Study On Antioxidant Activity In Vivo And Vitro,and Protective Effects On Acute Liver Injury Of Peptide From Corbicula Fluminea

Oxidation is a relatively common activity occurring in living organisms.Reactive oxygen species（ROS）and reactive nitrogen species（RN）,as the main products of oxidation,play a dual role in physiological functions.The application of chemically modified antioxidants is restricted due to their potential harm to human health.Therefore,it is valuable to obtain natural antioxidants with high safety from animals and plants.Corbicula fluminea is a bivalve shellfish,widely distributed in Chinese inland waters,which has a high nutritional value.In this study,antioxidant peptides were extracted from Corbicula fluminea by separation and purification technology,and their protective effects on chemical acute liver injury were further studied.Firstly,the nutritional components and quality of the soft part of Corbicula fluminea were compared and analyzed.The results showed that the moisture content was 86.64%,the crude protein,crude fat and ash content in the dry sample were 62.80%,11.67%and4.82%,respectively.There were 17 kinds of amino acids（557.87 mg/g）in the samples,including 6 essential amino acids（206.77 mg/g）,accounting for 37.06%of the total amount of amino acids.Especially,the proportion of essential amino acids conformed to FAO/WHO recommended standards.Corbicula fluminea was also abundant in minerals,especially Cu and Zn elements,which are higher than common shellfish.In conclusion,Corbicula fluminea is a kind of mollusk with high nutritional value.The trypsin was chosen to hydrolyze the zing Corbicula fluminea,and the short peptide recovery rate,hydrolysis degree and DPPH free radical scavenging ability of hydrolysates were evaluated.Then,the hydrolysis process parameters were determined by single factor experiment and response surface optimization.The optimum hydrolyzing conditions of trypsin were in a 45°C environment,pH 7.5,solid-liquid ratio（w/v）1:7.5,enzyme dosage 2020 U/g.At this condition,the DPPH free radicals scavenging rate of hydrolysates was 62.22%.The hydrolysates were separated into three fractions with molecular weight of>10ku,10 ku-5 ku and<5 ku by ultrafiltration method,and the t the fraction with<5 ku showed the highest antioxidant activity in vitro.The four fractions ofⅠ,Ⅱ,ⅢandⅣwere obtained by Sephadex G-25 gel chromatography,and cell experiments showed that the fractionⅠhad the strongest ability to protect HepG-2 cells against H₂O₂-induced oxidative stress.Then,the fractionⅠwas separated into nine fractions by DEAE anion exchange chromatography,and the F_Ⅰ-3 had the highest DPPH radical scavenging capacity.Finally,the target polypeptide X was purified by RP-HPLC.Its molecular weight was 988.3 u and its amino acid sequence was KGPAPFYPL.The liver injury mice model induced by CCl₄ was used to study the effects of peptide on enhancing antioxidative ability of defense system,inhibiting inflammatory response and hepatocyte apoptosis.The results showed that the peptide had no toxic effect on mice,and could reduce liver index,improve spleen index,reduce ALT and AST contents in serum.Meanwhile,peptide administration could increase SOD and CAT activity in liver,and reduce MDA content.HE staining,immunohistochemistry and TUNEL staining showed that the peptide could significantly improve liver tissue damage,reduce the production of inflammatory factors,and inhibit hepatocyte apoptosis.In conclusion,the peptides obtain from Corbicula fluminea hydrolysate have protective effects on liver injury of mice.