Differerces In Antioxidant,Anti-aging Activities Of Astaxanthin Isomers And Molecular Mechanism Involved

Astaxanthin is a kind of carotenoid with strong antioxidant activity,which plays an important role on the anti-aging activity and maintains the normal physiological function of the body.There are many double bonds in the molecule of astaxanthin and there are different geometric isomers in nature.Astaxanthin is predominantly trans in nature.Bescides,in processing,all-trans astaxanthin is easy to convert into a different cis isomers.At present,the study on the antioxidant activity of different geometric isomers of astaxanthin has not yet been conclusive,and its anti-oxidation and and anti-aging activity is not clear.Therefore,the in vitro antioxidant chemistry evaluation method,Caenorhabditis elegans and RNA-seq were used to reveal the antioxidant,anti-aging activity differences and mechanism of them among 9-cis,13-cis and all-trans astaxanthin.It plays an important role in both revealing the structure-activity relationship of astaxanthin and guiding people to a reasonable diet of astaxanthin.The main research contents and results are as follows:(1)Transformation,identification and separation and purification of different astaxanthin isomers.The all-trans astaxanthin was subjected to cis isomers by solvent heating and iodine irradiation.HPLC-APCI-MS and Q values were used to identify isomers which were commonly used at home and abroad.The result confirmed that the cis astaxanthins was 9-cis and 13-cis astaxanthin.They were purified by preparative liquid phase to obtain 9-cis astaxanthin and 13-cis astaxanthin monomer with purity of 97% and 88%.(2)Evaluation on antioxidant activity in vitro of different geometric isomers.DPPH free radical scavenging,ABTS free radical scavenging and ORAC were adopted to evaluate the antioxidant activity of different isomers of astaxanthin.There was no significant difference in scavenging DPPH free radicals between different geometric iso mers of astaxanthin(p>0.05).9-cis astaxanthin was significantly higher than all-trans and 13-cis astaxanthin in the removal of ABTS free radicals(p<0.05)and its total antioxidant capacity index(ORAC)was significantly higher than that of 13-cis astaxanthin(p<0.05).Consolidate the above results,the order of antioxidant activity of astaxanthin in different isomers was: 9-cis> all trans> 13-cis.(3)Comparison of antioxidant and anti-aging activities of Astaxanthin with different isomers.Evaluation of antioxidant and anti-aging activity by N2 Caenorhabditis elegans model under normal culture conditions and oxidative damage environment to evaluate the effects of life span,physiological indexes,antioxidant markers and astaxanthin configuration in vivo.Under normal culture conditions,the average life of 9-cis,13-cis and all-trans was improved by 59.39%,24.99% and 30.43%,respectively.There was a significant difference in the lifespan of each experimental group(p<0.05).There was no significant difference in physiological index(fecundity,sinusoidal frequency and mobility)between different isomers of astaxanthin(p>0.05),but there was a significant difference in the swing frequency of the head(p<0.05).Compared with the control group,9-cis and all-trans astaxanthin isomers of astaxanthin could significantly reduce the intestinal lipofuscin(p<0.05),all-trans and 9-cis astaxanthin significantly reduced the accumulation of ROS in Caenorhabditis elegans(p<0.05),and was significantly better than that o f 13-cis(p<0.05),but they have no significant difference(p>0.05).The activity of SOD and CAT in Caenorhabditis elegans was significantly increased by different geometric isomers(p<0.05).Different geometric isomers of astaxanthin in C.elegans can still maintain the original configuration.In the oxidative damage environment,There was no significant effect of astaxanthin on the survival rate of Caenorhabditis elegans(p>0.05).Compared with the control group,9-cis and all-trans astaxanthin could significantly reduce the accumulation of ROS in C.elegans,which respectively decreased by 56.05% and 15.07%.In addition,the 9-cis astaxanthin group and other experimental groups have significantly different(p<0.05).Taken together,the order of antioxidant and anti-aging activity of astaxanthin in vivo was 9-cis> all-trans> 13-cis,which was consistent with the results of in vitro experiments.(4)Antioxidant and anti-aging mechanism of different geometric isomers in C.elegans.The effects of three different isomers of astaxanthin on gene expression and metabolic pathway of C.elegans were studied by RNA-seq.In the group of 9-cis,substantial changes in the expression level of 227 genes were detected(187 up-regulated,40 down-regulated).In the group of 13-cis,substantial changes in the expression level of 315 genes were detected(166 up-regulated,150 down-regulated).In the group of all-trans,substantial changes in the expression level of 482 genes were detected(336 up-regulated,146 down-regulated).There were 4 genes correlated with antioxidant and aging phenotypic of 23 common significant differentially expressed genes which were phenotypically related to Longevity regulating pathway and oxidoreductase pathways.These genes revealed that astaxanthin may be involved in the regulation of oxidative reduction in C.elegans,to play a role in extending life.At the same time,different isomers of astaxanthin in the antioxidant and anti-aging mechanism also exists a certain difference.Through the KEGG pathway analysis,different isomers of astaxanthin is enriched in the longevity regulation pathway and pathways of antioxidant enzymes,but their action sites are not the same.Compared to 9-cis astaxanthin,13-cis and all-trans are involved in encoding more Cytochrome P450 genes.Compared to other groups,13-cis group have some genes encoding peroxidase,such as dhrs-4,F59F4.1,daf-22 and so on.But the significant differences genes of 13-cis did not participate in C.elegans longevity regulation pathway.Compared with 9-cis and 13-cis,all-trans astaxanthin groups were different in the longevity pathway.fat-5 is associated with lipid metabolism,and pha-4 is associated with dietary restrictions whichcan reduce damage in the peroxide conditions and extend the life of C.elegans.