Ultra high-Performance Liquid Chromatography (UHPLC) method development and validation for the identification of oxidized product of Epigallocatechin-3-Gallate (EGCG).

OBJECTIVE: In the present study we reported oxidation of epigallocatechin-3-gallate (EGCG) and validation of oxidized product by a validated ultra high-performance liquid chromatography (UHPLC) method. METHODS: Two hundred milligrams of EGCG was oxidized in 5 mL of hydrogen peroxide (H2O2) and was identified by a validated UHPLC method with precision and robustness. Confirmation of parameters like C-H stretching and mass was carried out using infrared spectroscopy and mass spectroscopy, respectively. Identification of oxidized EGCG (O-EGCG) was done by UHPLC. RESULTS: The infrared spectroscopy chromatograms observed less intensity C-H stretching as compared to O-EGCG. The mass of EGCG and O-EGCG were 459.09 and 915.16, respectively. Structure elucidation revealed a loss of one proton in O-EGCG as compared to EGCG. Validation of the developed method was specific, with linear correlation coefficient 0.9981 and 0.9917, respectively for EGCG and O-EGCG, the accuracy rate of 95.2%-99.6% for EGCG, and 99.18%-101.5% for O-EGCG. CONCLUSION: Together, the results of this study demonstrate the formation of a dimer also the UHPLC method developed for identification of both EGCG and O-EGCG is validated as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.

Assessment of Antioxidant, Immunomodulatory Activity of Oxidised Epigallocatechin-3-Gallate (Green Tea Polyphenol) and Its Action on the Main Protease of SARS-CoV-2-An In Vitro and In Silico Approach.

Owing to the instability of Epigallocatechin Gallate (EGCG), it may undergo auto-oxidation and form oxidised products or dimers. In the present study, we aimed to evaluate the therapeutic effects, including antioxidation and immunomodulatory action, of the Oxidised Epigallocatechin Gallate (O-EGCG) as compared to native EGCG and the action of these compounds on main protease (Mpro) docking against SARS-CoV-2. HCT-116 (Human Colon Cancer) cell lines were used to estimate the total antioxidant capacity and lipid peroxidation levels and pro-inflammatory markers (human IL-6, IL-1ß, TNF-α). Further, molecular docking analysis was performed by AutoDock and visualised in Discovery studio. Improved antioxidant capacity of O-EGCG was observed, and there was a significant decrease in the inflammatory markers (IL-1ß, IL-6, and TNF-α) when O-EGCG was applied as compared to EGCG. The O-EGCG was shown to be strongly associated with the highest docking score and active site residues of IL-1, IL-6, and TNF- α, as well as the Mpro of SARS-CoV-2, according to in silico approach. The in vitro and in silico analyses indicate an improved therapeutic action of the oxidised form of EGCG. The effective inhibitory action of O-EGCG against SARS-CoV-2 suggests further exploration of the compound against COVID-19 and its efficacy. However, in vivo studies and understanding of the mechanism of action of O-EGCG may yield a better opinion on the use of O-EGCG and future human clinical trials.