Hideki Kawamura

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.

Plantagoside, a novel α-mannosidase inhibitor isolated from the seeds of Plantago asiatica, suppresses immune response

A hot-water extract from the seed of Plantago asiatica showed a potent inhibitory activity against jack bean alpha-mannosidase, and a flavanone glucoside, plantagoside, was isolated as the inhibitor. Plantagoside was a specific inhibitor for jack bean alpha-mannosidase (IC50 at 5 microM) and appeared to be a non-competitive inhibitor of the enzyme. Whereas, negligible or weak inhibitory activities were observed for beta-mannosidase, beta-glucosidase, and sialidase tested. Plantagoside also inhibited alpha-mannosidase activities in mouse liver lysosomal and microsomal fractions, and the enzyme inhibitory activity in microsomal fraction was enhanced in the presence of glucosidase inhibitor, castanospermine. Plantagoside suppressed antibody response to sheep red blood cells and concanavalin A induced lymphocyte proliferation which was measured by [3H]thymidine incorporation.

A novel type of B-cell mitogen isolated from juzen-taiho-to (TJ-48), a Japanese traditional medicine

Juzen-taiho-to (TJ-48), a Japanese traditional medicine, is known to have various immunological activities including the induction of B-cell proliferation. We investigated the properties of the pectic polysaccharide fraction of TJ-48 (F-5-2) which is most active in the proliferation of spleen cells. To an extent equal to that of TJ-48, F-5-2 induced the proliferation of B-cells, particularly those holding both sIgM and sIgD. The proliferation induced by F-5-2 was T-cell independent and macrophage dependent. The macrophages could be substituted for a soluble factor(s) secreted from the macrophages but not for IL-1. Generally, B-cell mitogens are known to induce the proliferation of B-cells and subsequently differentiation into plasma cells. However, although F-5-2 induced the B-cell differentiation, it arrested their development in the intermediate stage of the B-cell differentiation. The B-cells induced by F-5-2 produced IgM antibody in response to IL-6 and an antigen (SRBC) but not IgG antibody. F-5-2 induced the expression of IL-6R not only on IgM+ and IgG+ B-cells but also on IgD+ B-cells. These results suggest that F-5-2 is a new type of B-cell mitogen.

Effects of Medicinal Plants on Hemopoietic Cells

It has been found that Juzentaihoto (TJ-48), one of the traditional Japanese kampo (herbal) medicines, improves the general condition of cancer patients receiving chemotherapy and radiation therapy. We analyze how TJ-48 elicits such effect, and show that oral administration of TJ-48 accelerates recovery from hemopoietic injury induced by radiation and the anti-cancer drug mitomycin C. The effects are found to be due to its stimulation of spleen colony-forming units. Based on the present findings, we propose that the administration of TJ-48 should be of benefit to patients receiving chemotherapy, radiation therapy or bone marrow transplantation.

Pectic polysaccharide from roots of Glycyrrhiza uralensis: Possible contribution of neutral oligosaccharide in the galacturonase-resistant region to anti-complementary and mitogenic activities

Digestion with endo-alpha-(1-->4)-polygalacturonase liberated the enzyme-resistant region (PG-1c) as an active site of the anti-complementary and mitogenic pectic polysaccharide (GR-2IIc) from Glycyrrhiza uralensis. Partial acid hydrolysis of PG-1c resulted in acidic oligosaccharides, and methylation analysis and GC-MS analysis of the acidic oligosaccharides suggested that PG-1c comprised a rhamnogalacturonan core such as -->2)-Rha-(1-->4)-GalA-(1-->2)-Rha-(1-->4)-GalA-(1-->-->4)-GalA-(1-->4) as the acidic moiety. Degradation of uronic acids by lithium decreased the anti-complementary and mitogenic activities of PG-1c. Although the products from PG-1c were still active, the methylglycoside of alpha-L-Rha-(1-->4)-alpha-D-GalA-(1-->2)-alpha-L-Rha-(1-->4)-alpha-D-Gal A did not show both activities. The products obtained by the lithium degradation from PG-1c gave fractions containing various neutral oligosaccharide-alditols. Among these fractions the longest and the short oligosaccharide-alditol fractions had relatively potent anti-complementary activity, whereas all oligosaccharide-alditol fractions expressed weak but significant mitogenic activity. GC-MS analysis indicated that the short oligosaccharide-alditol fraction contained various kinds of di- to tetrasaccharide-alditols. However, malto-oligosaccharide-alditols, and malto-, isomalto-, and laminari-oligosaccharides did not show anti-complementary and/or mitogenic activities, and these results suggested that certain neutral carbohydrate chains in PG-1c were responsible for the expression of mitogenic activity as well as anti-complementary activity of PG-1c.

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.