Structural characterization and protective effect of gallic acid grafted O-carboxymethyl chitosan against hydrogen peroxide-induced oxidative damage

Abstract

In this study, gallic acid (GA) was grafted onto O-carboxymethyl chitosan (O-CMCS) by a free radical-mediated method. The synthesized GA grafted CMCS (GA-g-CMCS) was characterized by several instrumental methods and the antioxidant activity of the conjugate was evaluated by in vitro and cellular assays. Results showed the grafting ratio of GA-g-CMCS was 60.8 mg GAE/g. Thin layer chromatography and UV-vis, Fourier-transform infrared and proton nuclear magnetic resonance spectroscopic analyses all confirmed GA was successfully grafted onto O-CMCS. Meanwhile, covalent linkage formed between the amino group of O-CMCS and the carboxyl group of GA via amide bond. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and the reducing power of O-CMCS were remarkably improved by grafting with GA. Cellular assays showed GA-g-CMCS was non-toxic to RAW264.7 cells at 25-400 μg/mL. Moreover, GA-g-CMCS had a protective effect against hydrogen peroxide (H₂O₂)-induced oxidative damage in RAW264.7 cells. As compared with H₂O₂-treatment alone, the pretreatment of the cells with 100, 200 and 400 μg/mL of GA-g-CMCS significantly increased cell viability, reduced apoptosis and intracellular reactive oxygen species production, and improved membrane integrity and intracellular antioxidant enzyme (superoxide dismutase, catalase, glutathione peroxidase) activity. Our results suggested GA-g-CMCS could be developed as a novel antioxidant.

Keywords: Antioxidant; Carboxymethyl chitosan; Conjugate; Gallic acid; Hydrogen peroxide; RAW264.7 cells.