Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, China; School of Pharmaceutical Science of Shanxi Medical University, Taiyuan, Shanxi, China.
Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan, Shandong, China.
J Ethnopharmacol. 2020 May 10;253:112679. doi: 10.1016/j.jep.2020.112679. Epub 2020 Feb 23.
Gardenia Fructus (GF), a traditional Chinese medicine for clearing heat and purging fire, has been reported to use to treat thrombotic related diseases, but the antithrombotic components are not clear.
To develop efficient research methods for discovering some representative antithrombotic compounds of GF.
AB line zebrafish induced by arachidonic acid (AA) was used as a fast and trace-sample-required valuation model for antithrombptic effect of GF samples. Among nine samples of GF from different production areas, two samples with the largest difference in bioactivity were selected for downstream analysis. High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS) was applied to detect compounds in the GF samples. And herbal metabolomics and grey correlation analysis (GCA) were used to identify crucial compounds with potential antithrombotic activity. Then the bioactivity of those important compounds was verified on the zebrafish model. Network pharmacology was used to explore the protein targets and signaling pathways of these compounds.
Among the GF samples, S1 (Huoshan City, Anhui Province), and S6 (Jichun City, Hubei Province), significantly differed in thrombus inhibiting bioactivity. HPLC-Q-TOF/MS identified a total of 614 compounds in each GF sample. 19 compounds were selected as important potential variables from metabolomics data by orthogonal partial least squares discriminant analysis (OPLS-DA). And 10 compounds among them were further found to be positively correlated with the antithrombotic bioactivity of GF by GCA. Finally, 3 compounds in them, geniposide, citric acid, and quinic acid, were confirmed as representative antithrombotic chemical markers of GF. Using network pharmacology analysis, some key protein targets, such as proto-oncogene tyrosine-protein kinase Src (SRC) and cyclin-dependent kinase 2 (CDK2), and some signaling pathways were found to supply powerful evidence about antithrombotic mechanisms of three compounds and GF.
This research have succeeded to discover and identify three representative antithrombotic compounds of GF using an efficient integrated research strategy we established, an Omics Discriminant-Grey Correlation-Biological Activity strategy. The antithrombotic chemical makers we found could also contribute to provided more accurate index components for comprehensive quality control of GF.
栀子（GF）是一种传统的清热解毒中药，已被用于治疗血栓相关疾病，但抗血栓成分尚不清楚。
开发有效的研究方法，以发现 GF 的一些代表性抗血栓化合物。
使用 AA 诱导的 AB 系斑马鱼作为快速、微量样本要求的 GF 样品抗血栓作用评估模型。在来自不同产地的 9 个 GF 样品中，选择生物活性差异最大的两个样品进行下游分析。高效液相色谱-四极杆飞行时间质谱联用（HPLC-Q-TOF/MS）用于检测 GF 样品中的化合物。并采用草药代谢组学和灰色关联分析（GCA）鉴定具有潜在抗血栓活性的关键化合物。然后在斑马鱼模型上验证这些重要化合物的生物活性。网络药理学用于探索这些化合物的蛋白靶点和信号通路。
在 GF 样品中，S1（安徽省霍山县）和 S6（湖北省鸡春市）在血栓抑制生物活性方面有显著差异。HPLC-Q-TOF/MS 鉴定出每个 GF 样品中共有 614 种化合物。正交偏最小二乘判别分析（OPLS-DA）从代谢组学数据中选择了 19 种重要的潜在变量。其中 10 种化合物通过灰色关联分析（GCA）进一步发现与 GF 的抗血栓生物活性呈正相关。最后，从中确定了 3 种化合物，京尼平苷、柠檬酸和奎宁酸，作为 GF 的代表性抗血栓化学标志物。通过网络药理学分析，发现 SRC 和 CDK2 等一些关键蛋白靶点以及一些信号通路，为三种化合物和 GF 的抗血栓机制提供了有力的证据。
本研究采用我们建立的一种有效的综合研究策略，即“组学判别-灰色关联-生物活性”策略，成功发现和鉴定了 GF 的三种代表性抗血栓化合物。我们发现的抗血栓化学标志物也有助于为 GF 的全面质量控制提供更准确的指标成分。
