Masumi H. Sakurai

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.

Orally administered decoction of Kampo (Japanese herbal) medicine, "Juzen-Taiho-To" modulates cytokine secretion and induces NKT cells in mouse liver.

The effects of orally administered decoction of Juzen-Taiho-To (JTT; Si-Quan-Da-Bu-Tang in Chinese) on cytokine production in hepatic lymphocytes were studied in mice. JTT was found to increase interferon gamma (IFN-gamma), as well as interleukin-4 (IL-4), IL-5 and IL-6 secretion from stimulated hepatic lymphocytes, whereas IL-2 secretion was reduced. The number of IFN-gamma- and IL-4-spot forming cells (SFC) were not changed by administration of JTT. These results suggest that modulation of cytokine secretion by JTT might not be due to changes in the number of cytokine secreting cells within liver lymphocytes. CD4/CD8 ratio and alphabeta/gammadelta T cell receptor (TCR) ratio in hepatic lymphocytes were not changed. However, flow cytometric analysis revealed that the population of CD3 positive intermediate cells in NK positive cells (NKT cells) was increased after oral administration of JTT. The population of CD3int IL-2Rbeta+ cells was also increased. The induction of NKT cells by JTT was reduced by injection of 2-chloroadenosine. JTT enhanced transcription of IL-12 mRNA in liver. From these results, it may be concluded that a rise in NKT cell population contributes, at least partially, to the modulating effect of JTT on cytokine production in liver lymphocytes, and macrophages. The production of IL-12 in liver may also contribute to this NKT induction.

Complement activating galactan chains in a pectic arabinogalactan (AGIIb-1) from the roots of Angelica acutiloba Kitagawa☆

Abstract The complement activating (anti-complementary) arabinogalactan (AGIIb-1), isolated from the roots of Angelica acutiloba Kitagawa, was digested with exo-β- d -(1→3)-galactanase after a preceding digestion with exo-α- l -arabinofuranosidase. Gel filtration of the digested products gave a high molecular weight fraction (HMW) which could not be further digested, and an oligosaccharide fraction (LMW). The latter originated from exterior β- d -(1→3)-galactan chains of AGIIb-1. HMW showed similar potent anti-complementary activity as arabinofuranosidase-digested AGIIb-1 (AF-AGIIb-1), but LMW did not show any activity. HMW was composed mainly of Ara and Gal in addition to small proportions of Rha, Xyl, GlcA and GalA. Methylation analysis indicated that HMW comprised terminal Ara∝, terminal Gal and 3,6-linked Gal as well as 4-linked Gal, 4-linked GalA and terminal GlcA. Further digestion of HMW with endo-β- d -(1→4)-galactanase gave a polymeric fraction (GN-1) and oligosaccharidefraction (GN-2). Although most of β- d -(1→4)-galactosyl chains were liberated from HMW by this galactanase digestion, anti-complementary activity of GN-1 was not changed significantly in comparison with that of HMW. Controlled Smith degradation of HMW gave four kinds of fractions. Among them, the fraction (CSD-1) having the largest molecular-weight showed a relatively potent anti-complementary activity, and it was mainly composed of a (1→3)-galactan. Exo-β- d -(1→3)-galactanase digestion of CSD-1 gave six fractions (CSD-1-1~CSD-1-6). The polymeric fraction (CSD-1-1) and the oligosaccharide fractions (CSD-1-4 and 1-5) had potent anti-complementary activity. Methylation analysis indicated that CSD-1-1, 1-4 and 1-5 consisted mainly of terminal, 6-linked and 3,6-linked Gal. CSD-1-4 and CSD-1-5 were also mainly composed of 3-linked Gal. These results indicates that complement activating ability of AGIIb-1 is expressed by the inner galactan chains.

Antigenic epitope for polyclonal antibody against a complement activating pectin from the roots of Angelica acutiloba Kitagawa

Abstract A polyclonal antibody (anti-AR-2IIb-IgG) was developed against a complement activating pectin (AR-2IIb) which was purified from the roots of Angelica acutiloba Kitagawa. Neutral oligosaccharide–alditols were released from the “ramified” region (rhamnogalacturonan core possessing side chains rich in neutral sugars, PG-1) by lithium-degradation. The mixture significantly inhibited the reactivity of anti-AR-2IIb-IgG to PG-1 or AR-2IIb. Gel filtration and HPLC analysis indicated that the mixture contained two kinds of long oligosaccharide–alditols in addition to several kinds of shorter oligosaccharide–alditols, and both the long oligosaccharide–alditols had significant inhibitory activity against the reactivity of the antibody to PG-1. However, the short oligosaccharide–alditols had no effect. Methylation analysis suggested that one of the long oligosaccharide–alditols consisted of a β-(1→3,6)-galactan-like structure. The inhibitory activity of PG-1 against the reactivity of the antibody to PG-1 decreased significantly on exo -α- l -arabinofuranosidase digestion followed with exo -β- d -(1→3)-galactanase digestion, but not by exo -α- l -arabinofuranosidase or exo -β- d -galactosidase digestion. Although β- d -(1→6)-galacto-di- to tetra-saccharides showed no inhibitory activity, a mixture of oligosaccharides liberated from PG-1 by exo -β- d -(1→3)- galactanase digestion, still showed inhibitory activity. The oligosaccharide consisting mainly of terminal, 3- and 4-linked and 3,6-branched Gal, terminal Ara f and 4-OMe-GlcA inhibited the reactivity of the antibody to PG-1.

Galactose-containing polysaccharides from Dictyostelium mucoroides as possible acceptor molecules for cell-type specific galactosyl transferase.

Dictyostelium discoideum has polysaccharides that accept galactose residues by the action of cell-type-specific galactosyl transferase. This paper describes partial purification of the major galactose-accepting polysaccharide that was isolated from the related strain, Dictyostelium mucoroides and proposes its plausible carbohydrate sequences. The most potent acceptor activity was observed in the neutral and Ricinus communis agglutinin (RCA-60) bound galactosaminoglycan, consisting of galactose (Gal) and galactosamine (GalN). However, the acceptor polysaccharides are highly heterogeneous in the reactivity with RCA-120 and in molecular mass. The peak fraction was analyzed by (1)H- and (13)C-NMR studies, methylation analysis, beta-D-galactosidase digestion, controlled Smith degradation and reducing terminal analysis. Based on these results, we tentatively propose the following novel type of the common carbohydrate sequences as the acceptor polysaccharides. [abstract - see text].

Hemopoietic Stem Cell-stimulating Ingredients in Kampo (Japanese Herbal) Medicine “Juzen-Taiho-To”

We have previously found that one of the kampo (Japanese herbal) medicines, Juzen-Taiho-To (TJ-48), accelerates recovery from hemopoietic injury induced by radiation and an anti-cancer drug. n-Hexane-soluble substances from TJ-48 showed significant stimulatory activity on the proliferation of hemopoietic stem cells in vitro Chromatographic separation and spectrometric identification using NMR and GC-MS revealed that the active fraction of TJ-48, which contained fatty acids such as oleic, linoleic and linolenic acids, accelerated stem cell proliferation. Oral administration of oleic acid to mitomycin C-treated mice enhanced CFU-S counts on day 14 to twice the control group. When the fatty acid composition of TJ-48 was compared with other kampo medicines, the same active fatty acids were detected even in other kampo prescriptions which had not been found to accelerate recovery from hemopoietic injury, but in different ratios. Although not all kampo medicines tested showed the stimulatory activity, their fatty acid fractions did. These results suggest that hemopoietic stimulation by TJ-48 might be the result of the combined effect of the active unsaturated fatty acids and other hydrophilic ingredients.