Preparation Of Acaudina Molpadioides Collagen Peptide,evaluation Of Antioxidant Activity And Mechanism Of Action

Acaudina molpadioides are widespread in the eastern coastal areas of China,and their body walls contain a large amount of collagen,which is a high-quality raw material for the preparation of collagen peptides.In this subject,the collagen peptide was prepared by microwave-assisted alkaline protease hydrolysis of collagen from Acaudina molpadioides.Its antioxidant activity and mechanism were evaluated and explored in vivo and in vitro.The main experimental results are as follows:(1)The collagen of Acaudina molpadioides was extracted by enzymatic method and its physiochemical properties were identified.The results show that the collagen of Acaudina molpadioides had a maximum ultraviolet absorption peak at 232 nm,and 5main amide bands were observed in the infrared spectrum.The isoelectric point of collagen was about p H 4.25 by zeta potential analysis.The collagen was hydrolyzed by alkaline protease under microwave assisted to prepare the collagen peptides.The results show that 250 W was the best microwave power based on the OH scavenging rate.The OH scavenging rate of collagen peptides prepared under this power increased from70.3%to 96.2%,and the content of oligopeptides(Mw?1 k Da,AMP)also increased by30.2%.(2)AMP revealed the good OH and DPPH scavenging activities,with scavenging rates of 93.1%and 41.2%,respectively.The oxidative damage model of RAW264.7 cells was established by H₂O₂ treatment and used to evaluate the antioxidant activity of AMP and its mechanism.The results show that AMP pretreatment could promote the proliferation of RAW264.7 cells,reduce the levels of reactive oxygen species(ROS)and malondialdehyde(MDA),and increase the activities of glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)in the cells.Western blot experiments show that AMP could inhibit the phosphorylation of JNK,ERK and p38,and up-regulate the expression of p-PI3K and p-AKT proteins,thereby activating the PI3K/AKT pathway.In addition,AMP could significantly increase Nrf2,HO-1 and NQO1 protein expression,and reduce Keap1 protein expression,thereby activating Keap1/Nrf2-ARE pathway.(3)The acute liver injury of mice was establised by CCl₄ treatment in order to evaluate the antioxidant activity of AMP in vivo and explore its mechanism.The results show that the liver of AMP and V_E pretreated mice brighter than model group,and their cell morphology tended to be normal.Furthermore,AMP could significantly reduce the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and MDA,improve catalase(CAT),SOD and GSH-Px activities in mice.Western Blot experiment show that AMP could inhibit the phosphorylation of JNK,ERK and p38,up-regulate the expression of p-PI3K and p-AKT protein,thereby activating PI3K/AKT pathway.In addition,AMP could significantly increase the protein expression of Nrf2,HO-1 and NQO1,and reduce Keap1,thereby activating the Keap1/Nrf2-ARE pathway.In summary,AMP activated the Keap1/Nrf2-ARE signaling pathway by activating the PI3K/AKT signaling pathway.Up-regulation of antioxidant enzymes could significantly reduce the level of oxidative stress,leading to the inhibition of the phosphorylation of the MAPKs signaling pathway.Our results show that AMP revealed the antioxidative activity via PI3K/AKT,Keap1/Nrf2-ARE and MAPKs signaling pathways.