Study On The Anti-oxidation Activity,Simulated Digestion And Fermention In Vitro Of Gallic Acid Grafted O-carboxymethyl Chitosan

O-carboxymethyl chitosan(O-CMCS)is a kind of water-soluble derivative of chitosan with excellent viscosity,non-toxic biocompatibility and biodegradability,which has good applications in the field of food engineering prospect.However,the biological activity of O-CMCS is relatively low,so it is necessary to modify its structure to expand its application in the food field.In this study,gallic acid(GA)was grafted onto O-carboxymethyl chitosan(O-CMCS)by using ascorbic acid/hydrogen peroxide(AA/H2O2)redox couple as free radical initiator.The structural characteristics,antioxidant activity,simulated digestion and fermentation characteristics in vitro of the synthesized GA grafted CMCS(GA-g-CMCS)were mainly studied systematically.The results of this study are of great significance for improving the antioxidant activity of O-CMCS and broadening its application in the field of functional foods(1)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.The result of Folin-Ciocalteu colorimetric method showed that the grafting ratio of GA-g-CMCS was 60.8 mg GAE/g.GA-g-CMCS only presented a yellow spot at the starting line of the thin layer chromatography silica gel plate without.migration,indicating that the sample did not contain free gallic acid.Fourier-transform infrared spectrum showed a new absorbtion peak at 1640 cm-1,corresponding to the stretching vibration of C=O of the amide bond,indicating that GA was mainly grafted to O-CMCS through the amide bond.Proton nuclear magnetic resonance spectrum of GA-g-CMCS presented a new proton peak at 7.1 ppm,which corresponds to the phenyl proton signal of GA.X-ray diffraction pattern showed that GA-g-CMCS was amorphous.The observation results of scanning electron microscope further showed that GA-g-CMCS presented a variety of morphology such as round,sheet-like and rod-like,which indicated that GA had successfully grafted onto O-CMCS.(2)The in vitro and cellular antioxidant activity of GA-g-CMCS was evaluated.The results of in vitro experiments showed that the 1,1-diphenyl-2-picrylhydrazine radical scavenging activity and reducing power of O-CMCS were significantly improved by grafting with GA.Cellular assays showed GA-g-CMCS was non-toxic to RAW264.7 cells at 25-400μg/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 apoptosisand 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.(3)The effects of salivary,gastric juice and intestinal juice on GA-g-CMCS were studied by simulating human digestive system.Results showed that the average molecular weight of O-CMCS decreased significantly,while the average molecular weight of GA-g-CMCS was not significantly changed.Thin layer chromatography also showed GA-g-CMCS didn’t release the grafted GA group after in vitro simulated digestion.Scanning electron microscope showed O-CMCS was degraded into round,sheet-like and rod-like morphology.By contrast,the surface morphology of GA-g-CMCS was almost unchanged.The high-throughput sequencing of mouse intestinal microbiota was performed by Illumina second-generation sequencing technology to investigate the effect of GA-g-CMCS on intestinal microbiota fermentation in vitro.Results showed that GA-g-CMCS could improve the diversity of intestinal microbiota,significantly increase the abundance of Firmicutes and Bacteroides,and inhibit Fusobacteria.In addition,GA-g-CMC S could promote the growth of Subdoligranulum,Blautia and Bacteroides,significantly reduce the growth of Fusobacterium,and promote the production of short-chain fatty acids.Therefore,GA-g-CMCS could modulate intestinal microecology.