A multidimensional integrated strategy for identifying anti-thrombotic quality markers(Q-Markers) in Taohong Siwu decoction through combined analysis of chemical profiling, bioactivity, and pharmacodynamic efficacy.

ETHNOPHARMACOLOGICAL RELEVANCE: Taohong Siwu Decoction (TSD), originating from the Qing Dynasty medical text Gynecology Ice Mirror, is a representative formula for treating blood stasis syndrome, with the effects of promoting blood circulation, nourishing blood, and resolving stasis. Following this lead, modern research has revealed that the formula exerts antithrombotic effects through multiple pathways, thereby providing a critical pharmacological basis for its function of "promoting blood circulation and removing blood stasis". However, the pharmacodynamic material basis underlying this efficacy awaits further clarification.

AIM OF THE STUDY: This study aims to establish a multidimensional spectrum-effect correlation model for TSD by integrating its chemical composition with relevant biological activity and antithrombotic efficacy. The goal is to identify quality markers (Q-Markers) that are closely associated with its effects of promoting blood circulation and resolving blood stasis.

MATERIALS AND METHODS: UPLC-QqQ-MS/MS was used to quantify 31 components in TSD. Its biological activity was assessed via in vitro anticoagulant and antiplatelet assays, and efficacy via a zebrafish thrombosis model. The study employed grey relational analysis (GRA) and backpropagation neural networks (BP-ANN) to analyze component-activity, activity-efficacy, and component-efficacy correlations. It also constructed a TSD antithrombotic component-target-pathway network via bioinformatics and molecular docking, identifying potential active components. Validation via zebrafish models and quantitative real-time polymerase chain reaction (qRT-PCR) confirmed TSD's multi-component and multi-target interactions, ultimately determining its antithrombotic Q-Markers.

RESULTS: Thirty-one compounds across 15 TSD batches were quantified. Systematic cluster analysis categorized the batches into four groups, with Rehmannia glutinosa (Gaertn.) Libosch. ex DC. root quality identified as the main factor affecting TSD's quality consistency. The study confirmed TSD's in vitro anticoagulant and antiplatelet aggregation activity, as well as its satisfactory thrombus inhibition rate in zebrafish models. By combining GRA and BP-ANN, the study identified 19 key components; through molecular docking and experimental validation, six compounds were screened out: rehmannioside D, amygdalin, paeoniflorin, ferulic acid, hydroxysafflor yellow A, and chlorogenic acid. These six components exhibited strong binding affinity to thrombosis-related targets including GAPDH, IL-6, AKT1, CASP3, and EGFR, and were finally confirmed as Q-Markers for TSD's blood circulation-promoting and stasis-resolving efficacy.

CONCLUSIONS: Via multidimensional spectrum-effect correlation analysis, this study identified TSD's blood circulation-promoting Q-Markers. These findings lay a foundation for TSD's quality standards/clinical application and provide a reference for traditional Chinese medicine (TCM) constituent-pharmacology research.