Central Laboratory, Changchun Normal University, Changchun, P. R. China.
J Sep Sci. 2018 Dec;41(24):4458-4468. doi: 10.1002/jssc.201800691.
Puerariae Flos is the flower of Puerariae Radix, which is a common Chinese herb containing numerous isoflavones in all parts of the flower. Standard methods for screening and isolating isoflavones are typically labor intensive and time consuming. In this study, a new assay based on ultrafiltration with liquid chromatography and mass spectrometry was developed for the rapid screening and identification of ligands for α-glucosidase, xanthine oxidase and lactate dehydrogenase in the extract of Puerariae Flos. Three isoflavones were identified as α-glucosidase inhibitors, three isoflavones were identified as lactate dehydrogenase inhibitors, and no specific binding ligands were identified for xanthine oxidase in the extract. Subsequently, specific binding ligands, puerarin, genistin, and tectorigenin (purities were 90, 60, 99, and 91.73%, respectively), were separated by high-speed counter-current chromatography. The partition coefficient values of the target compounds and resolutions of peaks were employed as indicators and the solvent system and mobile phase flow rate were optimized for two-stage separation. An optimized two-phase solvent system comprised of ethyl acetate/ethanol/water (4:0.5:3, v/v/v) was successfully used to isolate the three compounds from Puerariae Flos. The monomer compounds isolated, collected, and purified by high-speed counter-current chromatography were analyzed by high-performance liquid chromatography, resulting in the isolation of three targeted compounds. The chemical structures of all three targeted compounds were individually identified by ultra high performance liquid chromatography with high-resolution mass spectrometry. The results demonstrate that ultrafiltration with liquid chromatography and mass spectrometry combined with high-speed counter-current chromatography is not only a powerful tool for screening and isolating α-glucosidase and lactate dehydrogenase inhibitors in complex samples, but also a useful platform for identifying bioactive compounds for preventing and treating diabetes and stroke.
野葛花是野葛的花，野葛是一种常见的中国草药，其花的各个部分都含有多种异黄酮。筛选和分离异黄酮的标准方法通常劳动强度大，耗时较长。在这项研究中，建立了一种基于超滤的新方法，结合液相色谱和质谱，用于快速筛选和鉴定野葛花提取物中α-葡萄糖苷酶、黄嘌呤氧化酶和乳酸脱氢酶的配体。鉴定出三种异黄酮为α-葡萄糖苷酶抑制剂，三种异黄酮为乳酸脱氢酶抑制剂，在提取物中未鉴定出黄嘌呤氧化酶的特定结合配体。随后，通过高速逆流色谱法分离特定的结合配体，葛根素、大豆苷元和染料木黄酮（纯度分别为 90%、60%、99%和 91.73%）。目标化合物的分配系数值和峰的分辨率用作指标，优化溶剂系统和流动相流速进行两阶段分离。成功地使用优化的两相溶剂系统，乙酸乙酯/乙醇/水（4:0.5:3，v/v/v），从野葛花中分离出三种化合物。通过高速逆流色谱法分离、收集和纯化的单体化合物用高效液相色谱法进行分析，结果分离出三种目标化合物。通过超高效液相色谱-高分辨率质谱法对三种目标化合物的化学结构进行了单独鉴定。结果表明，液相色谱-质谱联用超滤与高速逆流色谱相结合，不仅是筛选和分离复杂样品中α-葡萄糖苷酶和乳酸脱氢酶抑制剂的有力工具，也是鉴定用于预防和治疗糖尿病和中风的生物活性化合物的有用平台。
