJWWT" with the use of macroporous adsorption resin column,7 groups of fractions were isolated,determined the content ratio among total polysaccharides,total flavonoids and total saponins in each group of fractions.HK-2 cells was cultured under high glucose conditions in vitro,components at different concentration and total extract were added,after incubated for 24h,the cellular supernatant was collected,The malondialdehyde(MDA)content is determined by thiobarbituric acid method and superoxide dismutase(SOD)content is determined by xanthine oxidase method;Use Western Blot to detect the expression of α-SMA of HK-2 cells after incubated for 24h.Results:The optimized extraction process parameters:extract twice by water decocting method,solid-liquid ratio 1:10、1.5h,solid-liquid ratio 1:8、1h,respectively;the optimized adsorption clarifier method parameters:solid-liquid ratio 1:1,adsorption clarifier component A dosage 7%,adsorption clarifier component B dosage 3.5%,temperature 60℃.7 groups of fractions were obtained from the refined decoction eluted by ethanol at varying concentrations through macroporous adsorptive resins,The content ratio of total polysaccharides,total flavonoids and total saponins from group 1 to group 4 is 1:0.012:0.067、1:0.098:0.072、1:1:0.238、1:0.727:0.273,no polysaccharide was found in group 5、6、7;The content ratio of total flavonoids and total saponins from group 5 to group 7 is 1:0.833、1:0.974、1:0.3.Compared with the normal group,there was no significant difference in the content of MDA of Osmotic pressure control group,and the content of MDA in high glucose group(HG)was increased(P<0.05);Compared with HG group,the content of MDA in the medication administration group was decreased,there were significant difference in the partial adminstration concentration from group 1 to group 5 and all the adminstration concentration of group 6、7、8(P<0.05).Compared with NG group,there was no significant difference in the the activity of SOD in HG group,and the content of SOD in HG group was declined(P<0.05),aside from group 6,the content of SOD of other medication administration group increased,extreme significant difference was found from partial adminstration concentration of group 2 to group 4(P<0.01).The experiment of Western Blot showed,a-SMA protein of NG group had micro expression,compared with NG group,there were no significant difference found in MAN group in the expression of α-SMA,compared with NG group,the expression of α-SMA in HG group increased(P<0.01);compared with HG group.The expressin of α-SMA in adminstration group was inhibited,the protein content increased(P<0.01).Conclusion:The extration and refining process of "QJWWT" is stable and appliable,acting on the high glucose-induced HK-2 cell,isolated effective component can enhance the cellular SOD activity,decrease the production of MDA,and downregulate the expression of α-SMA in HK-2.Indicate that the effective part of this decoction can ameliorate renal fibrosis and improve the renal function through inhibiting the oxidative stress in high glucose-induced HK-2 cell and the differentiation from renaltubular epithelial cells to mesenchymal cells.