Rhamnogalacturonan II dimers cross-linked by borate diesters from the leaves of Panax ginseng C.A. Meyer are responsible for expression of their IL-6 production enhancing activities

Abstract

Leaves of Panax ginseng C.A. Meyer contain larger amounts of thiobarbituric acid (TBA)- positive material, suggested to be rhamnogalacturonan II (RG-II), than the roots. Starting from leaves, three different polysaccharides, GL-RI, GL-RII and GL-RIII, were isolated from polysaccharide subfraction GL-5 by subsequent anion-exchange and size-exclusion chromatography. Sugar and methylation analyses indicated that the three polysaccharides consisted of the same substitution patterns of 2- O-methyl-fucose, 2- O-methyl-xylose, apiose, 3- C-carboxy-5-deoxy- L-xylose (aceric acid, AceA), 3-deoxy- D- manno-2-octulosonic acid (Kdo), and 3-deoxy- D- lyxo-2-heptulosaric acid (Dha), being characteristic monosaccharides of RG-II, but no other pectic components. Another RG-II (GL-4IIb2) has also been isolated without endo-polygalacturonase digestion from leaves of P. ginseng, and turned out to be a macrophage Fc receptor expression enhancing polysaccharide [K.-S. Shin, H. Kiyohara, T. Matsumoto, and H. Yamada, Carbohydr. Res., 300 (1997) 239–249]. GL-4IIb2 and GL-RIII had relatively potent interleukin-6 (IL-6) production enhancing activity of macrophages, however, GL-RI and GL-RII had negligible and weak enhancing activities, respectively. Partial acid hydrolysis (0.1 M trifluoroacetic acid, 40°C, 24 h) of GL-RI, GL-RII and GL-RIII gave in each case a large-size fraction (AH-1), an intermediate-size fraction (AH-2) and a short oligosaccharide fraction (AH-3). EIMS and FABMS analyses indicated that AH-3 consisted of Rha-(1→5)-Kdo and Ara-(1→5)-Dha, and that AH-2 comprised non-, mono- and di-acetylated AceA-containing hexa- to nona-saccharides. Also for AH-1 no significant structural differences were observed among the polysaccharides, but the presence of microheterogeneity is suggested. The AH-1 fractions did not show any IL-6 production enhancing activity. Size-exclusion HPLC indicated that GL-RIII mainly comprised RG-II of higher molecular mass (12,000), and that GL-RI and GL-RII mainly contained RG-II of lower molecular mass (7000). Boron content and 11B NMR analyses indicated that the higher molecular weight polysaccharide in GL-RIII was mainly present as a RG-II dimer cross-linked by borate diesters. Dissociation of the RG-II dimer containing borate diester in GL-RIII to the monomer by mild acid treatment significantly decreased its IL-6 production enhancing activity whereas re-dimerization of dissociated GL-RIII recovered the enhancing activity. Artificial re-formation of the RG-II dimer containing borate diester in GL-RI also potentiated the IL-6 production enhancing activity. The present observation is a first report of biological activity related to the RG-II dimer.