高速逆流色谱分离纯化桑葚花色苷及其抗氧化活性

Separation and Purification of Anthocyanins from Mulberry Fruit by High-Speed Counter-Current Chromatography and Their Antioxidant Activity

以桑葚为原料,在经过大孔树脂纯化的基础上,通过高速逆流色谱技术对桑葚花色苷进行分离纯化,结合紫外-可见光谱、高效液相色谱-质谱联用及核磁共振技术对分离纯化得到的组分进行鉴定,同时测定桑葚花色苷粗提取物和分离得到的花色苷组分对脂质体抑制率和1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-trinitrophenylhydrazine,DPPH)自由基清除能力。最终确定正丁醇-甲基叔丁基醚-乙腈-水-三氟乙酸(2∶2∶1∶5∶0.01,V/V)为两相溶剂体系,以上相为固定相、下相为流动相,在主机转速850 r/min、流速2 mL/min、检测波长254 nm条件下进行分离纯化,最终得到4个分离组分(组分Ⅰ、Ⅱ、Ⅲ、Ⅳ)。经鉴定发现组分Ⅳ为非花色苷类,组分Ⅰ、Ⅱ和Ⅲ分别为飞燕草素-3-葡萄糖苷、矢车菊素-3-葡萄糖苷和天竺葵素-3-葡萄糖苷,其含量和纯度分别为17.4、33.7、9.8 mg/100 mg和92.27%、94.05%、90.82%。桑葚花色苷粗提物以及组分Ⅰ、Ⅱ和Ⅲ对抑制脂质体过氧化和DPPH自由基清除率的半抑制浓度分别为(0.77±0.02)、(0.34±0.02)、(0.55±0.04)、(0.68±0.01)g/L和(0.40±0.01)、(0.16±0.01)、(0.22±0.01)、(0.35±0.03)g/L。

In this study, anthocyanins from mulberry fruit were separated and purified by sequential macroporous resin adsorption chromatography and high-speed counter-current chromatography(HSCCC), and were identified by UV-Vis spectroscopy, high performance liquid chromatography-mass spectrometry(HPLC-MS) and nuclear magnetic resonance. We further evaluated the inhibitory effects of the crude extract and the purified components on lipid peroxidation and 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH) radical. The HSCCC was performed using a two-phase solvent system composed of n-butanol, methyl tert-butyl ether, acetonitrile, water and trifluoroacetic acid(2:2:1:5:0.01, V/V) with the upper phase as the stationary phase and the lower phase as the mobile phase under the conditions of rotation speed of 850 r/min, flow rate of 2 mL/min and detection wavelength of 254 nm. Finally, four peaks(componentsⅠ, Ⅱ, Ⅲ and Ⅳ) were displayed on the HSCCC chromatogram. Component Ⅳ was identified as non-anthocyanin, while components Ⅰ, Ⅱ and Ⅲ as delphinidin-3-glucoside, cyanidin-3-glucoside and pelargonidin-3-glucoside with purities of and 92.27%, 94.05% and 90.82%, respectively and their contents in the crude extract were 17.4, 33.7, 9.8 mg/100 mg, respectively. The 50% inhibiting concentration of the crude extract, and components Ⅰ, Ⅱ and Ⅲ were(0.77 ± 0.02),(0.34 ± 0.02),(0.55 ± 0.04) and(0.68 ± 0.01) g/L for inhibiting lipid peroxidation and(0.40 ± 0.01),(0.16 ± 0.01),(0.22 ± 0.01) and(0.35 ± 0.03) g/L for scavenging DPPH radical, respectively.