蓝莓花青素酰化产物的稳定性及诱导肝癌细胞凋亡的机制研究

The property of anthocyanins were unstable and influenced by the pH, temperature and media.The common technology for stabilization anthocyanin include adding antioxidants,microcapsule,dextrin inclusion,but the effect was not significant.This program was by drawing galloyl acyl group into molecular structure of anthocyanin that seperated and purified from blueberry,to obtain modified products-acylated anthocyanin. On the basis of the effect of light, pH,temperature and other factors on the color of acylated products, using laser light scattering method to determinate the molecular weight of acylated anthocyanin,mass spectrometry, infrared, nuclear magnetic resonance to detect the structural changes. By the establishment of sub-acute rat liver cancer model, to explore the protection and oxidative stress effect of the acylated anthocyanins on liver injury in rats.Using improved MTT assay ( WST-8 method) to further observe the inhibitory action of acylated products to the growth of cell of hepatoma,by propidium iodide staining to detect changes in cell cycle, Annexin V- FITC/PI flow cytometry to detect the level of apoptotic cells, focusing microscope and Western blotting to observe and detect cell autophagy. With antioxidant activities in scavenging free radicals of acylated anthocyanins, discussing the inhibiory effect and the possible mechanism of anthocyanins on human hepatoma cell from the antioxidant aspects.The study will provide some experimental evidences and theoretical basis for establishing the evaluation system of blueberry anthocyanins stability, as well as clinical applications.

花青素性质不稳定, 易受食品体系中pH、温度和介质的影响。目前常用的花青素稳定化技术包括添加抗氧化剂、微胶囊化 、糊精包合等，但效果并不显著。本项目拟通过在分离、纯化的蓝莓花青素的分子结构上引入没食子酰基, 获得改性产物-花青素酰化物。在探讨光照、pH、温度等因素对酰化物颜色影响的基础上，利用激光光散射法测定花青素酰化物的分子量, 质谱、红外、核磁检测其结构变化。通过建立亚急性大鼠肝癌模型，探索花青素酰化物对肝损伤大鼠的保护和氧化应激作用。并进一步采用改良MTT 法( WST-8法)观察酰化产物对肝癌细胞的生长抑制，碘化丙锭单染色研究细胞周期改变，Annexin V- FITC/PI双染流式细胞术检测细胞凋亡水平，聚焦显微镜观察和Western blotting分析细胞自噬发生。结合花青素酰化物体外清除自由基抗氧化活性，从抗氧化角度探讨其对肝癌细胞的抑制作用及其可能作用机制。

蓝莓花青素具有重要的生理功能和开发应用前景，但其性质不稳定, 易受体系中pH、温度和介质的影响。常用的花青素稳定化技术包括添加抗氧化剂、微胶囊化、糊精包埋等，但效果并不显著。本项目通过将分离、纯化的蓝莓花青素的分子结构上引入没食子酰基, 获得改性产物花青素酰化物，利用红外、紫外可见光谱检测其结构变化。为确定酰化位点，以花青素中含量最为丰富的矢车菊素-3-O-葡萄糖苷为基础原料，优化其酰化方法，利用高效制备型液相色谱进行分离纯化；结合傅里叶红外光谱扫描和液相色谱质谱联用仪进行结构测定，对比酰化前后物质的稳定性和亲脂性。通过建立亚急性大鼠肝癌模型，探索花青素酰化物对肝损伤大鼠的保护和氧化应激作用。采用MTT法观察酰化产物对肝癌细胞的生长抑制，结合花青素酰化物体外清除自由基抗氧化活性，探讨其对肝癌细胞的抑制作用。研究结果表明：修饰后得到的没食子酰化花青素总酚含量为179.7±8.7 mg/g，约为酰化前的3倍。矢车菊素-3-O-葡萄糖苷的酰化率为31%左右，酰化位点为糖苷键的伯醇羟基。酰化产物的油水分配系数为7.98，明显高于酰化前的0.24，表明酰化后矢车菊素-3-O-葡萄糖苷的亲脂性明显增强，且稳定性、抗氧化性均有增加。酰化前后的蓝莓花青素对Hep G2、Caco-2肿瘤细胞均具备增殖抑制效果，且酰化后的抑制效果更明显。本研究为实现花青素更为广泛、有效的综合利用提供可能。