Preparation Of Proteins From Chlorella Vulgaris And The Effect On Oxidative Stress And Glucose Metabolism

The incidence rate of type 2 diabetes is increasing year by year,and it shows a typical trend of younger age,which seriously affects the health problems of human beings,and at the same time,they are faced with huge economic burden and social pressure.All the current therapeutic drugs have side effects,so it is of practical significance to search for natural products with hypoglycemic effect as a treatment.CVP was used as the research object to explore the optimal extraction method and conditions,measure the antioxidant activity in vivo and in vitro,detect the expression of genes related to oxidative stress to explore the possible mechanism of the antioxidant effect,and conduct a preliminary study on the hypoglycemic effect to explore the CVP become potential natural hypoglycemic drugs.The main research results are as follows:1.Taking the extraction rate of CVP as the index,five methods of alternate freeze-thaw,hot water extraction,ultrasonic extraction,alkaline solution extraction and ultrasonic combined with alkaline solution extraction were compared.It is found that the extraction method of ultrasonic combined with alkaline solution is the best.According to the results of single factor experiment,Plackett-Burman experiment and Box-Benhnken experiment,the best extraction conditions of CVP were obtained.The ratio of material to liquid was 1:47,the extraction temperature was 40?,and the mass fraction of Na OH was 2%.On this condition,the extraction rate of CVP could reach53.42±1.25%.2.In vitro antioxidant activity of CVP was determined by colourimetry.The results showed that CVP could improve the reducing power and superoxide free radical scavenging rate,with concentration dependence,but the effect was lower than that of Vc at the same concentration.CVP had scavenging effect on ABTS free radical and DPPH·,and the effect of Vc was better than that of CVP under the same concentration.Under the condition of light,the formation of hydroxyl radicals was promoted,but under the condition of avoiding light,it had the scavenging ability and with concentration dependence,but the effect was lower than that of Vc.CVP had the effect of enhancing the metal chelation ability,had the concentration dependence,and the effect was better than that of EDTA at the same concentration.C.elegans was used as a model organism to determine the antioxidant capacity of CVP in vivo.Results showed that it could improve the enzyme activity of SOD,GR,GSH-Px,CAT,and the content of GSH,and reduce the content of MDA and ROS,all of which were concentration-dependent,but the effect was lower than that of Vc.3.The lifespan of C.elegans under oxidative stress induced by 0.01 mol/L H₂O₂or 83.3 mg/m L methyl purpurin was detected,and the expression levels of antioxidant-related genes in c.elegans under oxidative stress induced by 77.15 mg/m L methyl purpurin were determined by real-time PCR.The results showed that CVP could prolong the lifespan of C.elegans under oxidative stress.Real-time PCR results showed that CVP could increase the expression level of sod-1,sod-2,sod-3,sir-2.1 and skn-1 genes in C.elegans under oxidative stress.4.Hep G2 cells were used to explore the conditions for the establishment of insulin resistance model and determine the stability.It was concluded that the optimal conditions for the establishment of bovine insulin were 10^-6mol/L for 36 h and could be maintained for 36 h.With 100?g/m L,200?g/m L and 300?g/m L of CVP or metformin to develop IR-Hep G2 after 36 h,founded that both of them could relieve insulin resistance,promote the absorption and utilization of glucose by cells,improve glycogen synthesis,have hypoglycemic effect,and the effect of metformin is better than that of CVP under the same concentration.However,CVP had less inhibitory effect on the proliferation of IR-hep G2 cells.Therefore,CVP may be developed as a potential new natural medicine for the treatment of insulin resistance in diabetic patients.