Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; DICP-CMC Innovation Institute of Medicine, Taizhou 225300, China.
Pathology, Fudan University Shanghai Cancer Center, Shanghai 200237, China.
J Chromatogr A. 2019 Sep 13;1601:224-231. doi: 10.1016/j.chroma.2019.04.073. Epub 2019 May 1.
Flavonoid glycosides are widespread in herbs and often used as medicines and nutraceuticals because of good bioactivities and low toxicities. However, due to their structural complexity and diversity, isolation of flavonoid glycosides and evaluation of their bioactivities are still highly challenging. To solve this problem, a new method for separation and preparation of novel flavonoid glycosides from Lobelia chinensis Lour (L. chinensis) was developed. To avoid the interference of non-flavonoids, a solid phase extraction method was used to selectively enrich the flavonoids from the total extract. Based on hydrophilic and hydrophobic properties of the flavonoid chemical structure consisting of sugar residue and diphenylpropane (CCC) skeleton, a structure-guided method development strategy was employed to design a 2D-HILIC × RPLC system in the first time. After optimization of chromatographic conditions, high selectivity and symmetric peaks of flavonoids were obtained on a zwitterionic Click XIon column and a polar-modified Atlantis T3 column. Based on these two columns, a 2 D-HILIC × RPLC system was constructed and successfully enlarged from the analytical level to the preparative level. In the first dimension, 20 fractions were obtained with good peak shapes at high sample loading. In the second-dimensional preparation, nine compounds were isolated and identified. Seven of them were novel flavonoid glycosides, lobelitin A-G, and two other known compounds were linatin and diosmin, respectively. Their target activities were evaluated via label-free cell phenotypic assays. Four novel flavonoid glycosides lobelitin A-D were found to have agonistic activities at G protein-coupled receptor 35 (GPR35). These results demonstrated that this method was effective to orthogonally separate flavonoids at the preparative level, especially for novel active flavonoid glycosides. The discovery of flavonoid glycosides with novel agonistic activity on GPR35 also sheds light on the mechanisms of action of L. chinensis relevant to its clinical application.
类黄酮糖苷广泛存在于草本植物中，由于具有良好的生物活性和低毒性，常被用作药物和营养保健品。然而，由于其结构的复杂性和多样性，类黄酮糖苷的分离和生物活性评价仍然极具挑战性。为了解决这个问题，开发了一种从半边莲（Lobelia chinensis Lour）中分离和制备新型类黄酮糖苷的新方法。为了避免非类黄酮的干扰，采用固相萃取法从总提取物中选择性富集类黄酮。基于糖基和二苯丙烷（CCC）骨架组成的类黄酮化学结构的亲水性和疏水性，首次采用结构导向的方法开发策略设计了 2D-HILIC × RPLC 系统。优化色谱条件后，在两性离子 Click XIon 柱和极性修饰的 Atlantis T3 柱上获得了类黄酮的高选择性和对称峰。基于这两根柱子，构建并成功地将 2D-HILIC × RPLC 系统从分析水平放大到制备水平。在第一维中，在高样品加载量下获得了 20 个具有良好峰形的馏分。在二维制备中，分离并鉴定了 9 种化合物。其中 7 种为新型类黄酮糖苷，分别为 lobelitin A-G，另外两种为已知化合物 linatin 和 diosmin。通过无标记细胞表型测定法评估了它们的靶标活性。发现 4 种新型类黄酮糖苷 lobelitin A-D 对 G 蛋白偶联受体 35（GPR35）具有激动活性。这些结果表明，该方法在制备水平上能够有效地正交分离类黄酮，特别是对于新型活性类黄酮糖苷。发现具有新型激动活性的类黄酮糖苷对 GPR35 的作用机制也为半边莲的临床应用提供了启示。
