Preparation Of Antioxidant Silk Fibroin Film Via Enzymatic Modification

As a natual polymer, silk fibroin has the characteristic of good mechanical behavior and biocompatibility, and it has a wide range of applications in the fields of medicine and tissue engineering. Catechin belongs to the natural polyphenolic compounds with antioxidant effect, it has a scavenging effect on variety of free radicals. Catechin could be enzymatically grafted onto the silk fibroins and endow silk fibroins with anti-oxidation property, which undoubtedly might impart the additional function to the fibroin-based products.In the present work, the actions of tyrosinase on the oxidation of three kinds of catechins were investigated. To explore the effects of different reaction time, enzyme concentration and oxygen concentration on the catalytic reaction, the oxidation mechanism of catechin was investigated by using LC-MS. The changes of molecular weight and the number of amino groups in the silk fibroins, before and after incubating with catechin and tyrosinase were examined by means of SDS-PAGE, HPLC and 1H-NMR methods. DPPH and ABTS assays were used to evaluate the antioxidative effects for the catechin-treated fibroin membrane. The biocompatibility for the modified fibroin membrane was also investigated according to NIH/3T3 cell viability.The results indicated that catechin could be enzymatically oxidized by tyrosinase, subsequently formed reactive o-quinones and some catechin derivatives of dimers and trimers. After incubating ε-PLL with catechin, the characteristic structure of benzene was detected in the infrared spectras of the formed products, indicating that catechin could be efficiently grafted onto the model amine compound of ε-PLL. The results from SDS-PAGE and HPLC revealed the molecular weight of silk fibroin was increased after incubating with catechin, which was also verified by the data of decreased number of amine groups from 1H-NMR analysis. After incubating the silk fibroin membrane with 1 U/mL tyrosinase and 0.3 mM catechin at 20～30℃ and pH7, antioxidant ability was obtained. Meanwhile, extending incubation time led to an obvious improvement of antioxidant ability. After washing for 8 h, the oxidation resistance of modified silk fibroin membranes was slight higher than that of the control, implying that the durability of the obtained antioxidant ability was acceptable. As can be seen from TG-DTG curve, the thermodynamic property for the modified silk fibroin did not noticeably change compared to the blank. Cell viabilities for the modified fibroin membrane and the control were all above 80%, indicating that modified fibroin membranes have good biological compatibility and the potential applications in medical fields and tissue engineering.