暗褐脉柄牛肝菌子实体多糖的分离纯化、结构表征及体外降血糖活性

Extraction,Purification,Characterization and in Vitro Hypoglycemic Activity of Polysaccharides from Fruiting Body of Phlebopus portentosus

为研究暗褐脉柄牛肝菌多糖的结构特性及生物活性,本研究以暗褐脉柄牛肝菌子实体为研究对象,采用水提醇沉法制备粗多糖,Sevag试剂脱蛋白,大孔吸附树脂脱色,DEAE-52纤维素柱色谱分离,丙烯葡聚糖凝胶S-400HR纯化制备均一多糖。采用高效凝胶渗透法测定分子质量、PMP柱前衍生法结合高效液相色谱测定单糖组成、紫外光谱分析是否含蛋白或核酸、红外光谱分析官能团种类、核磁共振波谱初步分析多糖的结构,扫描电子显微镜观察多糖的形貌。利用α-葡萄糖苷酶抑制实验筛选出活性较好的多糖组分并进一步研究其酶动力学,初步阐明暗褐脉柄牛肝菌多糖的体外降血糖作用机制和类型。结果表明,筛选的4种大孔树脂(HP 2MGL、HP 20、SP 850、SP 825)中,SP 825大孔树脂综合脱色效果最好;活性组分(PPP-0)重均分子质量为31 059 Da,含有α和β构型糖苷键,是一种由半乳糖Gal、葡萄糖Glc、岩藻糖Fuc、甘露糖Man组成的杂多糖;体外降血糖活性实验表明粗多糖和3种均一多糖组分均对α-葡萄糖苷酶有抑制作用,其中PPP-2抑制率最高,PPP-0次之,且在酶动力学上属于竞争性可逆抑制。本研究可为暗褐脉柄牛肝菌多糖在降血糖相关功能食品和药品方面的开发提供理论依据。

In order to study the structural properties and biological activities of the polysaccharides of Phlebopus portentosus,water-soluble polysaccharides from the fruiting body of P.portentosus were extracted by hot water extraction followed by ethanol precipitation,deproteinized using Sevag reagent,decolorized using macroporous adsorption resin,and purified to homogeneity by successive chromatography on DEAE-52 cellulose and Sephacryl S-400HR columns.The molecular mass of the purified polysaccharides was determined by high performance gel permeation chromatography(HPGPC),and the monosaccharide composition by high performance liquid chromatography(HPLC)following pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone(PMP).Ultraviolet(UV)absorption spectroscopy was used to examine whether the polysaccharide samples contained proteins or nucleic acids,and the types of functional groups,structure and morphology were characterized by infrared spectroscopy,nuclear magnetic resonance(NMR)spectroscopy and scanning electron microscopy(SEM),respectively.The polysaccharide with highα-glucosidase inhibitory activity was selected to evaluate its inhibition kinetics onα-glucosidase.Furthermore,its in vitro hypoglycemic mechanism and inhibitory type were clarified.The results showed that among four macroporous resins(HP 2MGL,HP 20,SP 850 and SP 825)tested,SP 825 macroporous resin had the best decolorization efficiency.The fraction PPP-0 had a weight average molecular weight(mw)of 31059 Da.Moreover,it was a heteropolysaccharide mainly composed of galactose,glucose,fucose and mannose that were linked byα-andβ-glycosidic bonds.In vitro hypoglycemic experiments showed that the crude polysaccharide and the three purified polysaccharide fractions all exhibited inhibitory activity on α-glucosidase, among which PPP-2 had the highest inhibitory rate, followed by PPP-0. The inhibition was competitive and reversible. This study could provide a theoretical basis for the development of functional foods with hypoglycemic activity and hypoglycemic agents from P. portentosus polysaccharides.