2.6. Determination of Antiglycation Activity

Antiglycation activity of the plant extracts was determined using the bovine serum albumin assay with slight modification [23]. Bovine serum albumin (Sigma Aldrich) (500 µl) was incubated with glucose (400 µl) and plant extracts (100 µl). Phosphate buffer saline (100 µl) was used as the sample control and Arbutin (100 µl) (Sigma Aldrich) as the reference standard. A negative control constituting BSA (500 µl), phosphate buffer saline (400 µl), and plant extracts (100 µl) was included. The reaction mixture was allowed to proceed at 60°C for 72 hours and terminated by addition of 10 µl of 100% (w/v) trichloroacetic acid (TCA) (Sigma Aldrich). The TCA added mixture was kept at 4°C for 10 minutes and thereafter centrifuged for 4 minutes at 13000 rpm. The precipitate was redissolved in alkaline phosphate buffer saline (pH 10) and quantified for relative amount of glycated BSA, based on fluoresce intensity in 96-well plates using a microtiter-plate multimode detector (Promega-Glomax Multidetection System). The excitation and emission wavelength used were at 370 nm and 440 nm, respectively. Five concentrations of each sample were analysed in triplicate. Percentage inhibition was calculated using the formula provided below and the sample concentration required for 50% inhibition of BSA glycation was calculated: