Xue Hongkun, Zhu Xiaohan, Tan Jiaqi, Fan Linlin, Li Qian, Tang Jintian, Cai Xu
Key Laboratory of Particle & Radiation Imaging, Ministry of Education, Department of Engineering Physics, Tsinghua University, No. 30 Shuangqing Road, Haidian District, Beijing 100084, China.
Academy for Advanced Interdisciplinary Studies, Peking University, No. 5 Yiheyuan Road, Haidian District, Beijing 100871, China.
Foods. 2021 Mar 1;10(3):509. doi: 10.3390/foods10030509.
An efficient strategy for the selection of active compounds from blueberry based on counter-current fractionation and bioassay-guided separation was established in this study. Blueberry extract showed potential α-glucosidase inhibitory activity. After extraction by different solvents, the active components were enriched in water. The water extract was divided into six fractions via high-speed counter-current chromatography to further track the active components. Results indicated that the α-glucosidase inhibition rate of F4 was remarkable higher than the others. Cyanidin-3-glucoside (C3G) with a purity of 94.16% was successfully separated from F4 through column chromatography, and its structure was identified by ultraviolet spectral, Fourier-transformed infrared spectroscopy, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, H nuclear magnetic resonance (NMR), and C NMR. The interaction mechanism between C3G and α-glucosidase was clearly characterized and described by spectroscopic methods, including fluorescence and circular dichroism (CD) in combination with molecular docking techniques. C3G could spontaneously bind with α-glucosidase to form complexes by hydrogen bonds. The secondary structure of α-glucosidase changed in varying degrees after complexation with C3G. The α-helical and β-turn contents of α-glucosidase decreased, whereas the β-sheet content and the irregular coil structures increased. Molecular docking speculated that C3G could form hydrogen bonds with α-glucosidase by binding to the active sit (Leu 313, Ser 157, Tyr 158, Phe 314, Arg 315, and two Asp 307). These findings may be useful for the development of functional foods to tackle type 2 diabetes.
本研究建立了一种基于逆流分馏和生物测定导向分离从蓝莓中筛选活性化合物的有效策略。蓝莓提取物显示出潜在的α-葡萄糖苷酶抑制活性。经不同溶剂萃取后，活性成分富集于水相中。通过高速逆流色谱法将水提取物分为六个馏分，以进一步追踪活性成分。结果表明，F4的α-葡萄糖苷酶抑制率显著高于其他馏分。通过柱色谱法从F4中成功分离出纯度为94.16%的矢车菊素-3-葡萄糖苷（C3G），并通过紫外光谱、傅里叶变换红外光谱、高效液相色谱-电喷雾电离-串联质谱、氢核磁共振（NMR）和碳核磁共振对其结构进行了鉴定。结合分子对接技术，通过荧光和圆二色性（CD）等光谱方法明确表征并描述了C3G与α-葡萄糖苷酶之间的相互作用机制。C3G可通过氢键与α-葡萄糖苷酶自发结合形成复合物。与C3G复合后，α-葡萄糖苷酶的二级结构发生了不同程度的变化。α-葡萄糖苷酶的α-螺旋和β-转角含量降低，而β-折叠含量和不规则卷曲结构增加。分子对接推测C3G可通过与活性位点（Leu 313、Ser 157、Tyr 158、Phe 314、Arg 315和两个Asp 307）结合与α-葡萄糖苷酶形成氢键。这些发现可能有助于开发用于治疗2型糖尿病的功能性食品。
