超声辅助双水相提取蓝莓花色苷工艺优化及其抗肿瘤活性研究

Optimization of ultrasound assisted aqueous two-phase extraction of anthocyanins from blueberry and its antitumor activity

采用超声辅助双水相提取蓝莓花色苷,通过单因素实验和正交实验确定最佳的提取工艺;采用MTT法测定蓝莓花色苷提取物(Blueberry anthocyanins extracts,BAEs)对HepG2细胞存活率的影响;利用Hoechst 33342对HepG2细胞进行染色,通过激光共聚焦显微镜观察细胞形态;采用流式细胞分析仪测定不同浓度BAEs对HepG2细胞凋亡率和胞内ROS水平的影响。结果表明超声辅助双水相提取蓝莓花色苷最优工艺条件为:超声功率300 W、乙醇体积分数24%、硫酸铵质量分数18%、料液比(1∶30)g/mL和提取时间60min。在此条件下,花色苷得率为(2.14±0.05)mg/g。此外,BAEs可显著抑制HepG2细胞存活率、提高细胞核的荧光强度、细胞凋亡率和胞内ROS水平,进而发挥较好的抗肿瘤活性。本研究结果为花色苷提取提供一种具有发展潜力的提取方式,并为天然抗肿瘤药物的开发提供重要的物质基础。

Anthocyanins from blueberry were extracted by ultrasound assisted aqueous two-phase extraction method.The single factor and orthogonal experiments were applied to optimize the extraction process.The MTT assay was used to evaluate the effect of blueberry anthocyanins extracts(BAEs)on the survival rate of HepG2 cells.HepG2 cells were stained with Hoechst 33342 and observed by laser confocal microscope.The effects of different concentrations of BAEs on the apoptosis rate in HepG2 cells and ROS levels were determined by flow cytometry.It was found that the optimum extraction conditions were:ultrasound power 300 W,ethanol concentration 24%,ammonium sulfate mass fraction 18%,material to liquid ratio(1∶30)g/mL,and extraction time 60 min.Under these conditions,the yield of anthocyanins was(2.14±0.05)mg/g.In addition,BAEs could significantly inhibit the survival rate of HepG2 cells,improve fluorescence intensity of the nucleus,the rate of apoptosis and the levels of intracellular ROS.Therefore,it had antitumor activity.The results provide a potential method for anthocyanin extraction and an important reference for the development of natural antitumor drugs.