Stabilization of Natural Antioxidants by Silk Biomaterials

The stabilities of three natural antioxidants, vitamin C (VC), (−)-epigallocatechin gallate (EGCG), and curcumin, in silk films were examined and mechanisms of stabilization were elucidated. The antioxidants were physically incorporated into three types of silk films: as-cast, dried from hydrogels, and methanol-treated. Films were stored at 4, 37, and 45 °C for 30 days in phosphate-buffered saline, pH 7.4, along with controls consisting of free antioxidants. Incorporation of antioxidants did not significantly change film morphology or secondary structure. When stored at 4 °C, all samples showed similar antioxidant activities (percent scavenging) at different time points, determined by the colorimetric 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. At higher temperatures, VC in the as-cast film, EGCG in the as-cast and dried hydrogel films, and curcumin in the methanol-treated films retained more than 50% scavenging activity after 14 days of storage, significantly higher than the other samples. Interaction between antioxidants and silk, as well as degradation of the antioxidants, was investigated by fast-performance liquid chromatography (FPLC) and high-pressure liquid chromatography (HPLC), with an aim of understanding the mechanisms of silk-based stabilization. Binding of antioxidant molecules to hydrophobic or to hydrophilic/hydrophilic boundary regions of silk, depending on the chemical properties of the antioxidant, may account for the observed stabilization effects. The data can help guide further engineering of antioxidant-functionalized silk biomaterials.