Structure and properties of the glucans isolated from Ganoderma lucidum NSC B

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1 Structure and properties of the glucans isolated from Ganoderma lucidum NSC B aniline blue glucan -(1 3)-D- (Ganoderma lucidum) (GLPS) -1,3-D- aniline laminarin lentinan Krestin blue 0 50 g/ml Abstract Aniline blue is a common carbohydrate dye used in light microscopic examination. It specifically binds to glucans. This study exerted this specificity to develop peptone potato dextrose broth a quantitative fluorescence spectroscopic method to determine the content of -(1 3)-D-glucans in a sample of Ganoderma spp. The glucans used in this study includes glucan isolated from G. lucidum fruiting body and three commercial available glucans, laminarin (from Laminaria digitata), lentinan (from Lentinula edodes), and hexosamines uronic acids Krestin (from Trametes versicolor). All of glucans examined reacted to aniline blue and 75% 17% showed a linear relationship between 5 3fluorescence intensity and glucan 13 C-NMR concentration in the range of 2 to 50 g/ml. glucan size Simple sugars and α-glucans, glucose, exclusion chromatography maltose, dextrin and starch, used, as growth 1 multi angle laser light scatter

2 medium of mycelium fermentation did not interference with the determination. Other complex medium components, malt and yeast extract, peptone, and potato dextrose broth caused fluoresencent intensity loss and the interference of the medium should be compensated, if those components were included in the examining system. The -(1 3)-D-glucan isolated from fruiting body of G. lucidium was purified by using quaternary ammonia selective precipitation, protease, amyloglucosidase digestion, and alcohol precipitation. The major sugar component was glucose(75%) and galactose (17%) with small amount of arabinose (5 ) and mannose( 3 ). The glucosidic linkage was -(1 3) revealed by 13 C-NMR analysis. The molecular size distribution of major fraction was measured by 0.3N NaOH 1N HCl ph ml using size-exclusion chromatography 0.2ml aniline blue equipped with multi-angle laser-light scatter 1mg/ml 2 detection. Glucan from Ganoderma 395 nm mycelium fermentation was isolated with 495 nm same extraction procedure. The chemical composition and fluorescent forming 2N properties of both glucans were found to be identical. The determination of molecular size distribution of fermented glucan is still CARBOSep CHO-682 ongoing. Keywords: Ganoderma lucidum, -1,3-Dglucan, aniline blue, fluorescence spectroscopy polysaccharides (Transgenomic, NE, USA) ml/min NMR MeSOd6-D 2 O 6 1 v/v H 13 C -NMR Bruker DMX-600 NMR (Ganoderma lucidum) spectrometer Kim [1-5] [7] (1 6) protease glucosamylase [6] (fruiting body) (mycelium)

3 hexosamine uronic acid Folin-Lowry method 75% [6] 1 H-NMR 13 C-NMR laminarin curdlan lentinan aniline blue sodium carbonylbis (4-(phenyleneamino) [7] benzenesulfonate)[8] 1 H-NMR 395nm 495nm [9] laminarin lentinan PSK 4 95 (Ganoderma lucidum) protease (GLPS) aniline blue glucosamylase 0 50 g/ml hexosamine uronic acid hexosamine uronic acid Folin- Lowry method 6 glucose, maltose, dextrin, starch, yeast extract malt extract peptone potato dextrose broth (PDB) aniline blue (GLPS) - (1 3)-D- aniline blue aniline blue assay assay peptone potato dextrose broth 3

4 antitumor activities of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agric. Biol. Chem : Ganoderma lucidum 7. Kim, Y. T., Kim, E. H., Cheong, C., 2 Williams, D. L., Kim, C. W., and Lim, S. S-180 T Structural characterization of ,1 6 -linked glucans using NMR 3. spectroscopy. Carbohyd. Res. 328: ICR Thistlethwaite, P., Porter, I., and Evans, N Photophysics of the aniline blue fluorophore: a fluorescent probe showing specificity toward D-glucan. J. Phys. Chem. 90: Hikino, H., Ishiyama, M., Suzuki, Y., and Konne, C Mechanisms of hypoglycemic activity of ganoderan B: aglycan of Ganoderma lucidum fruit bodies. Planta Med. 55: Ukai, S., Kiho, T., Haro, C., Kuruma, I., and Tanaka, Y Anti-inflammatory effect of the polysaccharides from fruit bodies of several; fungi. J. Pharmacobio- Dyn. 6: Sone, Y., Okuda, R., Wada, N., Kishida, E., and Misaki, A Structures and 9. Wood, P. J., and Fulcher, R. G Specific interaction of aniline blue with D-glucan. Carbohyd. Polym. 4: D-Glucan aniline blue assay 4

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