瓦布贝母内生真菌WBS020多糖理化性质和抗氧化活性

Preliminary Physicochemical Characterization and Antioxidant Activity of Polysaccharide Fraction from Endophytic Fungal WBS020 Originated from Fritillaria unibracteata var. wabuensis

以一株分离自药用植物瓦布贝母的内生真菌Fusarium tricinctum WBS020为材料,对其胞外多糖进行研究。首先采用多种有机溶剂对WBS020发酵液进行脱脂除杂,用醇沉法获取粗多糖。随后用大孔吸附树脂法除色素,蛋白酶-Sevag联用法除蛋白质,半透膜法除盐等小分子物质,纤维素DEAE-52和Sephadex G-150柱色谱分离法获取纯化多糖。再用HPGPC-ELSD色谱法分析纯化多糖的均一性和平均相对分子质量,用PMP衍生法结合HPLC-DAD色谱法分析单糖组分,用UV、FT-IR分析多糖的部分理化性质。最后利用DPPH和ABTS自由基清除活性及亚铁离子螯合能力评价多糖的抗氧化活性。结果表明,经过一系列的提取分离纯化,得到一个胞外多糖主成分WBS020EPS1-2,其为含有少量结合蛋白质的纯度在95%以上的均一多糖,平均相对分子质量约为3.137×104,主要由甘露糖,鼠李糖,葡萄糖,半乳糖和阿拉伯糖以摩尔比0.43∶0.13∶10.00∶1.92∶0.11组成,同时,还含有一个未知成分,且单糖成分主要为α-构型链接的吡喃糖;此外,多糖WBS020EPS1-2对DPPH自由基清除活性较弱,而对ABTS自由基清除能力和亚铁离子螯合能力则较强,IC50分别为1.93 mg/mL和4.52 mg/mL。

The exopolysaccharide extracted from the fermentation broth of endophytic fungal Fusarium tricinctum WBS020 originated from Fritillaria unibracteata var. wabuensis(FUW) was studied. At first,the crude polysaccharide was precipitated with ethanol from the fermentation broth after being degreased by organic solvent,and further were preliminary purified by macroporous resin methods for decoloration,proteases in combination with Sevag reagent methods for deproteinization,equilibrium dialysis methods for desalination in vitro,the DEAE cellulose DE-52 anion exchange chromatography and sephadex G-150 column chromatography methods for the separation of polysaccharide. Subsequently,homogeneity average relative molecular weight and monosaccharide composition of the purified polysaccharide were studied by the methods of high-performance gel permeation chromatography coupled with evaporative light scattering detection(HPGPC-ELSD),PMP(1-phenyl-3-methyl-5-pyrazolone) precolumn derivatization with high performance liquid chromatography coupled to photodiode array detector(PMP-HPLC-DAD),respectively. And partial physicochemical properties were also analyzed by ultra violet(UV) spectra and fourier transform infrared(FT-IR) methods. Finally,the antioxidant activities were determined by DPPH radical scavenging assay,ABTS radical scavenging assay and ferrous ion chelating activity assay. Results showed that WBS020 EPS1-2,as the main exopolysaccharide,was obtained after the extraction,separation and purification. WBS020 EPS1-2 was homogeneous exopolysaccharide with95% purity and contained a small quantity of binding proteins,and was comprised of mannose,rhamnose,glucose,galactose and ara binose with a molar ratios of 0.43 ∶0.13 ∶10.00 ∶1.92 ∶0.11.Besides,there was a unknown monosaccharides. The monosaccharide in WBS020 EPS1-2 was a pyranose,which was connected by an α-glycosidic linkage. The average relative molecular weight of WBS020 EPS1-2 was approximately 3.137×104. In addition,exopolysaccharide WBS020 EPS1-2 had a weak DPPH radical scavenging activity,while possessed a strong ABTS radical scavenging effect and a good ferrous ion chelating activity with the IC50 value of 1.93 mg/mL and 4.52 mg/mL,respectively.