Isolation, Structural Investigation And Antioxidant Activity Of Pine Cone Polysaccharides

Pinus koraiensis (family Pinaceae) is widely distributed in northeasternChina. It has been used as the traditional Chinese medicine for a long time and beenrecorded in some books such as the Compendium of Materia Medica. Pine cones arethe fruits of Pinus plant, which have strong effects on eliminating phlegm, dispellingwind, removing phlegm and preventing asthma. They are of great value forutilization. According to the report, the water extracts from pine cones haveanti-oxidation, anti-bacteria and anti-tumor effects. However, in the modernmanufacturing, pine cones are usually discarded as wastes, which leads to thewasting of resources. In our study, the polysaccharide was extracted and purifiedfrom the pine cones of P. koraiensis and its structural characterization was alsoinvestigated in this thesis. Additionally, the antioxidant activities of different pinecone polysaccharides were also studied. All the information provided here willcontribute to better understanding of pine cone polysaccharides about theirstructural characterization and antioxidant activites, and be of great help for theirapplication as novel medical plant products. The results are shown as following.Three polysaccharide fractions were isolated from pine cones of P. koraiensis,P. armandii and P. sylvestris var. mongolica by water extraction and ethanolprecipitation, and then purified by trichloroacetic acid and activated carbon.According to the instrumental and chemical analysis, their physicochemicalproperties, such as neutral sugar content, uronic acid content, monosaccharidecomposition, polyphenol content, amino acid composition and molecular weightwere investigated. The result showed that they were all compounds, including uronicacids about50%, neutral sugar about40%. The content of amino acids andpolyphenol was less than2%. In the analysis of monosaccharide composition, itcould be seen that the molar ratios of monosaccharide components in PAP and PSPwere similar, whereas they were different from PKP.The polysaccharide fractions were extracted from pine cones of P. koraiensisby water extraction and ethanol precipitation, and then purified by trichloroaceticacid and activated carbon. And then the purified polysaccharide PKP-A-3b wascollected through30%ethanol precipitation, DEAE-52ion-exchange resin, andSephadex G-200column. The yield of PKP-A-3b was33%and it was proved to be asingle substance by HPLC-GPC. Its molecular weight was286.59kDa. Variouschemical and instrumental methods have been employed to study its structuralcharacterization and the results were as following: according to IR spectra and UVspectra, it did not contain binding ribose or protein, but uronic acid; the analysis of monosaccharide composition showed that it was composed of D-ribose, L-rhamnose,L-arabinose, D-xylose, D-mannose, D-glucose and D-galactose in the molar ratio of0.21:0.29:0.50:0.25:0.38:0.78:1.25; Periodate oxidation and Smith degradationof PKP-A-3b were performed to deduce the glycosidic linkage and the resultindicated that the glycosyl linkages of1→2,1→2,6,1→4and1→4,6might exist.And the presence of seven monosaccharides after Smith degradation implies thatthey might be linked in the form of1→3、1→3,6、1→2,3、1→2,4、1→3,4、1→2,3,4.1H NMR spectra showed that the anomeric protons of PKP-A-3b had both-and β-configurations. In the13C spectrum, there were mainly three signals from theanomeric carbons, which might be assigned to the→4)--D-GalpA-(1→、T--D-Galp-(1→and→2)--L-Rhap-(1→units, respectively. Signals at175.57and171.25were attributed to the acidic form of carboxyl group and estered carboxylgroup.The antioxidant capabilities of PKP, PAP and PSP were evaluated in this study.The results showed that: three polysaccharide fractions all had significantly potentscavenging ability on ABTS radicals and hydroxide radicals and their scavengingability decreased in the order of PKP> PAP> PSP. However, the scavenging abilityof ascorbic acid on both radicals was much better than all the three polysaccharidefractions. At30mg/mL, the scavenging rates of PKP on ABTS and radicals andhydroxide radicals were both above50%, which showed that PKP had the potentialto be developed as novel medical plant products. The different radical scavengingcapabilities of three polysaccharide fractions might be due to their differentchemical composition, such as uronic acid and monosaccharide composition.However, these polysaccharide fractions had little scavenging abilities onsuperoxide radicals. Furthermore, the antioxidant activities of5polysaccharidefractions precipitated with different concentrations of ethanol were also studied.The result showed that, they all were effective on scavenging ABTS radicals andhydroxide radicals and the increase in concentration of5polysaccharide fractionswas synonymous to an increase in scavenging capability. In the ABTS radicalscavenging assay, their scavenging ability decreased in the order of PKP-E>PKP-A> PKP-D> PKP-B> PKP-C, but all of them showed less effective radicalscavenging cabability than PKP. In the hydroxide radical scavenging assay, theirscavenging ability decreased in the order of PKP-E> PKP-A> PKP-B> PKP-D>PKP-C. PKP exhibited the less effective scavenging activity on hydroxyl radicalsthan PKP-E, but far more effective than PKP-D and PKP-E. PKP-A and PKP-B had the similar hydroxyl radicals scavenging capability with PKP, which demonstratedthat PKP-A could potentially be used as natural antioxidants and be worthy offurther investigation.