Study On The Preparation,Antioxidant And Emmunomodulatory Activities Of Acetylatedand Carboxymethylated Polysaccharides From Dimocarpus Longan Pulp

Object:To investigate the optimal conditions of acetylated and carboxymethylated derivatives from Dimocarpus longan polysaccharides. To determine the effect of acetylated and carboxymethylated longan polysaccharides on antioxidation in vitro. To study the effect of acetylated and carboxymethylated longan polysaccharides on immune activity both in vitro and in vivo. To illuminate the mechanism of immune regulation of acetylated and carboxymethylated longan polysaccharides, providing the scientific basis for industry development of longan polysaccharides.Methods:1. Crude polysaccharides were obtained with water-extraction and alcohol-precipitation method. Albumen was eliminated with proteinase hydrolization method. Pigments were decolored by H2O2. The total sugar content of crude polysaccharides was determined by phenol-sulfate acid method. 2. Homogeneous polysaccharide (LYP2) was further purified from crude polysaccharides by DEAE-cellulose (OH-) ion-exchange chromatography and Sephacryl S-300 chromatography.3. Acetylated derivative of Dimocarpus longan polysaccharides (Ac-LYP2) was prepared using acetic anhydride method. The synthetic conditions on the degree of substitution (DS) of the synthesized derivatives were optimized by response surface methodology.4. Carboxymethylated derivative of Dimocarpus longan polysaccharides was prepared using sodium hydroxide-monochloroacetic acid system. With chloroacetic acid concentration, reaction temperature and reaction time as the independent variables and carboxymethyl substitution degree of modified product as the response value, the modification conditions were optimized by response surface analysis. To determine the effect of acetylated and carboxymethylated longan polysaccharides on scavenging superoxide anion free radical, hydoxyl free radical, reducing capacity, lipid peroxidation and red cell hemolysis induced by H2O2.6. The immunosuppressive model in mice was established by intraperitoneal injection of cyclophosphamide (CY). Protective effects of Ac-LYP2 and CM-LYP2 were researched on immunosuppressive mice. SI (Spleen index) was used to evaluate the effect of Ac-LYP2 and CM-LYP2 on immune organs of immunosuppressive mice. Cellular immune function was determined using delayed-type allergy method (swelling ear method) induced by dinitrofluorobenzene (DNFB). The contents of hemolysin in the mice serum were detected to evaluate the humoral immune function. The contents of lysozyme in the mice serum and spleen were detected to evaluate nonspecific immune function. The contents of IFN-y and IL-4 were detected to evaluate the effect of Ac-LYP2 and CM-LYP2 on the balance of Th1/Th2.7. Immunoregulatory activities in vitro of Ac-LYP2 and CM-LYP2 were studied via spleen proliferation, macrophage phagocytosis, production of NO and cytokine secretion.Results:1. The yield of polysaccharides from Dimocarpus longan pulp was 7.22% and the sugar content was 67.85%. The removal rate of foreign protein reached 71.58%. A homogeneous polysaccharide (LYP2) was purified by 0.125 mol/L NaCl.2. The optimized conditions of acetylation were reaction time of 30 min, polysaccharides-to-acetic anhydride ratio of 10.2 and reaction temperature of 42 ℃. The degree of substitution was 0.443.3. The optimal conditions of carboxymethylation were chloroacetic acid of 1.2 mol/L, reaction temperature of 73 ℃ and reaction time of 3.2 h. The degree of substitution was 1.052.4. The antioxidant experiment in vitro show that LYP2, Ac-LYP2 and CM-LYP2 could scavenge hydoxyl free radical, and inhibit lipid peroxidation in liver tissue and red cell hemolysis. The concentration of eliminating 50% (EC50) of LYP2, Ac-LYP2 and CM-LYP2 on scavenging hydoxyl free radical was 5507.67 μg/mL (4.67 x 10-5 mol/L),702.41 μg/mL (5.45 x 10-6 mol/L) and 519.39 μg/mL (3.44 x 10-6 mol/L), respectively. The EC50 of LYP2, Ac-LYP2 and CM-LYP2 on scavenging superoxide anion free radical was 2219.05 μg/mL (1.88 x 10-5 mol/L),977.12 μg/mL (7.57 x 10-6 mol/L) and 489.70 μg/mL (3.24 x 10-6 mol/L), respectively. The concentration of inhitbiting 50% (IC50) of LYP2, Ac-LYP2 and CM-LYP2 on lipid peroxidation was 917.99 ug/mL (7.78 x 10-6 mol/L),646.04 μg/mL (5.01 x 10’6 mol/L) and 544.21 μg/mL (3.60 x 10-6 mol/L), respectively. The IC50 of LYP2, Ac-LYP2 and CM-LYP2 on red cell hemolysis was 620.82μg/mL (5.26 x 10-6 mol/L),380.11 μg/mL (2.95 x 10’6 mol/L) and 262.76 μg/mL (1.74 x 10-6 mol/L), respectively.5. LYP2, Ac-LYP2 and CM-LYP2 could improve the spleen index of immunosuppressive mice, significantly improve the ear swelling degree, rise HC50 and lysozyme content in the mice serum and spleen, increase the secretion of IL-4 and reduce the secretion of IFN-y in the mice serum. Furthermore, Ac-LYP2 and CM-LYP2 showed a stronger effect than LYP2 at the same concentration.6. The sort order of three polysaccharide components combined with LPS to promote B lymphocyte proliferation was CM-LYP2> LYP2> Ac-LYP2. The sort order of three polysaccharide components combined with Con A to promote T lymphocyte proliferation was Ac-LYP2> LYP2> CM-LYP2. LYP2, Ac-LYP2 and CM-LYP2 combined with LPS significantly improve the ability of macrophage cell on phagocytosis of neutral red, showing a dose-response relationship, and the sort order was Ac-LYP2> CM-LYP2> LYP2. LYP2, Ac-LYP2 and CM-LYP2 could promote peritoneal macrophages to secrete NO, IL-1β, TNF-a, and IL-12p40.Conclusions:1. Acetic anhydride method of optimizing the acetylation of longan polysaccharide is stable and feasible, which is an ideal method of acetylated modification.2. It is a kind of ideal structural modification method to synthesize carboxymethyl derivatives from longan polysaccharide using sodium hydroxide-MCA system.3. The introduction of acetyl and carboxymethyl is helpful to improve the antioxidant capacity from longan polysaccharides. The antioxidant capacity of Ac-LYP2 and CM-LYP2 is weaker than vitamin C starting from the mass concentration, but the antioxidant capacity of vitamin C is not as good as Ac-LYP2 and CM-LYP2 considering the amount of substance concentration.4. LYP2, Ac-LYP2 and CM-LYP2 can enhance specific immune function and nonspecific immune function in the immunosuppressive mice, they can also correct the disorder of Thl/Th2. The introduction of acetyl and carboxymethyl is beneficial to enhance immune ability.5. The in vitro results showed that LYP2, Ac-LYP2 and CM-LYP2 can not only promote the secretion of NO, IL-1β, TNF-a and IL-12p40, but also up-regulate proliferation of spleen lymphocyte and phagocytotic function of macrophages.