Abstract: Bamboo shavings, the outer or intermediate layer of bamboo stems, are the bulk of by-products produced in bamboo processing. In this study we investigated the isolation, chemical characterization, and immunostimulatory activity in vitro of the hemicelluloses from bamboo shavings. Shavings were first pretreated by steam explosion. The optimal pretreatment was found to be steam explosion at 2.2 MPa for 1 min. Following this pretreatment, the yield of hemicelluloses reached (2.05±0.22)% (based on the dry dewaxed raw materials), which was 5.7-fold higher than that of untreated samples. Bamboo-shavings hemicellulose (BSH) was then prepared by hot water extraction and ethanol precipitation from the steam-exploded shavings. Purification of BSH by anion-exchange chromatography of diethylaminoethanol (DEAE)-sepharose Fast Flow resulted in a neutral fraction (BSH-1, purity of 95.3%, yield of 1.06%) and an acidic fraction (BSH-2, purity of 92.5%, yield of 0.79%). The weight-average molecular weights (M_w) of BSH-1 and BSH-2 were 12 800 and 11 300 g/mol, respectively. Chemical and structural analyses by Fourier transform infrared spectroscopy (FT-IR), 1D (¹H and ¹³C) and 2D (heteronuclear single quantum correlation (HSQC)) nuclear magnetic resonance (NMR) spectra revealed that BSH-1 was O-acetylated-arabinoxylan and BSH-2 was O-acetylated-(4-O-methylglucurono)-arabinoxylan. BSH-1 had a higher content of acetyl groups than BSH-2. For the immunomodulatory activity in vitro, BSH and BSH-2 significantly stimulated mouse splenocyte proliferation while BSH-1 had no effect; BSH, BSH-1, and BSH-2 markedly enhanced the phagocytosis activity and nitric oxide production of the murine macrophage RAW264.7 in a dose-dependent manner. Our results suggest that the water-extractable hemicelluloses from steam-exploded bamboo shavings are naturally acetylated and have immunostimulatory activity.

竹茹中天然乙酰化半纤维素的分离、化学结构和免疫活性研究

[1]Boscá, L., Zeini, M., Través, P.G., et al., 2005. Nitric oxide and cell viability in inflammatory cells: a role for NO in macrophage function and fate. Toxicology, 208(2):249-258.

[2]Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72(1):248-254.

[3]Cao, L., Liu, X., Qian, T., et al., 2011. Antitumor and immunomodulatory activity of arabinoxylans: a major constituent of wheat bran. Int. J. Biol. Macromol., 48(1):160-164.

[4]Chen, Y., Duan, J., Qian, D., et al., 2010. Assessment and comparison of immunoregulatory activity of four hydrosoluble fractions of Angelica sinensis in vitro on the peritoneal macrophages in ICR mice. Int. Immunopharmacol., 10(4):422-430.

[5]Chen, Y., Zhang, H., Wang, Y., et al., 2014. Acetylation and carboxymethylation of the polysaccharide from Ganoderma atrum and their antioxidant and immunomodulating activities. Food Chem., 156(8):279-288.

[6]Dai, J., Wu, Y., Chen, S., et al., 2010. Sugar compositional determination of polysaccharides from Dunaliella salina by modified RP-HPLC method of precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone. Carbohyd. Polym., 82(3):629-635.

[7]Dubois, M., Gilles, K.A., Hamilton, J.K., et al., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem., 28(3):350-356.

[8]Ebringerova, A., Hromadkova, Z., Heinze, T., 2005. Hemicellulose. Adv. Polym. Sci., 186:1-67.

[9]Ebringerová, A., Kardo Ová, A., Hromádková, Z., et al., 2002. Immunomodulatory activity of acidic xylans in relation to their structural and molecular properties. Int. J. Biol. Macromol., 30(1):1-6.

[10]Fan, L., Ding, S., Ai, L., et al., 2012. Antitumor and immunomodulatory activity of water-soluble polysaccharide from Inonotus obliquus. Carbohyd. Polym., 90(2):870-874.

[11]Geng, Z.C., Sun, R.C., Sun, X.F., et al., 2003. Comparative study of hemicelluloses released during two-stage treatments with acidic organosolv and alkaline peroxide from Caligonum monogoliacum and Tamarix spp. Polym. Degrad. Stabil., 80(2):315-325.

[12]Gírio, F.M., Fonseca, C., Carvalheiro, F., et al., 2010. Hemicelluloses for fuel ethanol: a review. Bioresource Technol., 101(13):4775-4800.

[13]Gong, L., Huang, L., Zhang, Y., 2012. Effect of steam explosion treatment on barley bran phenolic compounds and antioxidant capacity. J. Agric. Food Chem., 60(29):7177-7184.

[14]He, M., Wang, J., Qin, H., et al., 2014. Bamboo: a new source of carbohydrate for biorefinery. Carbohyd. Polym., 111(20):645-654.

[15]Hoffmann, R.A., Geijtenbeek, T., Kamerling, J.P., et al., 1992. ¹H-N.m.r. study of enzymically generated wheat-endosperm arabinoxylan oligosaccharides: structures of hepta- to tetradeca-saccharides containing two or three branched xylose residues. Carbohyd. Res., 223:19-44.

[16]Ke, C., Qiao, D., Luo, J., et al., 2013. Immunostimulatory activity and structure of polysaccharide from Streptococcus equi subsp. zooepidemicus. Int. J. Biol. Macromol., 57(6):218-225.

[17]Kenealy, W., Horn, E., Davis, M., et al., 2007. Vapor-phase diethyl oxalate pretreatment of wood chips: Part 2. Release of hemicellulosic carbohydrates. Holzforschung, 61(3):230-235.

[18]Klinke, H.B., Thomsen, A.B., Ahring, B.K., 2004. Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl. Microbiol. Biotechnol., 66(1):10-26.

[19]Leopold, B., 1961. Chemical composition and physical properties of wood fibers. I. Preparation of holocellulose fibers from loblolly pinewood. TAPPI, 44(3):230-232.

[20]Ma, Y., Xing, Y., Mi, H., et al., 2014. Extraction, preliminary characterization and immunostimulatory activity in vitro of a polysaccharide isolated from Strongylocentrotus nudus eggs. Carbohyd. Polym., 111(1):576-583.

[21]Maekawa, E., 1976. Studies on hemicellulose of bamboo. Wood Res.: Bull. Wood Res. Inst. Kyoto Univ., 59:153-179.

[22]Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65(1):55-63.

[23]Nagata, J., Higashiuesato, Y., Maeda, G., et al., 2001. Effects of water-soluble hemicellulose from soybean hull on serum antibody levels and activation of macrophages in rats. J. Agric. Food Chem., 49(10):4965-4970.

[24]Peng, P., Peng, F., Bian, J., et al., 2011a. Isolation and structural characterization of hemicelluloses from the bamboo species Phyllostachys incarnata Wen. Carbohyd. Polym., 86(2):883-890.

[25]Peng, P., Peng, F., Bian, J., et al., 2011b. Studies on the starch and hemicelluloses fractionated by graded ethanol precipitation from bamboo Phyllostachys bambusoides f. shouzhu Yi. J. Agric. Food Chem., 59(6):2680-2688.

[26]Scheller, H.V., Ulvskov, P., 2010. Hemicelluloses. Annu. Rev. Plant Biol., 61(1):263-289.

[27]Sella Kapu, N., Trajano, H.L., 2014. Review of hemicellulose hydrolysis in softwoods and bamboo. Biofuel. Bioprod. Bior., 8(6):857-870.

[28]Song, H., Yang, R., Zhao, W., et al., 2014. Innovative assistant extraction of flavonoids from pine (Larix olgensis Henry) needles by high density steam flash-explosion. J. Agric. Food Chem., 62(17):3806-3812.

[29]Sukhbaatar, B., Hassan, E.B., Kim, M., et al., 2014. Optimization of hot-compressed water pretreatment of bagasse and characterization of extracted hemicelluloses. Carbohyd. Polym., 101(1):196-202.

[30]Sun, S., Cao, X., Xu, F., et al., 2014. Structure and thermal property of alkaline hemicelluloses from steam exploded Phyllostachys pubescens. Carbohyd. Polym., 101(1):1191-1197.

[31]Teleman, A., Lundqvist, J., Tjerneld, F., et al., 2000. Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing ¹H and ¹³C NMR spectroscopy. Carbohyd. Res., 329(4):807-815.

[32]Wang, Q.H., Shu, Z.P., Xu, B.Q., et al., 2014. Structural characterization and antioxidant activities of polysaccharides from Citrus aurantium L. Int. J. Biol. Macromol., 67(6):112-123.

[33]Wen, J., Xiao, L., Sun, Y., et al., 2011. Comparative study of alkali-soluble hemicelluloses isolated from bamboo (Bambusa rigida). Carbohyd. Res., 346(1):111-120.

[34]Yang, D., Zhong, L., Yuan, T., et al., 2013. Studies on the structural characterization of lignin, hemicelluloses and cellulose fractionated by ionic liquid followed by alkaline extraction from bamboo. Ind. Crop. Prod., 43(5):141-149.

[35]Yi, Y., Zhang, M., Liao, S., et al., 2012. Effects of alkali dissociation on the molecular conformation and immunomodulatory activity of longan pulp polysaccharide (LPI). Carbohyd. Polym., 87(2):1311-1317.

[36]Yu, Z., Xu, D., Lu, L., et al., 2016. Immunomodulatory effect of a formula developed from American ginseng and Chinese jujube extracts in mice. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 17(2):147-157.

[37]Zhao, T., Mao, G., Mao, R., et al., 2013. Antitumor and immunomodulatory activity of a water-soluble low molecular weight polysaccharide from Schisandra chinensis (Turcz.) Baill. Food Chem. Toxicol., 55(1):609-616.

[38]Zhao, W., Yang, R., Zhang, Y., et al., 2012. Sustainable and practical utilization of feather keratin by an innovative physicochemical pretreatment: high density steam flash-explosion. Green Chem., 14(12):3352-3360.

[39]Zheng, W., Zhao, T., Feng, W., et al., 2014. Purification, characterization and immunomodulating activity of a polysaccharide from flowers of Abelmoschus esculentus. Carbohyd. Polym., 106(1):335-342.

[40]List of electronic supplementary materials

[41]Table S1 ¹H NMR chemical shifts (relative to an internal H₂O, δ_H=4.70 ppm) of constituent monosaccharide residues in BSH-1 and BSH-2

[42]Table S2 ¹³C NMR chemical shifts (relative to an internal acetone-d₆, δ_C=30.50 ppm) of constituent monosaccharide residues in BSH-1 and BSH-2