Establishment Of A Model For Screening ARIs And Drugs Screening

Diabetes mellitus(DM) is a serious disease with severe complications. Polyol pathway plays an important role during the development of degenerative diabetic complications.Aldose reductase(AR) is key rate-limiting enzyme in polyol pathway,and it is the target enzyme for treatment of DM complications.Aldose reductase reduces excess D-glucose into D-sorbitol with the associated conversion of NADPH to NADP⁺.Available experimental data link D-glucose metabolism to long-term diabetic complications such as cataract,neuropathy, nephropathy,and retinopathy.Aldose reductase inhibitors should therefore be able to safely prevent or arrest the development of diabetic complications.Because of the scarce of suitable drugs for treatment of DM complications and the now existing drug Sorbinil and Tolrestat have some side effects,it is important for looking for new aldose reductase inhibitor with more safety and better effects.The aim of this study was to develop a convenient and reliable model for aldose reductase inhibitor screening.Base on this model,a number of drugs were screened.This study included three parts:1,In this study,we developed a simple and easy method for the extraction, isolation and purification of AR.Fresh calf lenses were homogenised in 3 volumes of phosphate buffer in grind.The crude extract was centrifuged at a high speed to remove insoluble material.Saturated ammonium sulfate was added to the supernatant fluid to 40%,50%and 75%saturation for centrifugal salt fractionation. The sediment obtained finally was dissolved in phosphate buffer,and then desalted with centrifugal ultrafiltration.Crude AR was identified with SDS polyacrylamide gel electrophoresis and the concentration of crude protein was determined by biuret method.2,We established a microamount model for screening aldose reductase inhibitor from the traditional Chinese medicines or naturally occurring drugs. NADPH is a coenzyme,which has strong reducibility.And so it is easily oxidized. The absorbance of enzymatic reaction system will decrease with the oxidation of NADPH.Base on this theory,the activity of AR can be determined by the oxidation of NADPH.A microamount model has been set up in 96 holes plate,using DL-glyceraldehydes as substrate,NADPH as coenzyme.The volume of enzymatic reaction was 300ul,including 0.03 mol/L the phosphate buffer solution,0.2 mol/L Li₂SO4,0.01mol/L 2-mercaptoethanol,0.25mmol/L NADPH,,3.5 mmol/L DL-glyceric aldehyde,3mg AR.The temperature of rection was 35℃.The unit of activity of AR was defined as a change in absorbance of 0.001 unit per minute.This model has the merit of high performance and high sensitiveness.Enzymatic rection system was stable and the activity of AR was 43.2 units after optimization, which could be used for screening drugs.3,Base on this model,a number ofextractives of medicines were screened. Results suggested that some samples can significantly inhibit the activity of AR.4,panax notoginseng(Burk.) F.H.Che has been chosen frome the some samples can significantly inhibit the activity of AR,I set up the process chart of preparation for panax notoginseng(Burk.) F.H.Che and concentration measurement of panax notoginseng.Results suggested that panax notoginseng(Burk.) F.H.Che can significantly inhibit the activity of AR.In this study,we developed a method for the extraction,isolation and purification of AR and a model for screening ARIs,which provided a way of screening the drugs for anti-diabetic complication.Base on this model,several Chinese medicinal materials were sreened and suggested inhibition on AR,which have perspective for being developed into drugs for.anti-diabetic complication.