红枣多糖的提取工艺及药理活性研究

Study on Extraction Technology and Pharmacological Activities of Polysaccharide from Ziziphus jujube

本研究以红枣多糖得率为指标,在单因素实验的基础上,利用Design-Expert8.0设计试验,采用响应面分析对红枣多糖超声辅助提取的条件进行了优化,随后以DPPH自由基、羟自由基清除能力为指标评价多糖的抗氧化效果、以α-淀粉酶和α-葡萄糖苷酶抑制率为指标证实红枣多糖具有降糖潜力,最后建立体外模型探究其降糖机制。首先对提取过程中主要影响多糖得率的水浴时间、料液比、超声时间和超声功率进行了单因素实验,同时利用响应面法进行优化分析。结果表明,红枣多糖最佳的提取条件为超声时间40 min,料液比1:20,超声功率80 W,水浴时间30 min,多糖得率达6.58%,与理论预测值6.71%一致。同时通过测定DPPH自由基、羟基自由基清除能力及还原力证实红枣多糖具有良好的抗氧化活性。此外,当多糖浓度达14 mg/mL时,对α-葡萄糖苷酶的抑制率为51.56%,而当浓度为4 mg/mL时对α-淀粉酶抑制率为28.43%,证实红枣多糖具有明显的降糖活性。在此基础上采用胰岛素抵抗HepG2细胞模型,以葡萄糖试剂盒测定及MTT法检测不同浓度的红枣多糖的葡萄糖消耗作用,结果表明当加药浓度为1.0 mg/mL时,细胞对葡萄糖的消耗量最大,GT/MTT达到模型组的154.2%。采用免疫印迹法(WB)检测胰岛素抵抗HepG2细胞中PI3K/Akt通路蛋白表达水平,确定红枣多糖可通过激活PI3K/Akt通路缓解胰岛素抵抗,发挥细胞保护作用。本研究对红枣多糖的提取方法进行优化,同时证实红枣多糖在体外具有抗氧化活性及降血糖活性,可作为一种潜在药食同源的抗氧化剂或功能性食品。

Polysaccharide from Ziziphus jujube Mill.(ZJP)was extracted by ultrasound-assisted extraction.Taking the extraction rate as the evaluation index,the extraction process of polysaccharide was optimized by Design-Expert8.0 and orthogonal experiment on the basis of single-factor test.Subsequently,DPPH free radical and hydroxyl free radical scavenging abilities were used as indicators to evaluate the antioxidant effect of polysaccharides,and the inhibitory rates ofα-amylase andα-glucosidase were used as indicators to prove that jujube polysaccharides had the potential of reducing blood sugar.Finally,an external model was established to explore its hypoglycemic mechanism.The optimum parameters were as follows:ultrasonic time of 40 min,solid-liquid ratio of 1:20,ultrasonic power of 80 W,water-bath incubation time of 30 min.Under these conditions the extraction rate of ZJP was 6.58%,which was consistent with the theoretical prediction value of 6.71%.Moreover,the antioxidant activity of ZJP was determined by DPPH free radical and hydroxyl free radical scavenging system and iron-reducing power.In addition,when the polysaccharide concentration reached14 mg/mL,the inhibition rate ofα-glucosidase was 51.56%,and when the concentration was 4 mg/mL,the inhibition rate ofα-amylase was 28.43%,respectively,confirming that jujube polysaccharide had obvious hypoglycemic activity.On this basis,the insulin resistance HepG2 cell model was used to detect the glucose consumption effect of different concentrations of jujube polysaccharides by glucose kit and MTT method.The results showed that when the concentration of the drug was1.0 mg/mL,the cell’s consumption of glucose was the largest,GT/MTT reached 154.2%of the model group.Western blotting was used to detect the protein expression level of PI3K/Akt pathway in insulin-resistant HepG2 cells,and it was determined that ZJP could alleviate insulin resistance and exert cytoprotective effect by activating PI3K/Akt pathway.In summary,the extraction method of jujube polysaccharide was optimized,and it was confirmed that jujube polysaccharide had anti-oxidant activity and hypoglycemic activity in vitro,and could be used as a potential anti-oxidant or functional food with a homology of medicine and food.