Deciphering Anticoagulant Active Substances and Mechanisms in Guanxinjing Formula Through Phenotype-Anchored Hierarchical Strategy and Topological Network Analysis

Anticoagulation is the cornerstone for preventing thrombosis in coronary heart disease patients with atrial fibrillation or a mechanical heart valve. Guanxinjing formula (GXJF) demonstrates anticoagulant effects, while its anticoagulant substances and mechanisms remain unclear. In this study, the chemical composition of GXJF was identified, establishing a chemical basis for the discovery of anticoagulant substances. Then, a phenotype-anchored hierarchical strategy (the formula, the medicinal materials, and the representative compounds) was developed to reveal active substances, especially synergy. Meanwhile, phenotype-based topological network analysis was employed to delineate the pivotal targets. Dynamic blood flow imaging analysis in zebrafish was used to evaluate the effect of active substances in vivo. This approach enables rapid identification of active substances by hierarchically eliminating interference from inactive components, uncovers potential synergistic effects, and accelerates target identification by focusing on phenotype-relevant protein networks. Pentagalloylglucose, (+)-catechin, senkyunolide A, and lithospermic acid were considered as the anticoagulant substances of GXJF. Notably, synergy between hydroxysafflor yellow A and anhydrosafflor yellow B were discovered. Topological network analysis and enzyme activity assays suggested that they exert anticoagulant effects by inhibiting coagulation factors Xa and Ⅱa. These findings provide support for the clinical application of GXJF and establish a foundation for its quality control.