【作者】 杨艳；

【导师】 刘万顺；

【作者基本信息】 中国海洋大学 ， 生物化学， 2005， 博士

【摘要】 机体在细胞有氧代谢过程中不断的产生自由基。在正常生理条件下,自由基不断的被清除。体内抗氧化防御体系包括酶解和非酶解两种机制。当异源性药物、毒物在体内的代谢过程中产生活性中间代谢产物,导致活性氧的过量产生,或者机体的防御体系受到损伤时,就会导致活性氧化应激状态。大量的研究资料表明氧化应激与很多疾病的发生密切相关。因此抗氧化剂及其作用机制的研究在医学上具有重要意义。还原型谷胱甘肽是机体最重要的非酶抗氧化剂,维持谷胱甘肽含量的稳态在很多病理过程中具有重要意义。L-半胱氨酸是提高GSH含量的主要途径,但是L-半胱氨酸在一定浓度时具有毒性而且容易被氧化,L-半胱氨酸前体化合物是有效的提高促进GSH合成,治疗氧化应激有关的疾病的有效手段。噻唑啉糖衍生物是L-半胱氨酸与糖类分子中的羰基缩合形成的环状化合物,它在体内能够通过非酶的水解反应释放出L-半胱氨酸,促进GSH的从头合成,提高GSH的含量,保护机体,具有广泛的生物学、医学功能。 壳寡糖是通过甲壳素脱乙酰得到的,它具有生物相容性、可降解性、无免疫原性、无毒和易吸收等特点,在药物学,食品等领域具有非常广泛的应用。近年来壳寡糖抗氧化功能的研究备受关住,但是在这方面的研究还不多,对其抗氧化机制尚未充分研究,而且关于壳寡糖降解产生的单糖—氨基葡萄糖的抗氧化功能,目前国内外尚未见报导。本实验利用L-半胱氨酸和氨基葡萄糖,N-乙酰氨基葡萄糖制备了两种新的噻唑烷酸糖衍生物（GlcNH₂Cys,GlcNAcCys）,对其熔点、比旋光、IR波谱和1H-NMR波谱进行了测定,结合两种已见报道合成的噻唑烷酸糖衍生物（GluCys,GalCys）的结构,比较并得出其结构为噻唑啉羧酸结构。 本实验通过体外、体内实验研究壳寡糖,氨基葡萄糖及其衍生物的抗氧化、肝保护功能。利用体外试验方法研究了氨基葡萄糖及其衍生物的自由基清除能力,还原力,金属络合能力以及对羟自由基诱导的生物大分子如脂类、脱氧核糖和蛋白质的氧化性损伤的保护作用。利用四氯更多还原

【Abstract】 Free radicals are constantly produced mostly as reactive oxygen species in aerobic cells. In physiological conditions, free radicals are removed by enzymic and nonenzymic antioxidant defenses including enzyme and antioxidant molecules. Once there is excessive generation of free radicals because of metabolism of exogenous toxins and/or impairment of endogenous antioxidants, oxidative stress occurs. Numerous evidences suggest that oxidative stress is closely related to pathological changes. So much interest focus on antioxidants investigation. Reduced glutathione plays critical roles in antioxidant defense. The homeostasis of GSH is very important in many pathological conditions. The most important and efficient way for GSH restoration is L-cysteine administration. But L-cysteine is toxic at functional concentration and is easily oxidized. The traditional prodrug design proved to be effective in stimulating GSH biosynthesis, counteracting oxidative stress with reduced toxicity. L-cysteine and aldoses can form thiazolidine derivatives through cyclo-condensation. Thiazolidine derivatives can be used as the precursor of L-cysteine, which can liberate L-cysteine and the sugars by non-enzymic hydrolysis. Their biological and pharmacological functions have been extensively studied.Chito-oligosaccarides （COS） can be prepared by deacetylation and enzymic hydrolysis of chitin, the second most abundant polymer in nature. The application of COS in pharmaceutical, food and other fields have been extensively studied because of good biocompatibility, degradation, non-toxicity and easy-absorbability. In recent years, studies have shown that COS and its derivatives have antioxidative activities both in vivo and in vitro. But further research needs to be done to better illustrate its antioxidant activity and mechanism. In this study, glucosamine （GlcNH₂） and N-acetyl-glucosamine （GlcNAc） was condensed with L-cysteine to form novel thiazolidine derivatives. The physical characteristics such as melting point, optical activity, IR spectrometer and ¹H-NMR were conducted. Based on the characterization of two previously reported thiazolidinederivatives, the results show that GlcNH2Cys and GlcNAcCys were 2-substituted-thiazolidine- 4（R）-carboxylic acids.In our study, the antioxidant of chitosan, GlcNH2, GlcNAc, and their thiazolidine derivatives were investigated in vitro and in vivo. Antioxidant activity such as free radical scavenging ability, reducing power, chelating effects and protective effects against oxidative damage to biomoleculars such as lipid, deoxyribose and protein were evaluated using in vitro hydroxyl radical generating system. The antioxidative, hepato-protective effects of COS and its derivatives on carbon tetrachloride/APAP-induced liver toxicity in mice and the possible mechanisms involved in their protection were also illustrated. UV radiation damage in closely related oxidative stress and protection of GlcNAcCys against UV radiation induced lipid and DNA damage in mitochondria and nuclei were studied to further apply GlcNAcCys as UV protector.Our data indicate that GlcNHa, GlcNI^Cys have strong in vitro antioxidative property. They can scavenge directly reactive free radicals like hydroxyl radical and superoxide anion, protect macromolecules including deoxyribose, protein and lipid against oxidative damage induced by hydroxyl radical. Pretreatment with COS and its derivatives （1.5 g/kg body weight per day） for 12 consecutive days before CCU challenge showed hepato-protective effects. Serum alanine and aspartate aminotransferase activities were effectively decreased. Hepatic malondialdehyde formation was inhibited and sulfhydryl contents, total antioxidant capabilities were restored. Liver metallothionein concentration was also significantly induced by pretreatment of COS and its derivatives. Genotoxicity as reflected by DNA fragmentation, however, was not mitigated by pretreatment of COS and its derivatives. APAP metabolism can cause severe GSH depletion, oxidative stress and subsequent liver toxicity. Thiazolidine derivatives administration （800 mg/kg body weight） 30 min after APAP challenge provide efficient protection as manifested by decreased serum enzyme activities, elevated sulfhydryl levels and total antioxidative capabilities and decreased malondialdehyde contents in liver. GlcNAcCys exhibit UV radiation protection function as it can inhibit lipid peroxidation in mitochondria and nuclei if pre-incubated toUV radiation.In conclusion, our results suggested that COS, glucosamine and thiazolidine derivatives have excellent in vitro and in vivo antioxidant properties. They promise to be candidates in oxidative stress-involved pathological conditions. Our work provide theoretical for further application of chitosan in biological, medical and nutritional fields.更多还原