Looking through a historical lens, attention to the stabilization of pharmaceutical proteins/peptides has been dramatically increased. Human insulin is the most challenging and the most widely used pharmaceutical protein in the world. In this study, the protein and coumarin as a plant-derived phenolic compound and two coumarin analogs with different moieties were investigated to evaluate the protein fibrillation and cytotoxicity. The obtained data showed that with a change in environmental pH, the behavior of the compounds on the process of insulin fibrillation will be changed completely. Coumarin (C1) and its hydrophobic analog, 7-methyl coumarin (C2), in an acidic environment, inhibit insulin fibrillation, change the oligomerization state of insulin and produce fibrils with notable lateral interactions with low cytotoxicity. However, negatively-charged 3-trifluoromethyl coumarin (C3) without significant changes in insulin structure and by altering the oligomerization state of the protein, slightly accelerates hormone fibrillation. Also, the compounds showed a disulfide protecting role during protein aggregation. Regarding the toxicity of the fibrils, it was observed that in addition to the secondary structures of proteinous fibrils, the ability to destroy the cell membrane is also related to the length of the fibrils and their degree of lateral interactions. By and large, this work can be useful in finding a better formulation for bio-pharmaceutical macro-molecules.
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