Preparation,Properties And Anti-Inflammatory Activity Of Citronellol Grafted Chitosan Oligosaccharide Derivatives

Chitosan oligosaccharide(COS)is the only alkaline amino oligosaccharide in nature with positive charge,which has good biocompatibility,water solubility,safety and easy absorption.In addition,COS also has anti-inflammatory,antioxidant and antibacterial activities.However,the anti-inflammatory activity of COS is limited.Chemical modification with molecules with anti-inflammatory activity is an effective way to improve the anti-inflammatory activity of COS and enhance the application value of COS.Plant essential oil is a natural plant extract,which has anti-inflammatory,antioxidant,immune regulation and other functional activities.However,its poor water solubility limits its application.This study selected chain terpenoids with high reactivity in essential oil.We used citronellol(Cit)to modify COS by N-alkylation to prepare novel COS derivatives,which improved the anti-inflammatory activity of COS and water solubility of Cit.Meanwhile,we used carrageenan(Carr)induced foot swelling in rats to study the anti-inflammatory activity and mechanism of COS derivatives.The main research contents and results are as follows:1.Preparation,characterization and properties of COS-g-Cit1-3.After bromination of Cit,COS-g-Cit was obtained by reacting Cit-Br with COS under the catalysis of triethylamine.The derivatives were characterized by UV-vis spectroscopy,FT-IR spectroscopy and ¹H NMR spectroscopy and the results indicated that Cit was successfully grafted onto COS.The derivatives with degree of substitution(DS)of0.167,0.200 and 0.182 were obtained by changing the molar ratio of reactants(Cit-Br:COS=2:1,4:1,6:1).In addition,thermogravimetry,X-ray diffraction and turbidity method were used to study the thermal stability,crystallinity and solubility of the derivatives.The results showed that the thermal stability of COS-g-Cit1-3 was better than that of COS,and the water solubility of COS-g-Cit1-3 was better than that of Cit.2.The safety and anti-inflammatory activity of COS-g-Cit1-3.The safety of COS-g-Cit1-3 was studied by upper-lower limit method and KM mice were given a single dose of COS-g-Cit1-3(2000 mg/kg).The results showed that the LD₅₀ of COS-g-Cit1-3 was more than 2000 mg/kg,and there was no abnormal behavior and death in mice.Compared with the control group,the difference of body weight gain,blood biochemical indexes and most organ coefficients in COS-g-Cit1-3 group were not significant(p>0.05).Histopathological results indicated that there was no obvious accumulation of inflammatory cells in kidney and liver.Carr induced acute inflammation model in SD rats was used to study the anti-inflammatory of COS-g-Cit1-3.The results showed that COS-g-Cit1-3 could inhibit foot swelling and inflammatory cell infiltration.The activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPx)and the content of glutathione(GSH)were significantly increased(p<0.01),while the contents of malondialdehyde(MDA)and nitric oxide(NO)were decreased.The above research showed that the anti-inflammatory activity of COS-g-Cit1-3(100 mg/kg)was better than that of COS and COS+Cit,which was dependent on the DS.3.The anti-inflammatory mechanism of COS-g-Cit1-3.ELISA was used to determine the content of cytokines in rat paw tissues.The results showed that COS-g-Cit1-3 could decrease the contents of pro-inflammatory cytokines TNF-?by increasing the contents of anti-inflammatory cytokines IL-4 and IL-10.q RT-PCR was used to detect the m RNA expression levels of inflammatory factors.The results showed that COS-g-Cit1-3 could down-regulate the expression levels of IL-1?,IL-6,i NOS,COX-2 and PGE₂m RNA by up-regulating the expression level of IL-33 m RNA.Western blot was used to determine the expression of NF-?B pathway related protein.The results indicated that COS-g-Cit1-3 could inactivate NF-?B signaling pathway via inhibiting the phosphorylation of NF-?B p65,I?B?and IKK?and the degradation of I?B?and IKK?.The above research showed that COS-g-Cit1-3 could alleviate Carr-induced inflammatory response in rats by inhibiting the expression of inflammatory mediators and NF-?B signaling pathway.Overall,the anti-inflammatory activity and thermal stability of the three COS derivatives were better than those of COS,which increased with the increase of DS,and the water solubility of COS-g-Cit1-3 was better than that of Cit.It will lay a theoretical foundation for the grafting of COS with essential oil and its wide application in various fields.