Insulin Amyloid Fibril Inhibition

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 λ_m 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)).