Study On Isolation, Purification, Structure And Biological Activity Of PKP-E From Pine Cones Of Pinus Koraiensis

The polysaccharides from pine cones of Pinus koraiensis (PKP) was investigatedby stepwise ethanol precipitation. Five fractions were isolated. PKP-E was chosen asthe material in the further tests by measuring the biological activities of the fivepolysaccharides. PKP-E was purified by DEAE-52and Sephadex G-100, and four mainhomogeneous polysaccharides were harvested. Then the structure and the bioactivitiesof the purified polysaccharides were investigated in the research. To the best of ourknowledge, no purification and structural characterization of polysaccharides from pinecones of P. koraiensis has been reported.(1) Five polysaccharide fractions named PKP-A,-B,-C,-D and-E weresuccessfully separated at final ethanol concentration of30%,50%,60%,70%and80%,respectively. Monosaccharide components analysis indicated that the five fractions allwere composed of L-rhamnose, L-arabinose, D-mannose, D-glucose and D-galactose.HPLC results revealed that the polysaccharides precipitated by higher concentration ofethanol solution had lower molecular mass. The content of D-galactose in the fivefractions were higher than other monosaccharides, while L-rhamnose, L-arabinose andD-galactose in PKP-E were much higher. The antioxidant activities of the five gradedfractions were studied on the basis of hydroxyl radical and ABTS radical scavengingtests. The five graded polysaccharide fractions exhibited good inhibiting power, andMTT tests in vitro showed the IC50of PKP-E were2070.0μg/mL.(2) The fraction PKP-E was successively purified by chromatography on DEAE-52and Sephadex G-100column, giving four major polysaccharide fractions termedPKP-E-1-1, PKP-E-1-2, PKP-E-2-1and PKP-E-2-2.(3) The structural characterization of purified fractions were studied by usingperiodic acid oxidation, smith degradation, methylation combined with HPLC, GC-MS,FT-IR and NMR spectra.The four purified fractions were heteropolysaccharides, composed of L-rhamnose,L-arabinose, D-mannose, D-glucose and D-galactose. The fractions were mainlyα-pyranose. No nucleic acid and no triple helix structure existed in PKP-E-1-1. A smallamout of protein and-O-were detected. The fractios were mainly linked by1â†'6or1â†'glycosidic bonds. α-L-Ara-(1â†',â†'2,6)-β-D-Man-(1â†',â†'4)-β-D-Glcp-(1â†'andâ†'6)-α-D-GalpA-(1â†'could be summarized by1D-and2D-NMR. Moreover,L-rhamnose, L-arabinose and D-galactose in PKP-E-2-1were much higher than otherpurified fractions.(4) According to the antioxidant essays, PKP-E-2-1was the most stable and active antioxidant. And the IC50on hydroxyl radical and ABTS radical were3.0and23.6mg/mL, separately.