Structural identification of active moiety in anti-tumor metastatic polysaccharide purified from fermented barley by sequential enzymatic hydrolysis

Abstract

The potent anti-cancer metastatic effects induced by the immunostimulatory activities of intravenously administered BF–I, an active polysaccharide purified from fermented barley, were previously evaluated. This study aimed to evaluate the anti-cancer metastatic effect of orally administered BF–I and elucidated the microstructure and active moiety of BF–I using sequential enzymatic hydrolysis. In experiments using Colon26-M3.1 carcinoma, prophylactic oral administration of BF–I potently suppressed lung cancer metastasis. Furthermore, gas chromatography-mass spectrometry analysis of partially methylated alditol acetate showed that BF–I comprised 15 different glycosyl linkages, including 4-linked Xylp (16.1%), terminal Araf (12.4%), 4-linked Glcp (32.8%), 3-linked Glcp (13.5%), 3,6-linked Glcp (1.5%), and 3,6-linked Galp (5.3%). These glycosyl linkage combinations are typical for arabinoxylan, yeast-derived β-glucan, barley-derived β-glucan, and type II arabinogalactan. To clarify the microstructure, BF–I was sequentially degraded via linkage-specific enzymatic hydrolysis using five enzymes. Furthermore, the sugar composition, glycosyl linkage, and reactivity with the β-glucosyl Yariv reagent were analyzed, along with sequencing based on tandem mass spectra of partially degraded fragment fractions. The results confirmed that BF–I comprises a mixture of four different types of polysaccharides (arabinoxylan, yeast-derived β-glucan, barley-derived β-glucan, and type II arabinogalactan) with similar molecular weights. Furthermore, different polysaccharides contribute significantly to cytokine (interleukin-6, -12, and tumor necrosis factor-α) secretion by peritoneal macrophages. Therefore, the potent anti-cancer metastatic activity of BF–I is proposed to be related to the immunostimulatory activities of the four different types of polysaccharides.