Hiroaki Kiyohara

Characterization of a biologically active pectin from Plantago major L.

Abstract PMII isolated from the leaves of Plantago major L. is a pectin type polysaccharide with anti-complementary activity. It is highly esterified and partly O-acetylated with regions of 1,4 linked polygalacturonic acid and at least two different hairy regions. The galactose side chains are linked to position 4 of rhamnose in the main chain. The structure of the galactan side chains is complex, but 1,3,6 linkages are dominating in one of the isolated hairy regions. Arabinose is attached to position 3 and 6 of galactose. In the other hairy region arabinose is attached to position 3 of galacturonic acid. De-esterification and de-acetylation do not alter the anti-complementary activity of PMII. Different parts of PMII were shown to have different activities. The smooth regions are only slightly active in contrast to the hairy regions which had significantly higher activity. The hairy regions of highest molecular weight (PVa) with 1,3,6 linked galactose side chains were found to be the most active fraction. The importance of arabinose for the activity seems to depend on the site of substitution. Removal of arabinose terminally linked to galactose increases the activity slightly while removal of arabinose linked to the galacturonic acid backbone decreases the activity.

STRUCTURAL CHARACTERIZATION OF A NEW ANTICOAGULANT FUCAN SULFATE FROM THE BROWN SEAWEED ECKLONIA KUROME

Methylation analysis of a fucose-containing, sulfated polysaccharide (C-II), which was isolated from the brown seaweed Ecklonia kurome and has a potent anticoagulant activity, showed the presence of 3-O- and 3,4-O-disubstituted fucopyranosyl residues in addition to small proportions of nonreducing, terminal fucofuranosyl and fucopyranosyl groups, and 2,3-di-O- and 2,3,4-tri-O-substituted fucopyranosyl and galactopyranosyl residues with various glycosidic linkages. Methanolysis of C-II gave several neutral oligosaccharide fractions in small proportions and two high-molecular-weight acidic fractions in large proportions. Methylation analysis of the low-sulfated acidic fraction showed that the proportion of 3-O-linked fucosyl residues increases and that of 3,4-O-disubstituted decreased as compared to C-II. Methylation and g.l.c.-m.s. analysis of the neutral oligosaccharide fractions showed the presence of Fuc-(1----3)-Fuc and a fucosyl trisaccharide, in addition to small proportions of Gal-(1----4)-Fuc, Fuc-(1----2)-Fuc, Fuc-(1----4)-Fuc, Fuc-(1----2)-Gal, and Fuc----Gal----Fuc. Methylated C-II was also desulfated by methanolysis, followed by remethylation with (2H3)methyl iodide, and most of (2H3)methyl groups were linked to O-4 of the 3-O-linked fucosyl residues. These results suggested a highly branched, new type of fucan sulfate containing a backbone of (1----3)-linked L-fucosyl residues having sulfate groups mainly attached to C-4.

Complement-activating polysaccharides from medicinal herbs

The complement system consists of over 20 serum proteins including nine com-plement components (C1 to C9) and their regulators, and is normally present in blood serum in an inactive form. The system is essential for the operation of the innate as well as the adaptive immune defence [1]. The complement proteins can be activated through three cascade pathways: by the classical pathway, by the alternative pathway and by the antibody-independent lectin pathway (Fig. 1). The activation of the classical pathway is initiated by immune complexes containing IgM and IgG antibodies, Cl binds to the Fc region of antibodies through subcomponent Cl q, then the complex activates the further complement system by the cascade mechanism. The alternative pathway is directly activated from C3 by microorganisms, protozoa, or some activators such as lipopolysaccharide through an antibody independent mechanism. Recently, an antibody-independent mannanbinding lectin (MBL) pathway has also been established as the third activation pathway of complement systems [2,3]. This pathway is initiated by the binding of serum MBL (carbohydrate binding component in complement-dependent antibacterial factor, RaRF [4]), which is structurally related to Cl q, to the surface carbohydrates and activated through two MBL-associated serine proteases. The MBL pathway is activated by specific carbohydrate structures of microorganisms including bacteria, yeast, parasitic protozoa and viruses, and exhibits antibacterial activity through killing mediated by the terminal, lytic complement components or by promoting phagocytosis [3].

B‐cell proliferation activity of pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L. and its structural requirement

Pectic polysaccharide fraction (BR‐2) containing pharmacologically active pectic polysaccharide, bupleuran 2IIc, which was prepared from a medicinal herb, the roots of Bupleurum falcatum L., was administered orally to C3H/HeJ mice for 7 consecutive days. Proliferative responses of spleen cells were enhanced in the presence of the purified pectic polysaccharide, bupleuran 2IIc, but another B‐cell mitogen, lipopolysaccharide (LPS) did not give a similar effect. In vitro studies using spleen cells showed that bupleuran 2IIc also stimulated lymphocytes, depleted of adherent cells or T cells. Bupleuran 2IIc treatment increased subpopulation of CD25+ and surface immunoglobulin M‐positive (sIgM+) lymphocytes. Non‐specific immunoglobulin secretion of spleen cells treated with bupleuran 2IIc was increased according to the culture time, and coexistence of interleukin‐6 (IL‐6) enhanced the secretion more than that of bupleuran 2IIc alone. These results suggest that bupleuran 2IIc proliferates B cells in the absence of macrophages, and the resulting activated B cells are then induced into antibody‐forming cells in the presence of IL‐6. Among the structural region of bupleuran 2IIc, ramified region (PG‐1), which consists of rhamnogalacturonan core rich in neutral sugar chain, showed the potent mitogenic activity suggesting it to be an active site. Mitogenic activity of bupleuran 2IIc was reduced in the presence of antipolysaccharide antibody (antibupleuran 2IIc/PG‐1‐IgG), which recognizes the ramified region of bupleuran 2IIc as the antigenic epitope. Mitogenic activity of bupleuran 2IIc was also reduced by the addition of β‐d‐GlcpA‐(1→6)‐β‐d‐Galp‐(1→6)‐d‐Galp or β‐d‐GlcpA‐(1→6)‐d‐Galp, which are a part of the epitopes of antibupleuran 2IIc/PG‐1‐IgG. These results suggest that the epitopes in bupleuran 2IIc act as active sites of the polysaccharide during mitogenic activity.

Structural characterisation of an anti-complementary pectic polysaccharide from the roots of Bupleurum falcatum L.

An anti-complementary pectic polysaccharide (BR-2-IIb), isolated from the roots of Bupleurum falcatum L., has an average molecular weight of 36,000 (gel filtration), and was subjected to methylation analysis before and after carboxyl-reduction, digestion with endo-polygalacturonase, base-catalysed beta-elimination, and partial acid hydrolysis. BR-2-IIb consisted mainly of galacturonic acid, arabinose, rhamnose, and galactose in the molar ratios 13.0:2.1:1.4:1.0 and contained a large enzyme-sensitive polygalacturonan region. The enzyme-resistant region (PG-1) was rich in neutral sugars and contained a backbone of 4-linked GalA and 2-linked Rha to which a highly branched arabinogalactan was attached to position 4 of some 2-linked Rha units. Partial acid hydrolysis of BR-2-IIb gave Ara-(1----3)-Ara, Ara-(1----4)-Arap, Ara-(1----5)-Araf, Ara-(1----6)-Gal, Gal-(1----4)-Gal, GalA-(1----2)-Rha, GalA-(1----4)-Rha, GalA----Rha----Rha, Gal----Rha----Rha, and GlA-(1----6)-Gal in addition to (1----4)linked oligogalacturonides. The anticomplementary activity of BR-2-IIb was enhanced by de-esterification, but carboxyl-reduction decreased the activity.

Heterogeneity and characterisation of mitogenic and anti-complementary pectic polysaccharides from the roots of Glycyrrhiza uralensis Fisch et D.C.

Two anti-complementary polysaccharide fractions (GR-2IIa and GR-2IIb), isolated from the roots of Glycyrrhiza uralensis Fisch et D.C., each gave five anti-complementary polysaccharides (GR-2IIa-1-5 and GR-2IIb-1-5) on h.p.l.c.; likewise, GR-2IIc gave two anti-complementary and mitogenic polysaccharides (GR-2IIc-1-2A and -2IIc-2) by gel filtration and h.p.l.c. GR-2IIc-1-2A showed the most potent anti-complementary activity. GR-2IIa-1-5 and GR-2IIb-1-5 contained 40-85% and 50-90% of GalA, respectively, in addition to Rha, Ara, and Gal. GR-2IIc-1-2A and -2IIc-2 mainly comprised Glc, Gal, GalA, and GlcA in addition to Rha, Fuc, Xyl, Ara, and Man. Methylation analysis and digestion with endo-alpha-(1----4)-polygalacturonase indicated that all of the polysaccharides contained polygalacturonan regions which were frequently methyl-esterified. GR-2II-a, -2IIb, and -2IIc gave enzyme-resistant fractions of large and intermediate sizes, in addition to oligogalacturonides. Each large fraction from GR-2IIa and -2IIb consisted mainly of Ara, Gal, and GalA, whereas the intermediate fractions were composed of small proportions of 2-Me-Fuc, 2-Me-Xyl, and apiose (Api), in addition to Rha, Ara, Gal, and GalA. The large fraction from GR-2IIc mainly contained Rha, Man, Gal, and GalA in addition to Fuc, Ara, Xyl, and Glc, whereas the intermediate fraction consisted of 2-Me-Fuc, 2-Me-Xyl, and Api, in addition to Rha, Ara, GalA, Fuc, Xyl, Man, Gal, and Glc. Base-catalysed beta-elimination followed by ethylation indicated that all the polysaccharides except GR-2IIc-2 contained a 4-linked uronic acid attached to position 2 of 2,4-linked Rha. Single radial gel diffusion, using the beta-D-glucosyl-Yariv antigen, indicated that GR-2IIa-1 and GR-2IIc-2 contained relatively large proportions of (1----3,6)-beta-D-galactan moieties. The anti-complementary activities of GR-2IIa-3, GR-2IIa-4, and GR-2IIb-4 decreased after de-esterification followed by digestion with endo-alpha-(1----4)-polygalacturonase. The large fractions from GR-2IIa-2IIc showed more potent anti-complementary activities than the original polysaccharide fractions, whereas the intermediate fractions and oligogalacturonides were inactive. The large fraction from GR-2IIc had more potent mitogenic activity than GR-2IIc, whereas the intermediate fraction and oligogalacturonides from GR-2IIc were inactive.

Structural characterisation of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa

An anti-complementary arabinogalactan (AGIIb-1), isolated from the roots of Angelica acutiloba Kitagawa, has been subjected to methylation analysis, digestion with alpha-L-arabinofuranosidase, controlled Smith-degradation, and partial acid hydrolysis. AGIIb-1 consisted of arabinose, galactose, rhamnose, galacturonic acid, and glucuronic acid in the molar ratios 1.8-2.2:1.0:0.2-0.3:0.2-0.4:0.1. AGIIb-1 contained mainly an arabino-3,6-galactan moiety, and most of the Ara was present as alpha-L-arabinofuranosyl residues in the non-reducing terminals and the highly polymerised and branched side-chains which were attached mainly to positions 3 and 6 of (1----6)- and (1----3)-linked Gal, respectively. Some Ara-containing chains were also attached to (1----4)-linked Gal residues. The 13C-n.m.r. data for AGIIb-1 showed that the Galp was beta. Mild acid hydrolysis of AGIIb-1 yielded several linear and highly branched arabino-oligosaccharides, a neutral arabinogalactan, and two acidic arabinogalactans. Some arabino-oligosaccharides contained a (1----4)-linked Arap at the reducing terminal. The neutral arabinogalactan contained (1----3)-, (1----4)-, and (1----6)-linked and 3,6-di-O-substituted Gal, whereas the acidic arabinogalactans contained, in addition, non-reducing terminal GlcA, (1----4)-linked GalA, and 2,4-di-O-substituted Rha. The anti-complementary activity was decreased when AGIIb-1 was partially hydrolysed with mild acid (10mM HCl, 100 degrees, 10 min), but treatment with exo-alpha-L-arabinofuranosidase markedly enhanced the activity.

Rhamnogalacturonan II from the leaves of Panax ginseng C.A. Meyer as a macrophage Fc receptor expression-enhancing polysaccharide

A complex pectic polysaccharide (GL-4IIb2) has been isolated from the leaves of Panax ginseng C.A. Meyer, and shown to be a macrophage Fc receptor expression-enhancing polysaccharide. The primary structure of GL-4IIb2 was elucidated by composition. 1H NMR, methylation, and oligosaccharide analyses. GL-4IIb2 consisted of 15 different monosaccharides which included rarely observed sugars, such as 2-O-methylfucose, 2-O-methylxylose, 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). Methylation analysis indicated that GL-4IIb2 comprised 34 different glycosyl linkages, such as 3,4-linked Fuc, 3- and 2,3,4-linked Rha, and 2-linked GlcA, which are characteristic of rhamnogalacturonan II (RG-II). Sequential degradation using partial acid hydrolysis indicated that GL-4IIb2 contained alpha-Rhap-(1-->5)-Kdo and Araf-(1-->5) Dha structural elements, an AceA-containing oligosaccharide, and uronic acid-rich oligosaccharide chains in addition to an alpha-(1 -->4)-galacturono-oligosaccharide chain. FABMS and methylation analyses suggested that the AceA-containing oligosaccharide was a nonasaccharide in which terminal Rha was additionally attached to position 3 of 2-linked Arap of the octasaccharide chain observed in sycamore RG-II. Component sugar and methylation analyses assumed that the uronic acid-rich oligosaccharides possessed a similar structural feature as those in sycamore RG-II. GL-4IIb2 had a larger molecular mass (11,000) than sycamore RG-II (approximately 5000).

Pinellic acid from the tuber of Pinellia ternata Breitenbach as an effective oral adjuvant for nasal influenza vaccine

This study describes the isolation, purification, characterization, and adjuvant activity of an orally active adjuvant substance from the tuber of Pinellia ternata, as an active herbal component of the traditional Japanese herbal (Kampo) medicine, Sho-seiryu-to (SST, Chinese name: Xiao-Qing-Long-Tang), which has been reported to show oral adjuvant activity for nasally administered influenza HA vaccine [Int. J. Immunopharmacol. 16 (1994) 605]. The active compound was identified as 9S, 12S, 13S-trihydroxy-10E-octadecenoic acid using infrared spectra, proton magnetic resonance, mass spectrometry, and circular dichroism, and named pinellic acid. Oral administration of pinellic acid (1 microg) to BALB/c mice given primary and secondary intranasal inoculations of influenza HA vaccine (1 microg) enhanced antiviral IgA antibody (Ab) titers 5.2- and 2.5-fold in nasal and bronchoalveolar washes, respectively, and antiviral IgG Ab titers 3-fold in bronchoalveolar wash and serum. Intranasal administration of pinellic acid (1 microg) with influenza HA vaccine (1 microg) slightly enhanced antiviral IgG Ab titers in bronchoalveolar wash and serum but not antiviral IgA Ab titers in nasal and bronchoalveolar washes. Pinellic acid showed no hemolytic activity. The results of this study suggest that pinellic acid may provide a safe and potent oral adjuvant for nasal influenza HA vaccine.

Enhanced production of hematopoietic growth factors through T cell activation in Peyer’s patches by oral administration of Kampo (Japanese herbal) medicine, “Juzen-Taiho-To”

The effects of Juzen-Taiho-To (JTT), a Kampo (Japanese herbal) medicine, on Peyers patch cells mediated hematopoietic response were investigated. When the conditioned medium of Peyers patch cells obtained from C3H/HeJ mice, which had orally administered JTT for 7 consecutive days, was added to the culture medium for bone marrow cells, the cells significantly proliferated compared with that of mice receiving water alone. Oral administration of JTT for 7 days increased the population of activated T cells in Peyers patch. The mechanism of Peyers patch cells mediated proliferation was investigated in vitro. When Peyers patch cells from untreated C3H/HeJ mice were stimulated with various concentrations of JTT, and the resulting conditioned medium was used for stimulation of bone marrow cells, the cells were proliferated in a dose dependent manner. The contents of granulocyte-macrophage colony stimulating factor (GMCSF) and interleukin-6 (IL-6) in the conditioned medium from JTT stimulated Peyers patch cells were increased compared with those from controls, and the population of Gr-1 antigen positive cells in the bone marrow cell cultures also increased. These results demonstrate that orally administered JTT can activate T-cells in Peyers patch cells and enhance the production of colony stimulating factor(s), in which at least GM-CSF and IL-6 may partly contribute to this activity.