Initially, insulin solution was prepared with a final concentration of 0.5 mg/mL in phosphate buffer (50 mM, pH 7.4) in the presence or absence of 1 mM concentration of silibinin and trans-chalcone. The vials containing the insulin samples were then incubated for 24 h at 37°C on a stirrer rotating at 100 rpm (10 (link)). Since the Congo red marker binds specifically to amyloid fibrils, resulting in an increase in λm as well as in absorbance levels, Congo red absorption rate of incubated protein samples was assayed in the absence and presence of silibinin and trans-chalcone (1 mM). To do so, 500 μL of Congo red solution (20 μM in 5 mM potassium phosphate and 150 mM sodium chloride, pH 7.4) plus 25 μL of the sample was poured into a 0.5 mL microtube and filtered using a 2-μM pore syringe filter, then stirred and placed in the laboratory for 10 min until color stabilization. The Congo red absorption spectrum was then recorded at 600-400 nm using the Shimadzu UV-1800 spectrophotometer (11 (link)).
Free full text: Click here