A Comparative Study Of Activity And Safety Between EGCG And EGCG Auto-oxidation Products (EAOPs)

(-)-epigallocatechin-3-gallate,the major and most bioactive polyphenol found in tea,contains a number of ortho or meta phenolic hydroxyl groups.EGCG auto-oxidation can be realized by deprotonation reaction under alkaline conditions.EGCG auto-oxidation products(EAOPs)has been reported to exhibit same or limited bioactivity on Tca8113 and CT26 proliferation inhibition and cytotoxicity compared to native EGCG,comparable activity to produce reactive oxygen species and inhibit of thioredoxin reductase,enhanced capacity to deplete cysteine thiol groups.This study further compares the modified protein ability of EGCG and EAOP,as well as the safety in vivo.HPLC analysis indicated that the speed of EGCG auto-oxidation was dependent on time and concentration in vitro.On the other hand,UPLC-QQQ-MS/MS analysis showed that EGCG levels decreased time-dependently over 3 hours in plasma and liver.We inferred that EGCG is likely to exert its physiological function in the form of oxidized product in vivo.The structure of EAOP for 2 hours was identified by UPLC-ESI-MS and UPLC-MS/MS.We found that the EGCG is able to form GCG,theasinensin A(or D),dimer quinone and some other polyphenols.EGCG oxidation produces EGCG quinone,which covalently modifies cysteinyl proteins forming quinoprotein adduct and loss protein activity.The activity of EGCG-modified GAPDH protein increased with the prolongation of incubation time.Compared with EGCG,GAPDH protein modifiation of the long-time EAOPs gradually decreased.It can be concluded that the long-time EAOPs maintain the biological activity compare to native EGCG,but safer than EGCG in vivo.The effect of EGCG and EAOP-16 h on the survival of KM mice was compared in acute toxicity model.The result showed that the survival rate of EAOP group was slightly higher than EGCG,which means EAOP toxicity is weaker.What is more,the influences of EGCG and EAOP-16 h on the physiological characteristics of KM mice was compared in subacute toxicity model.Firstly,the weight loss of EAOP treated mice was lower than that of EGCG group.Secondly,ALT,AST and ?-H2 AX assay indicated the liver injury of mice treated with EAOP was lighter than EGCG group.Thirdly,EAOP possess equivalent response of Trx and Grx system to EGCG,including thioredoxin reductase(Trx R),thioredoxin(Trx),glutathione reductase(GR)and glutaredoxin(Grx).Fourthly,EAOP has a strong capacity to impact p53 pathway,and the response of various p53 target genes to the EAOP but not EGCG implies that the EAOP is more active than EGCG.Fifthly,EAOP has a remarkable ability to modify liver protein.Overall,EAOP showed reduced toxic reactions compared to EGCG.These insights into the action of EAOP may provide a novel route of EGCG usage.