Comparison Of Commonly Used Methods For Measuring Antioxidant Peptide Activity

Antioxidant peptide was a biologically active peptide that inhibited oxidative stress or had a function of scavenging free radicals.It belonged to a kind of cell penetrating peptide and had attracted the attention of researchers in recent years.How to determine the activitives of antioxidant peptides had become the focus of attention.In response to this problem,different antioxidant peptides were used to compare the commonly used chemical,cytological and animal assay methods.The main research contented included the following three aspects:Comparison of reproducibility of antioxidant peptide chemical evaluation methods.Glutathione was used to compare six chemical methods for the determination of antioxidant peptide.The coefficient of variation?CV?of antioxidant peptide activity determined by DPPH·method,ABTS⁺method,·O^2-method,·OH method,Fe³⁺reduction method and Cu²⁺reduction method were respectively 2.34%,4.90%,8.08%,34.81%,141.15%and 157.69%;the standard deviations were 0.36,0.05,5.89,10.21,0.48 and5.33,respectively.The coefficient of variation and standard deviations of DPPH·method were significantly lower than those of the other five methods,and had high reproducibility.There was no significant difference among the different researchers in the coefficient of variation of antioxidant activitves of glutathione determined by DPPH·method.At the same time,the activitives of 467 and 568 antioxidant peptides synthesized by different experimenters were compared by this method,and the results showed no significant difference.Comparison of CAA method and DPPH·method.The harmless concentration range of antioxidant peptides was determined to be 0.5-4.0?g/mL by cytotoxicity test.Further,the antioxidant activity of the antioxidant peptide was measured by a Cellular Antioxidant Activity?CAA?method.The order of antioxidant activity was the same as that of DPPH·,all of them were:glutathione>antioxidant peptide 467>antioxidant peptide 568,but the difference of antioxidant activity determined by CAA method was much smaller than DPPH·method.Comparison of animal experiments with DPPH·method.The mouse aging model was established by D-galactose,and different antioxidant peptides were administered to mice by intragastric and intraperitoneal injection.The results showed that both the intragastric group and the injection group,after 7 weeks of administration,all of them could significantly or significantly?P<0.05 or P<0.01?decrease the content of malonaldehyde?MDA?,increase the total superoxide dismutase?SOD?and glutathione?GSH?trace level in the serum and liver of D-galactose aging model mice,and there was a dose-dependent effect.The decrease rate of MDA,the increase rate of SOD and the increase rate of GSH in glutathione group were>antioxidant peptide 467>antioxidant peptide 568,that was,the antioxidant capacity in vivo was glutathione>antioxidant peptide 467>antioxidant peptide 568,the same order as DPPH·method,the difference was close to CAA method.Three methods were used to compare the soy protein antioxidant peptide and egg white antioxidant peptide of unknown structure,which was consistent with the above results.Therefore,animal experimental methods could accurately reflect antioxidant peptide activity in numerous assays,however,the cycle was long,the cost was high,and the operation was complicated;the CAA method based on cell level had the advantages of simple operation,short test time,less material consumption and strong repeatability,which was highly consistent with animal experimental results and was a good method to evaluate the activity of antioxidant peptides;DPPH·method could basically react with the sequence of antioxidant peptides although it had great difference with the former two methods in evaluating the activity of antioxidant peptides,because of its optimal reproducibility,DPPH·method could be used as the basic method for screening antioxidant peptides.