Purification And Structure Analysis Of The Polysaccharides From Panax Quinquefolius

Panax quinquefolius?P quinquefolius?,as a world-known herb,contains many active components,such as saponins,peptides and polysaccharides.It has been reported that P.quinquefolius polysaccharide has many bioactivities,such as immunomodulation,anti-tumor,antioxidant and hypoglycemic activities.But there is still a lack of sduty about the detailed structure of P.quinquefolius polysaccharides,which restricted the study about their biological activities and the relationship between structure-activity.In this study,P.quinquefolius polysaccharide was extracted by hot water and systematically fractionated by a combination of anion-exchange and gel permeation chromatographies.Furthermore,the macromolecular features and anti-tummer activities of these fractions were studied.A crude polysaccharide,referred to as WQP?for Water-soluble Quinquefolius Polysaccharides?,was obtained with the yield of 10.5%,after extracted by hot water,precipitated by 80%ethanol and deproteinated by Sevag reagent.Monosaccharide composition analysis by HPLC indicated that WQP contained Glc?43.3%?,GalA?33.8%?,Gal?9.5%?,Ara?9.3%?and Rha?2.0%?.WQP contained 85.5%total sugar,24.5%uronic acid and 0.9%protein.The crude polysaccharide WQP was first applied to DEAE-Cellulose column and seperated into two fractions,an unbound fration WQPN by water elution with a recovery of57.9%and a bound fraction WQPA by 0.5 M NaCl elution with a recovery of 32.5%.In the second run purification,WQPA was applied to DEAE-Cellulose column and eluted by a stepwise gradient of NaCl.With these elution steps,WQPA was seperated into five fractions,WQPA-N?16.0%?,WQPA-1?4.0%?,WGPA-2?37.6%?,WGPA-3?30.9%?and WGPA-4?16.0%?corresponding to 0.0,0.1,0.2,0.3 and 0.5 M NaCl elution,respectively.All collected acidic fractions from the DEAE-Cellulose column,WQPA-1^WQPA-4,were further separated on a Sepharose CL-6B column and six fractions were obtained,named WQPA-1-AG,WQPA-1-HG,WQPA-2-AG,WQPA-2-HG,WQPA-3-HG and WQPA-4-HG respectively.Gel permeation chromatography and high permeation chromatography showed that the two neutral fractions WQPN and WQPA-N were heterogeneous,while the six acidic fractions were homogenous,with molecular weights approximately ranging from 5.5×10³to2.3×10⁵Da.The major structural features of P.quinquefolius polysaccharide fractions,including monosaccharide composition,molecular weight,glycosidic linkage,were elucidated using high performance liquid chromatography,enzymolysis,Fourier transform infrared spectroscopy and ¹³C NMR.Analytical results showed that the neutral fractions WQPN and WQPA-N were starch-like glucans that can be totally hydrolyzed into small molecules with?-amylase.WQPA-1-AG mainly contained Glc,Gal and Ara,with Man,Rha and GalA as minor components.These monosacchrides were in the form of?-1,4-Glc,?-1,3-Ara,?-1,3,6-Gal and?-1,6-Gal glycosidic linkage in WQPA-1-AG,which indicated that it might be made up of glucan and arabinogalactan.WQPA-2-AG mainly contained Gal and Ara and might present in three linkage forms including?-?1-5?-Araf,?-?1-3?-Araf and?-?1-3,6?-Galp,which indicated that WQPA-2-AG belonged to arabinogalactan.WQPA-1-HG mainly composed of Glc and GalA and present in?-1,4-Glc and?-1,4-GalA forms,which indicated that it might be made up of glucan and homogalacturonan.WQPA-2-HG^WQPA-4-HG contained high amounts of GalA,72.3%,78.6%and 86.9%respectively.Combining the pectolytic hydrolysis and NMR analysis,it was deduced that these fractions were composed of a backbone of a linear-?-?1-4?-linked D-galacturonic acid with different degrees of methyl-esterification.On a preliminary bioactivity test,WQP?WQPN and WQPA exhibited antitumor activity against Carcinoma HCT-116 in vitro.Moreover,the antitumor activity was similar with each other.However,there was no antitumor activity against Carcinoma HT-29 in these three fractions.In this research work,the purification and macromolecular features of P.quinquefolius polysaccharide had been systemly studied.Furthermore,we also showed the antitumor activity of these fractions,which was helpful for the study of strcture-activity in P.quinquefolius polysaccharide.