Study On Extraction, Purification, Bio-Activity And Preliminary Structure Identification Of Porphyran Polysaccharide

Porphyran belongs to the class of Rhodophyta, order of Protoflorideae, family of Bangiales, genus of Porphyra. Porphyran distributes naturally in intertidal zone of Fujian, Guangdong and Zhejiang provinces coast areas. Recently, the wide study of component and value of health protection on Porphyra has discovered that Porphyran Polysaccharide (PP) and protein are main boi-active constituents. Porphyran Polysaccharide was the objective in this thesis. The extraction and purification of PP were studied. Then the anti-oxidation effect and evaluation of the immunomodulatory activity of main fraction were measured, the active fraction was identified finally. The monosaccharide composition and chemical structure of the active PP were identified.The main results showed as follows:1. The extraction technology of hot water extract, ultrasonic-assisted extract and Cellulase synergistic method under ultrasonic wave were established:Cellulase synergistic method under ultrasonic wave was used to extract the water-soluble polysaccharide from Porphyra, and was compared with the hot water extraction method and ultrasonic-assisted method. On the foundation of the single-factor tests, the porphyran polysaccharide extraction by hot water method was designed and its result was analyzed using Design-Expert 7.0 software. The relation model of the extraction rate with different operating variables is established. Normal probability plot of residual for the polysaccharide extraction and response surface for desirability function of the porphyran polysaccharide extraction with different factors were given in this paper. The results showed that the optimum conditions of Porphyran Polysaccharide with hot water extraction method were as follows:the extraction temperature of 85℃, extraction time of 2.3 h and the ratio of water to material of 49:1, and under such conditions, the yield could be up to 5.18% at one-time extraction. The optimal extraction condition obtained lately by orthogonal tests of ultrasonic-assisted extraction were as follows:extraction time of 30 minutes, the ratio of water to material of 50 : 1, the pH was 7.0. Under these conditions, the extraction rate was 6.78%. The optimum extraction conditions with Cellulase synergistic method under ultrasonic wave were as follows:ultrasonic-assisted extraction time of 10 minutes, the ratio of water to material powder (v/w) was 30, the extraction time was 50min with temperature at 50℃and pH 6.0. Under such conditions, the yield could be up to 11.56% at one-time extraction. The results showed that Cellulase synergistic method under ultrasonic wave was simple and had higher extraction yield.2. Separation and purification of PP and the evaluation of the physical and chemical properties of main PP fraction:This paper compared with deproteinized methods of Sevage and TCA. The Sevage method is more effective in removing protein. Porphyra polysaccharide (PP) was successively fraction by column chromatography on DEAE-52 cellulose. A lyophilized fraction of PP was chromatographed on a DEAE Cellulose-52 anion-exchange column to yield seven peaks, PP1, PP2, PP3, PP4, PP5, PP6 and PP7. The yields of PP1, PP2, PP3, PP4, PP5, PP6 and PP7 were 5.18%,11.21%,17.15%, 19.88%,4.77%,3.02% and 1.24%, respectively. The physical and chemical property studies showed that Porphyra polysaccharide was a kind of polysaccharide which doesn’t contain monosaccharide, starch.3. The anti-oxidation effect and evaluation of the immunomodulatory activities of PP, PP3 and PP4:The results showed that Porphyra polysaccharide of PP, PP3 and PP4 all exhibited strong scavenging activities on hydroxyl radical and superoxide and reducing power. Meanwhile, PP, PP3 and PP4 had scavenging effects on hydroxyl radical and superoxide anion radical; and had the inhibiting effects on lipid peroxidation of liver homogenate in vitro. The porphyra polysaccharide of PP, PP3 and PP4 also could protect liver from peroxidation induced by Fe2+ or H2O2, and inhibit the mitochondria swelling and hemolysis of red blood cells.In different antioxidant systems, PP, PP3 and PP4 had different antioxidant abilities. In addition, the separation fractions (PP3 and PP4) of Porphyra polysaccharide had a synergistic and direct effects on the Con-A-stimulated lymphocyte proliferation as well as a hypoxia regulation effect. It further suggested that PP3 and PP4 may be the bi- diretional immuno-modulatory effects of polysaccharide. We also found that the antioxidant ability and immune function of PP4 was better than PP3. This study will provide foundation of the relationship between function and structure for the further research.4. Preliminary study on the structure of PP:A lyophilized fraction of PP4 was chromatographed on a Sephadex G-100 gel anion-exchange column to yield only one peaks of PP4a. And its recovery was 38.8%. Determination with GC, Porphyra polysaccharide(PP4a) was a heteropolysaccharide which consisted of rhamnose, arabinose, galactose and glucose. It was indicated that PP4a contains a lot of 1â†'3 glycosidic bond, and maybe contains 1â†'2,1â†'2,6,1â†'6 glycosidic bond bit according to the experimental results of sodium periodate oxidation and Smith degradation. The CD spectrum showed that asymmetry, conformation and aggregation of PP could be influenced by metal ions.