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This study was focused on the detailed structural investigation of Dendrobium officinale 2,3-O-acetyl glucomannan. A purified Dendrobium officilae polysaccharide sample (DOP) was obtained by sequential thermo-stable α-amylase treatments and multiple freeze-thaw treatments. DOP had an Mw of 312 kDa, consisted dominantly of mannose and glucose in a molar ratio of 6.9:1, and did not have branches. Acetyl groups were found attached to the O-2 or O-3 position of some mannose residues of DOP based on 1 D 1H NMR, 2 D 1H-1H TOCSY, and 2 D 1H-1H COSY spectra. DOP was then treated with endo-β-mannanase to produce oligosaccharides rich in O-acetylated sugar residues (HDOP). Results of 1 D 1H, 1 D 13C, 2 D 1H-1H COSY, 2 D 1H-1H TOCSY, 2 D 1H-1H NOESY, 2 D 1H-13C HMQC, and 2 D 1H-13C HMBC NMR analyses indicated that majority of O-acetylated mannoses were mono-substituted with acetyl groups at O-2 or O-3 position. There were small amounts of mannose residues with di-O-acetyl substitution at both O-2 and O-3 positions. Minor levels of mannoses with 6-O-acetyl, 2,6-di-O-acetyl, and 3,6-di-O-acetyl substitutions were also identified. Furthermore, gradient ethanol precipitation method was used to fractionate DOP to produce four major fractions that were all O-acetyl glucomannans but with different M/G ratio, molecular weight, and intrinsic viscosity. A method based on 1 D proton NMR spectroscopy was developed to calculate the degree of substitution (DS) of O-acetyl group of four glucomannan fractions, and the result indicated that these fractions were different in the degree of O-acetyl group substitution at O-2 and O-3 positions. MALDI-tof-MS analysis of the four major fractions subjected to mannanase and cellulose hydrolysis showed that these fractions varied in the distribution pattern of oligosaccharides. Jinjiudi Biotechnology Co. LTD., Kunming, Yunnan, China |
