Biophysical insight into the binding mechanism of epigallocatechin-3-gallate and cholecalciferol to albumin and its preventive effect against AGEs formation: An in vitro and in silico approach

Abstract

Advanced glycation end products (AGEs) are produced non-enzymatically through the process of glycation. Increased AGEs production has been linked to several diseases including polycystic ovary syndrome (PCOS). PCOS contributes to the development of secondary comorbidities, such as diabetes, cardiovascular complications, infertility, etc. Consequently, research is going on AGEs-inhibiting phytochemicals for their potential to remediate and impede the progression of hyperglycaemia associated disorders. In this study human serum albumin is used as a model protein, as albumin is predominantly present in follicular fluid. This article focusses on the interaction and antiglycating potential of (−)-Epigallocatechin-3-gallate (EGCG) and vitamin D in combination using various techniques. The formation of the HSA-EGCG and HSA-vitamin D complex was confirmed by UV and fluorescence spectroscopy. Thermodynamic analysis verified the spontaneity of reaction, and presence of hydrogen bonds and van der Waals interactions. FRET confirms high possibility of energy transfer. Cumulative antiglycation resulted in almost 60 % prevention in AGEs formation, decreased alterations at lysine and arginine, and reduced protein carbonylation. Secondary and tertiary structural changes were analysed by circular dichroism, Raman spectroscopy and ANS binding assay. Type and size of aggregates were confirmed by Rayleigh and dynamic light scattering, ThT fluorescence, SEM and SDS-PAGE. Effect on cellular redox status, DNA integrity and cytotoxicity was analysed in lymphocytes using dichlorofluorescein (DCFH-DA), DAPI and MTT assay which depicted an enhancement in antioxidant level by cumulative treatment. These findings indicate that EGCG and vitamin D binds strongly to HSA and have antiglycation ability which enhances upon synergism.