桔梗多糖的复合酶提取、结构表征及抗氧化活性分析

Compound Enzyme Extraction of Platycodon grandiflorum Polysaccharides and Its Structure and Antioxidant Activity Characterization

目的:优化复合酶法提取桔梗多糖工艺,并初步分析桔梗多糖的结构和体外抗氧化活性。方法:分别考察酶添加量、料液比、酶解时间、酶解温度对桔梗多糖提取率的影响,采用响应面法进行提取条件优化,利用高效液相色谱法(HPLC)测定多糖的分子量和单糖组成,应用核磁共振(NMR)和扫描电镜(SEM)对多糖的糖苷键和形貌进行分析,并对多糖清除自由基的能力和还原力进行研究。结果:桔梗多糖的最佳提取工艺为纤维素酶、果胶酶、木瓜蛋白酶的添加量为2%,酶解时间90 min,料液比1:30 g/mL,酶解温度50℃,在此条件下,多糖实际提取率为9.01%±0.07%,多糖含量达92%±0.76%。纯化后得到桔梗多糖组分PGP-W-1,分子量为6.2 kDa,由甘露糖、鼠李糖、葡萄糖、半乳糖、木糖和阿拉伯糖按照摩尔比4.9:4.3:7.9:7.8:4.8:18.6组成,是一种具有α型糖苷键和β型糖苷键的吡喃型多糖。体外抗氧化试验显示PGP-W-1对DPPH自由基、ABTS+自由基和羟基自由基清除率IC_(50)值分别为2.14、2.25、0.78 mg/mL。结论:本研究优选出的桔梗多糖提取工艺切实可行,多糖提取效率高并表现出良好的体外抗氧化活性。

Objective:Optimize the extraction process of Platycodon grandiflorum polysaccharides by compound enzyme method,and preliminarily analyze its structure and in vitro antioxidant activity.Methods:Response surface methodology was used to optimize the extraction conditions with the extraction rate of polysaccharides as the index and the addition amount of enzymes,solid-liquid ratio,enzymolysis time and enzymolysis temperature as the factors.The molecular weight and monosaccharide composition of purified polysaccharides were analyzed by high performance liquid chromatography(HPLC),the glycosidic bonds and surface morphology of purified polysaccharides were analyzed by nuclear magnetic resonance(NMR)and scanning electron microscopy(SEM),respectively,and the free radical scavenging ability and reducing power of purified polysaccharides were evaluated.Results:The optimum extraction conditions were as follows,the addition of cellulase,pectinase and papain was 2%,the enzymolysis time was 90 min,the solid-liquid ratio was 1:30 g/mL,the enzymolysis temperature was 50℃.Under these conditions,the actual extraction rate of polysaccharides was 9.01%±0.07%,and the purity of polysaccharides was 92%±0.76%.The purified polysaccharides component PGP-W-1(6.2 kDa)was composed of mannose,rhamnose,glucose,galactose,xylose,and arabinose with a molar ratio of 4.9:4.3:7.9:7.8:4.8:18.6.NMR spectrum showed that PGP-W-1 was pyranose ring withα-andβ-glycoside bond.The IC50 values of the PGP-W-1 on DPPH free radicals,ABTS+free radicals and hydroxyl free radicals were 2.14,2.25,and 0.78 mg/mL,respectively.Conclusion:The optimized extraction process of Platycodon grandiflorum polysaccharide was feasible with high extraction efficiency and showed excellent antioxidant activity in vitro.