Purification And Characterization Of A Low Molecular Weight Polysaccharide From Panax Ginseng By Enzymatic Hydrolysis

Ginseng root is one of the most renowned Chinese herbal medicines and foods with numerous pharmacological effects,which are attributed to various bioactive components,including ginsenosides,ginseng polysaccharides,vitamins,and volatile oils etc.Among these components,ginseng polysaccharides have aroused widespread concerns and showed numerous biological activities,such as anti-inflammatory,anti-cancer and anti-oxidant etc.In the past few decades,many polysaccharides have been purified and structurally identified from ginseng,majority of which are high molecular weight,and almost no low molecular weight polysaccharides have been reported.In this study,a novel neutral non-starch polysaccharide(GP1A)was extracted using hot water from ginseng roots,followed by a combination of a-amylase and papain enzymatic hydrolysis,and purified by DEAE-52 and Sephadex G-100 chromatography column.Chemical and instrumental analysis was carried out to systematically elucidate the microstructure and molecular structure of GP1 A.The result is as follows:(1)Ginseng roots were extracted with hot water and then precipitated with ethanol to prepare the crude ginseng polysaccharide.The extraction ratio of raw material to water was 1:20,the extraction temperature was 80℃ and the extraction time was 3h.The extraction yield of the crude ginseng polysaccharide was 9.32%(w/w),among which the total sugar content,the starch content and the protein content was 53.14±1.59%,9.18 ± 0.13%and 1.53±0.04%(w/w),respectively.Starch and proteins in crude ginseng polysaccharides was hydrolyzed by a-amylase and papain,which led the starch was completely removed and proteins content reduced to less than 0.5%.Finally,after the hydrolyzed polysaccharide was purified by DEAE-cellulose ion exchange chromatography column and G-100 Sephadex gel column,the ginseng polysaccharide GP1A with a yield of 7.28%was obtained.(2)The physicochemical properties of GP1A were determined by UV-Vis spectroscopy,gel permeation chromatography,scanning electron microscopy,X-ray diffraction and Fourier infrared spectroscopy.The results indicated that GP1A didn’t contain nucleic acid and protein impurities;GP1A was a homogeneous polysaccharide with the average molecular weight of 1.025 kDa;GP1A had a typical polysaccharide honeycomb microstructure with low crystallinity,which indicated that GP1A was an amorphous polysaccharide;The FT-IR results showed that GP1A contained characteristic functional groups of polysaccharides,of which a-type pyranoside was the dominating glycosidic bond.According to the physiochemical analysis,GP1A was a low molecular weight neutral polysaccharide with an amorphous structure.(3)After degraded by the trifluoroacetic acid and then derivative by the silane reagent,the monosaccharide composition and content of GP1A was analyzed by gas chromatography-mass spectrometry(GC-MS)and gas chromatography(GC).The results showed that GP1A was mainly composed of glucose,galactose,mannose,xylose and arabinose,and the molar ratio of which was 90.62:2.01:7.14:1.05:0.20,respectively.The primary structure of ginseng polysaccharide GP1A was further identified by one-dimensional and two-dimensional nuclear magnetic resonance(NMR)spectroscopy,including 1H-NMR,13C-NMR,1H-1H correlation spectroscopy,heteronuclear single quantum correlation spectroscopy and heteronuclear multiple-bond correlation spectroscopy.In the 1H NMR spectrum,the signals of anomeric protons were in the regions ranging from δ 4.5-5.4 ppm,which suggested that GP1A contained both of a-type and β-type glycosidic.According to the 13C NMR,the signals in the range from δ 60-85 ppm correspond to the pyranose ring structure of GP1 A.Five anomeric proton signals at δ 5.31 ppm、δ 5.14 ppm、δ 4.88 ppm、δ 4.58 ppm and δ 4.56 ppm in the 1H NMR spectrum was in accord with the carbon signals at δ 100.38 ppm,δ 99.60 ppm,δ 98.25 ppm,δ 95.76 ppm and δ 95.75 ppm in the 13C NMR spectrum,respectively.Based on the GC-MS analysis of the monosaccharide composition,the five signals were assigned to α-D-glucose,a-D-galactose,β-D-xylose,β-D-mannose,and β-D-arabinose.According to the monosaccharide composition of GP1A and assignments of 1H and 13C chemical shifts of each glycosidic linkage,the precise structure of GP1A was drawn as below:In summary,a low-molecular-weight neutral ginseng polysaccharide was extracted by hot water,precipitated by ethanol,and then followed by a combination of enzymatic hydrolysis and column purification.The physicochemical properties and primary structure of the low-molecular weight ginseng polysaccharide was systematically evaluated.This study might promote a better exploitation and application for the extraction,physicochemical properties and biological activity of ginseng polysaccharides.