Systematical Analysis Of The Polysaccharides From Panax Ginseng C.A.Meyer And Their Immunological Activities

Panax ginseng C. A. Meyer (P. ginseng) has been used in China as traditional medicine for over 2000 years. P. ginseng contains many active components including ginsenosides, ginseng peptides and ginseng polysaccharides. It has been reported that P. ginseng polysaccharides have immunomodulating, anti-tumor, anti-adhesive, antioxidant and hypoglycemic activities. But there is still a lack of understanding of the components and structures of all the ginseng polysaccharides. To understand total ginseng polysaccharides better, in this paper, water-soluble ginseng polysaccharides were extracted from the roots of P. ginseng by hot water and precipitated by ethanol. Then, the polysaccharides were totally fractionated by a combination of ion-exchange and gel permeation chromatography to obtain a series of homogeneous polysaccharides. Further, the structural features of these fractions were studied by enzymolysis, chemical methods and instrumental analysis. The immunomodulative and antitumor activities were also studied in mice. The investigation results are as follows:The water-soluble polysaccharides were extracted from the roots of P. ginseng with hot water, precipitated by 80% ethanol and deproteinated using the Sevag method. The polysaccharide mixture, referred to as WGP (for Water-soluble Ginseng Polysaccharides), was obtained in a yield of 9.7% (w/w). WGP contained 93.8% total carbohydrates, 7.6% uronic acid, 75.1% starch and less than 1% protein. Sugar composition analysis by high performance liquid chromatography indicated that WGP consisted of glucose (77.9%), galactose (6.8%), arabinose (4.6%), mannose (1.0%), rhamnose (1.1%) and galacturonic acid (8.7%).WGP was separated on a preparative DEAE-Cellulose column into two fractions first: an unbound fraction (WGPN) by water elution and a bound fraction (WGPA) by 0.5 M NaCl elution. Then, WGPA was separated on a preparative DEAE-Cellulose column into five fractions WGPA-H, WGPA-1, WGPA-2, WGPA-3 and WGPA-4 corresponding to 0.0, 0.1, 0.2, 0.3 and 0.5 M NaCl elution, respectively. Finally, the acidic fractions WGPA-1～WGPA-4 were further separated on Sepharose CL-6B into six homogenous fractions WGPA-1-AG,WGPA-1-HG,WGPA-2-AG,WGPA-2-HG,WGPA-3-HG and WGPA-4-HG.The major structural features of ginseng polysaccharide fractions were elucidated using high performance liquid chromatography, enzymolysis, Fourier transform infrared spectroscopy and 13C-nuclear magnetic resonance spectrometer. The results showed that WGPN were heterogenous starch-like glucans that can be totally hydrolyzed into small molecules byα-amylase. WGPA-H was a mixture of starch-like glucans and arabinogalactans. WGPA-1-AG and WGPA-2-AG were homogenous arabinogalactans with molecular weight of 1.1×105和1.2×105, respectively. These two fractions contained galactose and arabinose as main sugars and glucose, mannose, rhamnose, galacturonic acid and glucuronic acid as minor components. Arabinose in these two fractions might presents in three linkage forms includingα-(1-5)-Araf,α-(1-3)-Araf and non-reducing terminalα-Araf. Galactose hadβ-(1-3)-Galp,β-(1-4)-Galp,β-(1-3,6)-Galp and non-reducing terminal linkage forms which usually exist in the pectic arabinogalactan. WGPA-1-AG and WGPA-2-AG contained both type-I rhamnogalacturonan (RG-I) domains and homogalacturonan (HG) domains. WGPA-1-HG～WGPA-4-HG were homogenous polysaccharide fractions with molecular weight approximately ranging from 3.5×103 to 6.7×104, respectively. These four fractions contained high amount of galacturonic acid (62.4%～92.1%) and a small amount of glucose, rhamnose, galactose and arabinose. Based on the results of pectinase hydrolysis and NMR analysis, WGPA-1-HG～WGPA-4-HG were composed of a backbone of a linear-α-(1-4)-linked D-galacturonic acid with different degrees of methyl-esterification (30%～0). The small amount of neutral sugars might constitute RG-I domains linked to homogalacturonan domains.Summarizing the results from our experiments and literatures, we could conclude that P. ginseng polysaccharides are composed of starch-like glucan and pectin. The pectin contains arabinogalactan and galacturonan .The detailed structural features of WGPA-2-AG were elucidated using periodate oxidation, Smith degradation, methylation and partial acid hydrolysis. The results indicated that WGPA-2-AG contained a backbone of homogalacturonan domain linked to rhamnogalacturonan domain. The side chains contain arabinans, galactans and arabinogalactans attached to rhamnogalacturonan domain. Arabinose residues were present in the linkage forms of T-, 1,3-, 1,3,5-Araf and galactose residues in the forms of T-, 1,3-, 1,6-, 1,3,6-Galp. 42.9% arabinosyl residues were at the non-reducing terminals.The immunological and anti-tumor activities of ginseng polysaccharides were studied in mice. Ginseng polysaccharides can stimulate both T and B spleen lymphocytes of mice, inhibite the growth of transplanted S180 tumor cells and promote the immune systems of tumor bearing mice. The neutral ginseng polysaccharide WGPN had better immunomodulative activities than the acidic polysaccharide WGPA.