Study On Isolation, Purification, Structure And Antioxidant Activity Of Polysaccharides From Pulp Tissue Of Litchi (Litchi Chinensis Sonn.)

Polysaccharide is widely found in plants, animals and microorganisms. Polysaccharides have functions of modulating immunity, reducing blood sugar and blood fat, antioxidation, anti-tumor, antivirus et al. Litchi (Litchi chinensis Sonn ) originated from China, called'king of fruit'in South China, is one of four famous fruits in Guangdong province. Little research has been reported on polysaccharide of litchi pulp in relation to its biological activity. To further develop litchi fruits processing, studies were carried on isolation and purification of polysacchride from pulp tissue of litchi, and its antioxidant activity on liver protection was also estimated.Twenty-one litchi cultivars were choosen in this project. The water-soluble polysaccharide content was determined. The results showed that Heiye had the higest polysaccharide content with the value of 15.72g/kg. Thus, Heiye was selected for the following trails.Five different desication methods were used to produce litchi nuts. The results showed that the polysaccharide content and antioxidant activity of litchi polysaccharide were steady. In this project, we choose oven-drying to produce litchi nuts.The extraction methods of litchi were systematacially studied,the methods of leaching with hot water, microwave-assisted extraction and ultrasonic-assisted extraction were investigated, respectly. The results showed that: the extraction yield of litchi extracted by hot water was 24.22%, the extraction yield of extracted by microwave-assisted was 24.26%, the extraction yield of extracted by ultrasonic-assisted was 24.32%. The extraction time of three extraction methods was different: hot water> ultrasonic-assisted> microwave-assisted. Three extraction methods had no effect on antioxidant activity of litchi polysaccharide. In this project, microwave-assisted extraction was choosen to extract the litchi polysaccharide.Precipitation of litchi polysaccharides with different ethanol concentration: LFP1 was obtained by precipitation extracts with 20% ethanol solution. The supernatant was added ethanol to concentration of 50% to precipitate LFP2. Then the supernatant was added ethanol to concentration of 80% to precipitate LFP3. LFP4 was obtained by precipitation extracts with 80% of ethanol solution directly. Then, LFP1, LFP2, LFP3 and LFP4 were gained after purification.LFP2 was chromatographed on a DEAE-52 anion-exchanging column to yield two major fractions:LFPC2 and LFPC3. LFPC2 and LFPC3 were subjected to further purification on a Sephadex G-100 gel filtration column. Two major fractions, LFPC₂a and LFPC₃ were collected.By analysis of gel permeation chromatography (GPC) and gas chromatography (GC), LFPC₂a, which had a average molecular weight of 43927 Da, composed of arabinose, ribose, galactose and glucose in the molar ratio of 1.67:2.15:1.54:1.00.The results of periodate oxidation and Smith degradation showed that the molar percentage ratio of (1→3)-glycosidic linkages, (1→2)-glycosidic linkages and (1→6)-glycosidic linkages was about 63.84%:30.16%:6.0%. By using the LSCM to study LFPC₂a behavior in the solution, the conformation could be changed by the pH value. By SEM, LFPC₂a was observed the smooth figure under high-powered microscope. Thermal analysis of LFPC₂a indicated that litchi polysaccharide had similar endothermic peaks in nitrogen and air.LFPC₃, which had a average molecular weight of 33723 Da, composed of arabinose, rhamnose, galactose and glucose in the molar ratio of 2.42:3.44:1.00:1.65. The polysaccharide absorption peak showed similar time and shape as the protein absorption peak, suggesting that LFP3 is likely a glycoprotein.In vitro antioxidant activity of LFP1, LFP₂, LFP3 and LFP4 showed that exhibited a dose-dependent free radical scavenging activity against DPPH radical, superoxide anions, hydroxyl radical, chelating ability and reducing power. Among the four different fractions, LFP₂ showed the strongest scavenging activity.The antioxidant activity of litchi polysaccharide LFP₂ for mouse liver protection was investigated by CC14-induced and ethanol-induced liver injury experiments. Results indicated that LFP₂ increased SOD activity of mouse liver and serum, and reduced the content of MDA by dose-dependent pattern. LFP₂ also increased mouse liver CAT and GSH-Px activities. In the experiment of CC14-induced mouse oxidative injury, ALT and AST activities of mouse liver increased significantly, and high and medium dossage LFP₂ could inhibit the enzyme activities of ALT and AST. Results of experiment of ethanol-induced mouse oxidative injury showed that, three dossage of LFP₂ could also inhibit the enzyme activities of ALT and AST. These results indicated that litchi polysaccharide could increase the activity of antioxidant enzymatic system and antioxidant ability of liver to protect from injury.