Separation, Purification And Antioxidant Activity Of Polysaccharides From Pueraria Lobata Endophytes

Pueraria lobata(Willd)Ohwi is a perennial deciduous vine in the family Leguminosae.Traditionally all parts of Pueraria lobata(Willd)Ohwi such as roots,stems,leaves and flowers are used in medicine,which is a traditional Chinese medicine with high levels of polysaccharides.Up to now,there has been an unprecedented increase interest in the polysaccharides.Many reports have indicated that polysaccharides have many potential biological and pharmacological activities,including beneficial immunomodulatory,antioxidant,anti-tumor,anti-virus,anti-inflammatory,hypoglycemic activities and so on.Endophyte are microorganisms that colonize living plant tissue without causing any immediate,overt negative effect.They have the potential to synthesize various bioactive natural products that may directly or indirectly be used as therapeutic agents against numerous diseases.Polysaccharides extracted from a variety of endophy can be a natural antioxidant.In our previous research,we found that endophyte Penicillium sp.RPS29 was rich in polysaccharides.In this study,we investigated the polysaccharides from Penicillium sp.RPS29,which was an endophytic fungus isolated from fresh root of an apparently healthy Radix puerariae grown in the XiangYu forest of Qinling&Bashan Areas(China).Extraction,purification and antioxidant activities of endophyte Penicillium sp.RPS29 polysaccharides(PSP)were investigated in the study.Then monosaccharide compositions and molecular weights of PSP-1*and PSP-2*were also investigated.The results of the experiment provided theoretical basis for the widely application of PSP in pharmacy and health care products.1.Box-Behnken design was employed to optimize the hot water extraction conditions.Response surface methodology showed that the optimal conditions of polysaccharides extraction were:extraction temperature 80.83℃,extraction time 2.50h and water/material ration 30.00,and considering the practical convenience,we adjust the optimal conditions to extraction temperature 80.0℃,extraction time 2.50h and water/material ration 30.00.Under these conditions,the yield of PSP reached 12.89±0.15%,which matched well with the predictive yield of 12.90%.2.In this study we removed protein from PSP by Sevag method,and used activated carbon adsorption.The crude PSP was fractionated into two fractions labelled as PSP-1*and PSP-2*,which were purified by chromatography of Cellulose DEAE-52 and Sephadex G-100 column.UV wavelength scanning results showed that the two components do not contain starch,protein,amino acids,peptides and nucleic acids and other impurity.3.The monosaccharides composition of them were measured by high performance liquid chromatography(HPLC).The results showed that the PSP-1*was consisted of D-ribose,L-rhamnose,D-xylose,L-arabinose,and their relative mole percentages of all the quantitative monosaccharides were29.83%,35.61%,23.11%and 11.45%respectively.The PSP-2*was composed of D-ribose,L-rhamnose,D-glucose,D-xylose with the mole percentages of 26.30%,28.59%,35.13%and 9.98%respectively.The average molecular weights of PSP-1*and PSP-2*were estimated to be 2.77 X104 Da and 5.38 X103 Da,respectively.4.We evaluated antioxidant activity of PSP-1*and PSP-2*by the scavenging ability of DPPH free radical,ABTS free radical,hydroxyl radical free radical,superoxide ion free radical and ferrous ion chelating activity in vitro.The results showed that PSP-1*,PSP-2*have a certain antioxidant activity.They could be used as natural antioxidant.The research will provide technical and theoretical support for the development of endophyte polysaccharides.