Chemical Characterization And Immunomodulatory Activity Of Polysaccharides From Bamboo Shavings

China is a large bamboo-producing country and develops well in bamboo processing. At present, the utilization of bamboo in China is at an important stage of transition from the aspects of production, life, industrial timber to health products. Bamboo shavings, the outer or intermediate layer of bamboo stems, are the bulk of by-products produced in bamboo processing industry. Currently, they have not been effectively utilized yet. In this work, our attention has been focused on the active polysaccharides from bamboo shavings. We systematically investigate the isolation and purification technology, structural characteristics and immunological activity of the polysaccharides from bamboo shavings. We aim to find a way for converting bamboo shavings as value-added materials, as well as develop the polysaccharides from bamboo shavings as novel immunomodifier. The main results of this work are summarized as follows.1) We employed the method of steam explosion pretreatment followed by hot water extraction to fractionate the polysaccharides from bamboo shavings. The effects of different steam explosion pretreatment conditions on the yield of polysaccharides were investigated. Results showed that the yield of polysaccharides increased firstly and decreased thereafter with increasing steam pressure and residence time, and it achieved a maximum (2.05%±0.22%) after steam explosion at 2.2 MPa for 1 min, which was 6.7 times that of the untreated sample (0.31%±0.04%). The results suggested that steam explosion pretreatment was effective to assist the extraction of polysaccharides from bamboo shavings.2) The obtained bamboo-shavings polysaccharides (BSP, purity of 90.2%) was then purified by anion-exchange chromatography of DEAE-Sepharose Fast Flow, resulting in two fractions, namely BSP-1 (purity of 95.3%, yield of 1.06%) and BSP-2 (purity of 92.5%, yield of 0.79%), respectively. The primary structures of BSP-1 and BSP-2 were studied by a combination of sugar analysis, GPC, FT-IR, GC-MS, 1D (1H and 13C) and 2D (HSQC, HMBC) NMR spectra. It could be speculated that BSP-1 (Mw: 12,800 g/mol) was O-acetylated arabinoxylan(AX) consisting of a linear (1â†'4)-β-D-xylopyranosyl backbone decorated with branches at O-2 of acetyl groups(8.4%) or at O-3 of a-L-arabinofuranosyl(2.2%) and acetyl groups(22.8%); BSP-2 (Mw:11,300 g/mol) was O-acetylated 4-O-methyl-glucuronoarabinoxylan(GAX) consisting of a linear (1â†'4)-β-D-xylopyranosyl backbone decorated with branches at O-2 of acetyl groups(7.0%) and 4-O-methylglucuronic acid units(6.4%) or at O-3 of α-L-arabinofuranosyl(3.2%) and acetyl groups(18.1%), additionally, about 1.4% of (1â†'4)-β-D-xylopyranosyl units were di-acetylated (at O-2 and O-3). The conformations of BSP-1 and BSP-2 were observed by atomic force microscopy (AFM). Results showed that BSP-1 looked like a ’fluffy thread’ in a 10 ug/mL solution, while BSP-2 looked like a ’ball’ at the same concentration; BSP-1 showed its single molecule stretching behavior at 1 μg/mL, with the backbone chain length being about 1400 run, the width being 20-70 nm, the height being 1.3～2.5 nm and the branched chain length being 300～400 nm; while BSP-2 had the backbone chain length of about 2200 nm, the width of 30-80 nm, the height of 1.0～2.8 nm and the branched chain length of 200～300 nm.3) The results of in vitro immunological tests are as follows. BSP-1 exerted no effect on mouse splenic lymphocyte proliferation, while BSP and BSP-2 could stimulate the lymphocyte proliferation alone or with concanavalin A(ConA,2.5μg/mL) or lipopolysaccharide(LPS,7.5μg/mL). BSP, BSP-1 and BSP-2 had no influences on RAW264.7 macrophage proliferation, but could significantly enhance the phagocytosis activity and NO production of RAW264.7 macrophage in both dose-dependent and time-dependent manners.4) The results of in vivo immunological tests are as follows. Oral administration of BSP(100 or 200 mg/kg body weight(BW)/day) for two weeks could significantly improve the immune function of cyclophosphamide(CPA)-induced immunodepressed mice, with equivalent effect to 50 mg lentinan (positive control medicine)/kg BW. BSP showed the following improvements in CPA-induced immunodepressed mice:(a) significantly increased the number of bone marrow nucleated cells, peripheral white blood cells, peripheral blood lymphocytes and its subset (B-cells), and decreased the micronucleated polychromatic erythrocytes in bone marrow; (b) significantly elevated the carbon clearance phagocytic index and serum levels of hemolysin and immunoglobulins, also enhanced the proliferative responses of splenocytes to ConA or LPS and splenic nature killer(NK) cell activity; (c) significantly up-regulated the mRNA expression of transcription factors T-bet/GATA-3 (Thl/Th2 switcher) together with the secretions of IL-2, IL-12, TNF-α, INF-γ/IL-4 (Thl/Th2 cytokines) by splenocytes. Additionally, oral administration of BSP alone at a dose of 200 mg/kg BW/day for two weeks could significantly enchance the immune function of normal mice, resulting in the up-regulation of T-bet/GATA-3 mRNA expression and IL-2, IL-12, TNF-α, INF-γ/IL-4 secretion by splenocytes, the enchancement of splenocytes proliferation, as well as the formation of serum hemolysin and immunoglobulins. The above findings suggest that BSP may have a favorable prospect as an ideal source of bio-active polysaccharides due to its good immunostimulatory activity in both immunodepressed mice and normal mice. In view of the abundance of bamboo and its by-products in China, the seeking of immuno-active polysaccharides from bamboo shavings may not only provide a new way for the diet control in immune related diseases, but also promote the development of bamboo shavings as highly-valued bv-products.