Separation, Purification And Bioactivity Of Polysaccharide From Pine Cone Of Pinus Koraiensis

Pinus koraiensis belong to Pine Family:"Pinaceae", which is the most important pine species in northeast of china. This paper initiate the current study with separation and purification of polysaccharide from P. koraiensis pine cone. The bioactivities of polysaccharide of pine cone were investigated after knowing the physicochemical properties and monosaccharide composition. Polysaccharide of pine cone was extracted by the way of hot water extraction, on the basis of single-factor tests, its technology was optimized by orthogonal experiment with 3 factors and 3 levels. The results showed that the optimum conditions is as follow: the extraction temperature is 100℃, the ratio plant material/water is 1:12 (w/v), the extraction time is 4 h. The extraction rate ofpolysaccharide of pine cone was up 15.2 mg·gP-1P. At the same time, using SPME-GC-MS technique to analyze the chemical composition of pine cone, and 66 compands were identified, which are mainly monoterpenes and sesquiterpenes. Systematic study on the purification technology of polysaccharide of pine cone, the way of enzyme and TCA were used in deproteinization, the way of HB2BOB2B and active carbon were used in decolorization. In the deproteinization assessment, the way of enzyme showed that the optimum conditions were pH=8.80, dosage of enzyme equal to 0.10%, the temperature is 100℃and the time is 2.0 h, and the deproteinization rate of polysaccharide was up to 83.42%. The TCA method showed that the optimum conditions are 1:3(v/v) sample solution/TCA ratio, 20 min shaking time and TCA concentration equal to 6%, and the deproteinization rate of polysaccharide was up to 72.41%. In the decolorization test, the way of HB2BOB2B showed that the optimum conditions were 6% of HB2BOB2B, the reaction time is 60 min, the optimal temperature is 80℃and pH value is 11, and the decolorization rate of polysaccharide was up to 95.50%. The way of active carbon showed that the optimum conditions were the dosage of active carbon of 1.5%, the adsorption time of 60 min, the temperature of 50℃and pH value of 3.0, which the decolorization rate of polysaccharide was up to 74.52%.The crude polysaccharides was purificated, its physicochemical property and purity was analyzed. Using TLC (Thin Layer Chromatography) and GC-MS technique to analyze the composition of polysaccharide of pine cone, Monosaccharide identified in Pinus koraiensis are Rha: Ara: Xyl: Man: Glu: Gal, and their ratio of molar is 1.34: 1.73: 0.39: 2.59: 3.61: 8.66. At last, the anti-bactia and anti-oxidant activity of polysaccharide were investigated respectively. In the antibacterial study, there is an inhibition effect of coliform, salmonella and shigella, the concentration of polysaccharide and effect of inhibition have obvious dose-respone correlation when applied to coliform and salmonella. In antioxidant study, the polysaccharide of pine cone has considerable free radical clearance, its ICB50B is about 28 mg·mL^-1, and the polysaccharide of pine cone also has Fe²⁺ chelating ability, its ICB50B is about 78 mg·mL^-1.