Study On Extraction, Purification, Structure And Antioxidative Activity Of Polysaccharides From Pinus Koraiensis Pinon Shell

Pinus koraiensis belong to Pinaceae, which is the most important pine speciesin northeast of China. Pine nut shell are often discarded or burned as the shell afterremoval of pine nuts. Modern pharmacological studies have shown that Pinuskoraiensis Pinon Shell Polysaccharides have several bioactivities, includinganti-tumor, antiviral and so on. So it will be very meaningful to study on extraction,isolation and purification, biological activities of active polysaccharide.In this paper, Pinus koraiensis Pinon Shell Polysaccharide were systematicstudied on the aspect of optimization of extraction, separation and purification by themembrane separation combine with chromatography, antioxidant activity,monosaccharide, primary structure and improvement of the determination method,the results of the study are as follows:1. In the present work, the determination method of polysaccharides contentthrough3,5-Dinitrosalicylic acid (DNS method) was improved significantly. TheDNS method has many outstanding advantages, however, it had significantdifferences whether the maximum absorption wavelength of the test solution wasselected as the determination wavelength or not (the reason of objectors is that thestrongly absorption of colour-developing agent has serious interference under thedetermination wavelength), the exploratory study was carried out on the side. In thispaper, targeted review about this side was reported for the first time, and thecorresponding experimental datas were offered.The experiment results show that the basic reason of the above determinationwavelength controversy was appropriate design of blank solution, not wavelength factors. Colour-developing agent was selected as contrast, then the interference ofcolour-developing agent that was caused by selecting distilled water as contrast waseffectively eliminated. It was not only unnecessary that choosing other determinationwavelength rather than the maximum absorption wavelength of the test solution, butalso easy to increase the analysis error. In this experiment, the appropriatewavelength of DNS determination method of Pinus koraiensis Pinon ShellPolysaccharides content was the maximum absorption wavelength of the testsolution500nm.Colour-developing agent was selected as contrast simultaneously.The results of choosing other determination wavelength or selecting distilled wateras contrast were clearly wrong. The results can also provide a reference for DNSdetermination method of other polysaccharides or reducing sugar content.2. To study the extraction technology of ploysaccharides from Pinus koraiensisPinon Shell, the effects of water ratio，decoction time，and extraction times on theyield of the polysaccharides were investigated．Then, the optimum conditions wereobtained through the orthogonal experiment. The conditions were as follows：theratio of material to water1:15(g/ml)，extracted3times for2h per time. The sequenceof factors influence on polysaccharide yield was: water ratio>extractiontimes>decoction time. Under these optimal conditions, the yield of thepolysaccharides reached2.9%．3. Via membrane separation purification, deproteinization with papain-sevageand macroporous resin decolorization, the total polysaccharides was obtained fromcrude polysaccharides which were prepared by the optimum extraction conditionsfrom Pinus koraiensis Pinon Shell. and it is established that a could realizeindustrialization process route.The membrane separation technology combine with the ion exchangechromatography were used to furter purified total polysaccharides and fivecomponents were obtained and respectively named PPSPⅠ-1、 PPSPⅠ-2、PPSPⅠ-3、and PPSPⅠ-4. A method for isolation and purification of the totalpolysaccharides was established4. The monosaccharide composition and primary structure of totalpolysaccharides and purified polysaccharide were analyzed.Chromatography analysis showed that total polysaccharides PPSPⅠ-b is aheteropolysaccharide, at least from rhamnose、xylose monosaccharide compositionof the six, the molar ratio of monosaccharide composition as follows: Rha: Ara: Xyl: Man: Glu: Gal=3.40:1.40:0.79:7.82:10.67:18.35;PPSP Ⅰ-1and PPSP Ⅰ-2wasmainly composed of rhamnose, arabinose, mannose, glucose and galactose, and theratio of each sugar differed from each polysaccharide；PPSPⅠ-4is composed withonly glucose. GC shows each sample also contains an unknown chromatographicpeak, it infer that maybe other monosaccharide.The IR spectra analysis showed the four ingredients(PPSPⅠ-1、PPSPⅠ-2、PPSPⅠ-3and PPSPⅠ-4) have the typical characteristic absorption peaks of apolysaccharide. The monosaccharides of each component were all pyranose, but theglycosidic bond differed from each other. Infrared spectrum(IR) displayed thatPPSPⅠ-1and PPSPⅠ-2only exited α-glycosidic bond, PPSPⅠ-3contained α andβ-glycosidic bond at the same time, PPSPⅠ-4only contained β-glycosidic bond. Inaddition, it was inferred that PPSPⅠ-2、PPSPⅠ-3and PPSPⅠ-4was a kind of acidpolysaccharide, This is consistent with the result from the DEAE-cellulose column.5. It was studied that the antioxidative activity in vitro of five fractionpolysaccharide PPSPⅠ, PPSPⅡ, PPSPⅠ-a, PPSPⅠ-b, PPSPⅠ-2, and theregularity of the results was obtained.The results showed that five kinds of polysaccharides were all have a certainantioxidant activity in vitro and the antioxidant activity increased with increasingconcentration. The different fractions of polysaccharides all have the similar trendsto scavenge OH and DPPH·, the scavenging abilities was: Vc>PPSPⅠ>PPSPⅡ>PPSPⅠ-a> PPSPⅠ-b> PPSPⅠ-2. The antioxidant activity was the strongest of thetotal polysaccharide PPSP Ⅰ by membrane separation, and the activity wasdecreased after the protein was removed, the effects of the polysaccharide bydecolorization was relatively weaker the PPSPⅠ-2was the weakest.In previous studies, the technology of deproteinization and decolorization wereusually used to purified polysaccharides. Above-mentioned result indicating that theantioxidant activity of the PPSP may be associated with the protein、pigment andother components. Polysaccharide should not bindly be deproteinized and decoloredWhen it was used as the antioxidative activity components.This thesis has a systematic study focused on the Pinus koraiensis Pinon ShellPolysaccharides and made obvious progress. Several content was studied for the firsttime, by the above results, Pine nut shell waste resources has the good developmentprospects, the further development of work worthy of attention.