In vitro glycation of albumin

BSA was glycated in accordance with the method described by McPherson et al. (1988) with some modifications. For evaluation of antiglycating property of plant extracts, BSA (1 mL, 10 mg/mL) was incubated along with aqueous plant extracts (1 mL) and fructose (1 mL, 250 mM) in potassium phosphate buffer saline (PBS), (2 mL, 200 mM, pH 7.4) containing 0.02% sodium azide, in the dark at 37 °C for 4 days in sealed tubes under sterile conditions (0.22 μ filter), hereafter referred to as ‘glycated sample’. Negative control (1 mL BSA +3 mL PBS) and positive control (1 mL BSA +1 mL fructose +2 mL PBS) were maintained under similar condition. After incubation, unbound fructose was removed by dialysis against PBS and dialysate was used for further analysis. All additions and analysis were performed in triplicates.