| Diabetes is a complex pathogenesis of metabolic diseases, with longer duration of diabetes, it should be complicated by concurrent diseases of the eye, kidney and other organs. As a a typical representative of ophthalmic diseases among all the complications, diabetic retinopathy (DR) is one of the main factors that cause blindness. Studies have shown that the polyol pathway hyperactivity is closely related to DR. Numerous clinical studies have shown that Fufang Xueshuantong Capsule has significant treatment effect on DR, but the material foundation of its efficacy is not very clear, so the production process can not be refined, USP quality standards is inadequate, then prevents its application in clinical and expansion in the international market. Therefore, based on the preliminary studies that our research team done, starting from the level of the effective component, aldose reductase (AR, the first rate-limiting enzyme of polyol pathway) was chosen as the target enzyme, this article aimed at studying inhibition of the main effective components of the Fufang Xueshuantong capsule and their different constituent ratio on AR. So as to make the material foundation of its efficacy more clear, provide a certain basis for its future development direction at the same time.The experiment was divided into four parts:1In this study,we developed a simple and easy method for extraction, separation and purification of the aldose reductase. With fresh SD rat lens as material, W:V (1:3) ratio of pH6.2(research about AR has shown that the best survival pH for AR was6.2) phosphate buffer solution was added, then was grinded in a mortar at the temperature of less than4℃, the crude extract was centrifuged at a high speed to remove insoluble material. Saturated ammonium sulfate was added to the supernatant, to40% saturation, centrifuged to get theupernatant.Repeat this step to make ammonium sulfate saturation reach50%,75%saturation respectively, so the protein was progressively purified. Finally the precipitate was dissolved with the same buffer, dialysed for12h in a refrigerator, the dialysis fluid was changed every4h. The protein content of the crude enzyme was determined by the classic measurement-Coomassie brilliant blue method.2The article established a method for determining the activity of AR by ultraviolet spectrophotometer method. With NADPH (characteristic absorption is at340nm) as a coenzyme, and DL-glyceraldehyde as substrate, DL-glyceraldehyde was reduced to glycerin by with the catalyzing of AR, on the other hand, NADPH was converted to NADP+which doesn’t have absorption at340nm. Therefore, the activity of AR can be determined based on the descent of absorbance rate. Enzymatic reaction conditions such as the concentration of NADPH, the amount of the enzyme, reaction temperature, the concentration of DL-glyceraldehyde were inspected respectively, the reaction system was optimized. The final total volume of the reaction system was1mL, the reaction system was as follows:AR crude enzyme (100μL),0.0156mmol/L NADPH,100mmol/L pH6.2phosphate buffer,400mmol/L of lithium sulfate,10mmol/L β-mercaptoethanol,0.2 mmol/L DL-glyceraldehyde and the reaction temperature was37℃. The activity of the enzyme was defined as a change in absorbance of0.001units under reaction temperature, namely enzyme activity unit=△OD X1000, AR activity was36units after optimizing, which can be used for the research.3According to the method we established, selected components from Fufang Xueshuantong capsule-notoginsenoside R1, ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rd ginsenoside Re, salvianolic acid B, cryptotanshinone, tanshinone I, astragaloside IV, harpagoside. On this basis we added ginsenosides Rg3, ginsenosides Rh1, ginsenoside Rh12, ginsenoside F1, ginsenoside F2, protopanaxadiol and protopanaxatriol, which were reported as metabolic components of PNS, there were17kind of components in total. Their inhibition on AR were studied, IC50of R1, Rgi, Rb1, Rd, salvianolic acid B, tanshinone I, cryptotanshinone, astragaloside IV, harpagoside, F1, F2, Rg3, Rh1, Rh2, protopanaxadiol, protopanaxatriol were0.180、0.109、1.01、0.15、0.799、0.0652、0.0742、0.315、0.00385、0.720、0.582、0.202、0.547、0.494、0.260、0.189μmol/mL respectively, Re had no significant effect, IC50of positive control-quercetin was0.205μmol/mL.4Select the main eight kinds of prototype components from the seventeen kinds of components:R1, Rg1, Rb1, salvianolic acid B, tanshinone I, cryptotanshinone, astragaloside IV, harpagoside. The experiments were arranged by orthogonal experimental design which had eight factors and three levels to determine the inhibition of different ratios of the components on AR. The results showed that:The4kind of components-R1, Rg1and tanshinone I had statistically significant impact on the inhibition rate, and the strength of impact was R1> Rg1> harpagoside> tanshinone I(P<0.05); the multiple linear regression equation we had established was:y=-0.116+8.86R1+6.48Rg1+3.00tanshinone I+0.773harpagoside, r2=0.912, P<0.01, the equation was credible; the optimum composition ratio was:R1, Rg1, Rb1,tanshinone I, cryptotanshinone, astragaloside IV, harpagoside=1.00:0.0925:3.33:2.22:0.0534:0.251:0.00753:0.599.This article established a practicalextraction and purification method of aldose reductase, measurement of protein content and activity of AR, then the main effective components of Fufang Xueshuantong capsule and different ratio of the components were chosen to be determined the inhibition on AR. We preliminary elaborated the material foundation of Fufang Xueshuantong capsule so as to make it better applied to clinical. |
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