Preparation Of Series Of Chitooligosaccharides Derivatives And Study Of Their Neuronal Protection Of PC-12 Cells

Recently, the gradually increased incidence of Alzheimer's disease brings serious economic and mental burden to patients, their families, and society. However, the current clinical drugs can only delay the development of the disease, and not completely cure Alzheimer's disease. And most of the drugs have many side effects. Therefore, it is essential to develop low toxic and effective drugs to prevent and treatment Alzheimer's disease. One of the main pathological features of Alzheimer's disease is substantial loss of nerve cells. Protection of neurons can effectively mitigate and treat Alzheimer's disease. Chitooligosaccharides is a natural alkaline amino sugar, and many studies have shown that chitooligosaccharides can protect nerve cells. Therefore chitooligosaccharides can be a promising agent in treating neurodegenerative diseases.Using enzymatic hydrolyzed chitooligosaccharides mixture as raw material, three series of chitooligosaccharides and its derivatives were prepared in this dissertation. Firstly, five monomers of chitooligosaccharides were isolated and purified by gel column chromatography, including glucosamine trimer, tetramer, pentamer, hexamer and heptamer. Then three peracetylated chitooligosaccharides monomers and three N-acetylated chitooligosaccharides monomers were prepared by chemical modification, includiong peracetylated chitobiose, chitotriose, chitotetraose and N-acetylated chitobiose, chitotriose, chitotetraose. These monomers were analyzed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), mass spectrometry (MS),infrared spectroscopy (IR), and nuclear magnetic resonance (NMR).The neuroprotective effects of three series monomers of chitooligosaccharides were evaluated by copper chloride and glutamate cell models. The results showed that three Peracetylated Chitooligosaccharides monomers exhibit highest neuroprotective activity among the monomers in glutamate cell model and they can increase cell viability from 40% to 90%. The Peracetylated Chitotetraose showed higher activity than the Peracetylated Chitobiose and the Peracetylated Chitotriose, and the medium dose (200?g/mL) was higher than the low dose (100?g/mL) and high-dose group (400?g/mL). The glucosamine hexamer also exhibited good neuroprotective effect in glutamate cell model and it increased cell viability by 16% compared to glutamate treated cells.Furthermore, the Peracetylated Chitooligosaccharides monomers inhibited accumulated glutamate-induced ROS, decreased PC 12 cell apoptosis rate, prevent the releasing of LDH, and enhance the mitochondrial membrane potential. The study indicated that the Peracetylated Chitooligosaccharides monomers prevented against glutamate-induced cell injury via inhibiting ROS formation in PC 12 cells.In conclusion, this study preliminary discussed the neuroprotective effects of three series monomers of chitooligosaccharides and made such compounds as candidates for the therapy of neurodegenerative diseases.