The Regulatory Mechanism Of Methylated Metabolites Of Chicoric Acid On Hydrogen Peroxide (H₂O₂)-induced Oxidative Stress In HepG2 Cells

Cichoric acid(CA)is a caffeic acid derivative extracted from natural plants,such as Echinacea purpurea and chicory,which has the biological and pharmacological properties,including anti-oxidant,anti-inflammatory,hypoglycemic and insulin resistance.Hence,CA has been widely used as a food antioxidant and immune system enhancer.However,the biological activity of plant polyphenols in the body depends on their pharmacokinetic properties,including absorption,distribution,metabolism and excretion.Study had shown that CA undergoes extensive metabolism in the gastrointestinal tract or liver before entering the body's circulation.Its metabolic pathways mainly include hydrolysis,reduction,methylation,sulfonation,glucuronidation,acetylation,isomerization,and deoxygenation.The actual compound that CA exerts its biological activity in the body may be the parent or its metabolite.Methylation could increase the lipophilicity and cell permeability of polyphenols,thereby increasing the bioavailability of polyphenols in the body and better exerting their biological activity functions.Our recent research elucidated that methylated metabolites of CA have a higher concentration in the blood.Compared with CA,its biological activity and molecular mechanism are still unclear.Based on this,this study prepared methylated metabolites of CA by in vitro incubation,explored the structure-activity relationship between its structure and antioxidant activity,and clarified its protective effect and molecular mechanism on cell oxidative stress damage.The main research contents and results are as follows:(1)In order to obtain the methylated metabolites of CA,the major methylated metabolites of CA were isolated from an in vitro co-incubation system by preparative high-performance liquid chromatography(HPLC)and identified their structures by liquid chromatography quadrupole time-of-flight mass spectrometry(LC-QTOF-MS/MS).(2)In order to explore the in vitro antioxidant activity of methylation metabolites of CA,the scavenging ability of M1 and M2 to free radicals and their inhibitory effects on protein damage and lipid peroxidation damage were determined.The results showed that M1 and M2 both showed stronger DPPH·,·OH and ABTS⁺·scavenging abilities compared with CA,and had a significant inhibitory effect on protein damage and lipid peroxidation damage.(3)The antioxidant activities of the methylated metabolites of CA were evaluated after H₂O₂-induced oxidative stress in Hep G2 cells.The results indicated that both M1 and M2had better antioxidant capacities than CA by increasing cell viability,improving mitochondrial function,and balancing cellular redox status and could reducing the phosphorylation levels of MAPK/p38,MAPK/JNK,and NF-?B/p65.In addition,M1 and M2 can exert their strong antioxidant activity by mediating the Keap1/Nrf2 pathway and regulating the expression level of a serious of downstream antioxidant enzymes.Overall,our findings indicated that M1 and M2 were more effective than CA in alleviating H₂O₂-induced oxidative stress,and the higher the degree of methylation,the stronger the antioxidant activity.The current research indicated that the methylated metabolites of CA may be the potential be the candidates that responsible for the biological efficacies attributed to CA.The present research helped clarify specific mechanism of action of CA in vivo and provided the basis for future research on potential application value of methylated metabolites of CA.