The Structures Of Water-extracted Ginseng Pectic Polysaccharides And Their Distribution In Cell Walls

Panax ginseng C. A. Meyer (P. ginseng) has been used in China as traditional medicine for more than 4,000 years. P. ginseng contains many active components including ginsenosides, ginseng peptide, oliogosaccharides and ginseng polysaccharides. Ginseng polysaccharides are composed of starch-like glucans and ginseng pectic polysaccharides. Ginseng pectic polysaccharides are one of the three main components of ginseng root cell walls. Ginseng pectic polysaccharides have a wide range of pharmacological and therapeutically actions. They act on the central nervous system, cardiovascular system and endocrine secretion, promote immune function and metabolism, possess bio-modulation action and also have anti-stress and anti-ageing activities. However, little is known about the distribution of ginseng pectin polysaccharides in cell walls and their fine structures. We have undertaken an immunochemical study of ginseng polysaccharides. Antibodies directed to cell wall polysaccharide epitopes are important tools for the immunochemical analysis of polysaccharides in situ and they can also be useful to identify structural features present in isolated polysaccharide fractions. A combination of enzyme treatment, anion-exchange and size-exclusion chromatographies are used to fractionate different pectic polysaccharide domains; then the structures of the isolated pectic polysaccharide domains were studied by enzymolysis, ELISA, chemical methods and instrumental analysis.Here we report a study of the use of the cell wall antibodies in immunofluorescence procedures to determine the distribution and contents of cellulose, hemicelluloses, pectic polysaccharides and protoglycan in ginseng roots. Primary cell wall polysaccharides in ginseng roots are made of crystalline cellulose, xyloglucan, mannan, rhamonogalacturonan I (RG-I), protoglycans and high-methyl homogalacturonan (HG). Secondary cell wall is mainly composed of xylan, extensin and high-acetylated HG. Arabinogalactan protein (AGP) glycan is present on the surface of starch granules, in secretory cavities and in primary cell walls, which indicate diverse functions of AGP in ginseng roots. The results also showed that HG masking the epitopes of hemicelluloses and pectic polysaccharides in situ is widespread phenomenon in the primary ginseng cell walls. HG could mask the epitopes of hemicelluloses indicates that there are linkages between HG and hemicelluloses although we could not deduce if it is covalent linkages or not. HG could mask the epitopes of arabinan and AGP glycans but not galactan indicate that when the polymers assemble into cell walls, HG and galactan are present on the surface of cell wall, while arabinan and AGP glycans are wrapped inside.In order to study the structure characteristics of water-extracted pectic polysaccharides, ginseng pectin were fractionated systematically and detailedly according to the previous study in our lab. WGPA-H, WGPA-1, WGPA-2, WGPA-3 and WGPA-4 were made according to the method in our lab. Then WGPA-1~WGPA-4 were separated by size-exclusion chromatography to obtain four HG fractions and four RG fractions. In order to get non-HG pectic polysaccharides, ginseng pectin WGPA was hydrolyzed by endo-polygalacturonase to remove the un-esterified HG domain, then the hydrolysate was separated by a combination of anion-exchange and size-exclusion chromatography to get five RG-I fractions (RG-I-1, RG-I-2, RG-I-3A, RG-I-3B and RG-I-4), two AG fractions (AG1 and AG2) and one HG fraction (HM-HG).The structural features of isolated pectic fractions were elucidated using enzymolysis, HPLC, ELISA and 13C NMR. Analytical results showed that WGPA-1-HG and WGPA-2-HG were composed of a linearα-1,4-linked D-GalA with different degree of methyl-esterification and acetylation, and a small amount of neutral sugars that existed in these fractions might constitute RG-I domains with type II arabinogalactan linked to HG domains. Both WGPA-3-HG and WGPA-4-HG contain un-esterified and methyl-esterified HG. RG-I-2, RG-I-3A, RG-I-3B and RG-I-4 contain the characteristic monosaccharide compositions of RG-I which are GalA, Rha, Gal and Ara. Fractions RG-I-2 and RG-I-3B were two similar fractions, which had small molecular weights (around 5 Kd) and low ratios of Rha/GalA (around 0.25). RG-I-2 and RG-I-3B contained highly esterified HG segments and the the side chains were very complexed with type I and type II arabinogalactan (AG) as side chains. RG-I-3A and RG-I-4 are high molecular weight fractions that have similar compositions including typical RG-I monosaccharides and NMR peaks indicating high acetylation. Results showed that these two fractions contained highly esterified HG segments and the side chains were AG. Immunochemical analysis was processed on the structure features of the isolated ginseng pectic fractions which showed some antibodies bound strongly on these fractions. The striking feature of the in situ localization of the epitopes (that were observed to be abundant in the isolated ginseng fractions) is their predominant occurrence in the regions of cortical secretory cavities of the roots. The other cell wall components such as hemicelluloses that were not detected in the isolated fractions were not abundantly detected at these cavities. So the water-extracted pectic polysaccharides originated from secretory cavities.Pectin related enzyme hydrolysis, partial acid hydrolysis, 13C NMR and ELISA were used to study the fine structures of RG-I-3A and RG-I-4. The results showed that the backbone of RG-I-3A consists of two segments: the four residues repeats [â†'4)-α-GalpA-(1â†'4)-α-GalpA(1â†'4)-α-GalpA-(1â†'2)-α-Rhap-(1â†']n and two residues repeats [â†'4)-α-GalpA-(1â†'2)-α-Rhap-(1â†']n. The backbone of RG-I-4 consists of the―smooth region‖ofα-(1â†'4)–GalpA and [â†'4)-α-GalpA-(1â†'2)-α-Rhap-(1â†']n as the―hairy region‖. The side chains of RG-I-3A and RG-I-4 are short and concentrated. All the side chains are attached to the O-4 of Rha in the backbone. 71% of Rha in RG- I-3A and 67% of Rha in RG-I-4 contained side chains. There are several types of side chains: small amount of [α-1,5-Araf]5-7; [α-1,5-Araf]1-4, most Ara is on the non-reduce terminate; less [β-1,4-Galp]4-6, more than 80% [β-1,4-Galp]1-4; less [β-1,3/1,6-Galp]1-2 which linked to the C-4 of Rha.