Separation and antioxidant activities of new acetylated EGCG compounds

Acetylation could improve the bioavailability of (-)-Epigallocatechin-3-Gallate (EGCG), but the relationship of substitution degree and antioxidant capacity of acetylated EGCG was unclear. The acetylated EGCG products were separated by preparation high performance liquid chromatography (HPLC). Two mono substituted acetylated EGCG, three substituted acetylated EGCG (T-AcE), eight substituted acetylated EGCG (E-AcE) and (-)-Epigallocatechin gallate (EGCG) were isolated. The 7-acetyl-EGCG (S7-ACEGCG) and 7-acetyl-EGCG (T-AcE) were identified for the first time. The antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities of EGCG decreased significantly after acetylation modification. The more EGCG acetylation modification sites, the lower the total antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities. The antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities of 5-acetyl-EGCG (S5-ACE) were higher than 7-acetyl-EGCG (S7-AcE). Combining all the results in this and previous studies, acetylation modification is not conducive to the performance of EGCG antioxidant capacity.


Antioxidant activity analysis
The antioxidant activities of Fr. 1-Fr. 4 solution samples at 500 μg/mL were studied with total antioxidant capacity (T-AOC) kit, Scavenging superoxide anion capacity Kit, and Scavenging scavenging hydroxyl free radical capacity kit (Nanjing Jiancheng Biological Engineering Institute, China).

Statistical analysis
Statistical calculations were performed using the SPSS 13.0 for windows release computer program.Results were expressed as mean ± standard deviation (SD).The paired samples two-tailed T-test was applied for the comparison between two groups.Significance was defined as P < 0.05.The statistical analysis of the antioxidant activity data between the means was carried out by one-way analysis of variance (ANOVA) using SPSS statistics 26.0, followed by Duncan's multiple range test, to compare the means for significant variation (p < 0.05).

Isolation and analysis of EGCG acetylation
The Fr. 1-Fr. 4 was analyzed by HPLC/ESI-MS.The molecular weight of Fr. 1 was 458.4 m/z; MS-base peak data (molecular ion peak [M-H]-) was 457.4 m/z; MS + base peak data (molecular ion peak [M + Na] +) was 481.4 m/z.The above analysis results show that it is EGCG (Fig. 1).
The molecular weight of Fr. 2 was 500.4m/z.MS + base peak data (molecular ion peak [M + Na] +) was 523.4 m/z.[2 M + Na] + peak data was 1023.8 m/z.MS-base peak data (molecular ion peak [M-H]-) was 499.4 m/z.[2 M-H]-peak data was 999.8 m/z.The molecular weight of EGCG was 458.4 m/z.After acetylation, the hydrogen atoms (molecular weight 1) of a -OH site of the EGCG molecular structure was substituted by a acetyl groups (molecular weight 43).Then the molecular weight of single substituted acetylated EGCG is 500.4m/z.So, Fr. 2 is a single substituted O-acetylated epigallocatechin gallate (SoEGCG).In the present study, another mono-acetylated EGCG with 501DA has been reported 5 .But the structure elucidation of mono-acetylated EGCG have not revealed.In this study, The infrared spectroscopy data of (a) SoEGCG was following: 3180 (-O-H), 1754 (-C=O), 1673 (-C=C), 1457, 1322 cm -1 (Fig. s1a).

Total antioxidant capacity of EGCG acetylating components
The total antioxidant capacity kit is mainly to determine the antioxidant ability by restoring Fe 3+ to Fe 2+ , which combine with phenanthroline for forming a stable complex.The active antioxidant group in molecular structure of EGCG is eight hydroxyl groups, which can not only remove hydroxyl free radicals and reactive oxygen species, decompensate peroxides, block the peroxidation chain, but also can coordinate with metal ions in order to remove metal ion catalysis.The total antioxidant capacities of EGCG, SoEGCG, ToEGCG and AcEGCG are shown in Fig. 4. The total antioxidant capacity of SoEGCG and AcEGCG were lower than EGCG.But The total antioxidant capacity of ToEGCG were higher than EGCG.The ToEGCG in this study, as identified by MS for the first time, had the three -OH site acylated with acetyl groups, and the remaining -OH site on the aromatic rings of EGCG may contribute to the superior antioxidant capacity of ToEGCG.Maintenance of the total antioxidant activity of SoEGCG, ToEGCG and AcEGCG suggest that these derivatives may be used as antioxidants in more application area.

Scavenging superoxide anion radical capacities of EGCG acetylating components
The scavenging superoxide anion radical capacities kit is mainly to determine the antioxidant ability by analyzing the reduction on superoxide anion, which generate in the simulated reaction process between medium yellow purine and xanthine oxidase.It is the customary method for antioxidant activity evaluation.The scavenging superoxide anion radical capacities of EGCG, SoEGCG, ToEGCG and AcEGCG are shown in Fig. 5.The www.nature.com/scientificreports/scavenging superoxide anion radical capacities of SoEGCG and ToEGCG were lower than EGCG.The scavenging superoxide anion radical capacities of AcEGCG were higher than SoEGCG, ToEGCG, and EGCG.The scavenging superoxide anion radical capacity of SoEGCG was higher than ToEGCG.The eight hydrogen atoms acylated with acetyl groups may contribute to the scavenging superoxide anion radical capacity of AcEGCG.

Scavenging hydroxyl free radical capacities of EGCG acetylating components
Scavenging hydroxyl free radical capacity kit, which measures the inhibition degree of absorbance decreasing at 536 nm by samples, is the customary method for antioxidant activity evaluation.The scavenging hydroxyl free radical capacities of EGCG, SoEGCG, ToEGCG and AcEGCG are shown in Fig. 6.The scavenging hydroxyl free radical capacities of SoEGCG, ToEGCG and AcEGCG were lower than EGCG.There may be a certain correlation between acetyl groups space conformation and antioxidant activity.The structure-activity relationship of EGCG acetylating components need further research.

Conclusions
The preparative liquid chromatography, LC/MS, NMR methods were used to isolate and identify the EGCG acetylating products in this study.The single substituted O-acetylated epigallocatechin gallate (SoEGCG), three substituted O-acetylated EGCG (ToEGCG), and peracetylated EGCG(E-AcEGCG) were isolated.5-Acetyl-(-)-EGCG and 7-Acetyl-(-)-EGCG were identified.Separation methods in this study, provide the reference for further separation system of EGCG acetylated product, and obtaining different substitution degree of EGCG acetylating components.There are eight hydroxyl groups in the molecular structure of EGCG, which of substitution site contain O-substitution site and C-substitution site.The molecular structure, configuration, conformation and other aspects of different substitution degree of EGCG acetylating components were not clear.The antioxidant activity of different EGCG acetylating components were different, which were affected not only by the substitution degree, but also by the substitution site.By comparing the total antioxidant activity of peracetylated EGCG and EGCG, it was found that the antioxidant activity of peracetylated EGCG was significantly lower than that of EGCG 17 .The peroxide value (POV) and P-anisidine value (AV) index were emploied to evaluate the antioxidant activity of EGCG and acetylated EGCG mixture 5 .The results of acetylation of EGCG by Zhu indicated that the antioxidant activity of acetylated EGCG mixture was lower than EGCG 9 .The lipophilicity of the tested compound was in the order tri-acetylated derivatives > di-acetylated derivatives > mono-acetylated derivatives 18 .Comparing with the previous studies, we found that the total antioxidant capacity, scavenging superoxide anion radical capacity, scavenging hydroxyl free radical capacity of SoEGCG were also significantly lower than EGCG.But the scavenging superoxide anion radical capacity of ToEGCG was significantly higher than EGCG.And the total antioxidant capacity of ToEGCG was significantly higher than EGCG.The scavenging superoxide anion radical of SoEGCG was higher than ToEGCG.
The possible reason is that the antioxidant activity is not only related to the number of substitution group, but also the space conformation.Though the poor solubility, easy conversion, low bioavailability of EGCG in utilization can be solved by acetylating, antioxidant activity of different EGCG acetylating components are influenced by different factors, e.g.stereo chemical structure, configuration.The influence mechanism still needs to be further discussed.

Figure 4 .Figure 5 .
Figure 4. Total antioxidant capacity with different EGCG acetylating separation fraction at 500 μg/mL.Results are expressed as percentage of control and are mean ± SE (n = 6).*P < 0.05, vs. the same concentration of EGCG treated groups.

Figure 6 .
Figure 6.Scavenging hydroxyl free radical antioxidant capacity with different EGCG acetylating separation fraction at 500 μg/mL.Results are expressed as percentage of control and are mean ± SE (n = 6).*P < 0.05, vs. the same concentration of EGCG treated groups.