Objective To analyze the correlation between the content changes of characteristic components and its antithrombotic effect in different proportions of the Danshen (Salviae Miltiorrhizae Radix et Rhizoma, SMRR)-Honghua (Carthami Flos, CF) herb pair, and optimize the compatibility ratio of SMRR and CF for promoting blood circulation and removing blood stasis. Methods Fingerprint profiles and quantitative determination methods for characteristic components were established for SMRR-CF herb pair at different ratios (1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, 5:1). The HPLC combined with a comprehensive scoring method was used to determine the optimal extraction process of the SMRR-CF herb pair. The anti-thrombotic activity of the SMRR-CF extracts at different ratios was measured using zebrafish as a model organism. The principal component analysis (PCA) method was used to integrate the content of characteristic components and the intensity of antithrombotic activity, and the optimal ratio of SMRR and CF was comprehensively evaluated from two aspects of composition and efficacy. Results The fingerprint profiles of the SMRR-CF herb pair in nine different ratios collectively identified 17 common peaks, among which nine characteristic components were characterized. These include hydroxysafflor yellow A (peak 2), luteoloside (peak 3), kaempferol-3-O-rutinoside (peak 4), quercitrin (peak 5), salvianolic acid B (peak 8), dihydrotanshinone I (peak 12), cryptotanshinone (peak 14), tanshinone I (peak 15), and tanshinone II_A (peak 17). The optimal extraction process was determined to be maceration with 10 times the amount of 70% ethanol for 1 h by reflux. The content determination showed that the characteristic components changed significantly after the compatibility of SMRR and CF compared with the single herb. When the SMRR-CF ratio was 1:5, decoction yielded the highest concentrations of salvianolic acids and tanshinones derived from SMRR. Conversely, ratios of 3:1, 4:1 and 5:1 produced the greatest amounts of hydroxysafflor yellow A, luteoloside and kaempferol-3-O-rutinoside from CF, indicating that co-decoction facilitates the extraction of bioactive constituents from both herbs. Pharmacodynamic evaluation revealed that the 3:1 ratio exhibited the strongest antithrombotic activity, achieving a thrombosis inhibition rate of 43.7%. Principal-component analysis of the “composition-effect” dataset ranked the comprehensive indices (F-values) as 3:1 > 4:1 > 5:1 > 2:1 > 1:1 > 1:5 > 1:4 > 1:2 > 1:3, confirming that the 3:1 ratio represents the optimal compatibility of the SMRR-CF pair. Conclusion In this study, chemical profiling and in vivo pharmacodynamic evaluation were integrated to elucidate the quantitative relationship between the dynamic changes of characteristic constituents and the antithrombotic efficacy of the SMRR-CF herb pair across different compatibility ratios. An optimal ratio was identified through a “composition-effect”-based comprehensive assessment. These findings not only establish a foundation for subsequent dose-effect investigations of this classic combination, but also offer a rational reference for determining prescription design in latent prescriptions.

Salviae Miltiorrhizae Radix et Rhizoma-Carthami Flos herb pair  /  fingerprint  /  antithrombotic effect  /  principal component analysis  /  compatibility optimization  /  dose-effect correlation  /  HPLC  /  hydroxysafflor yellow A  /  luteoloside  /  kaempferol-3-O-rutinoside  /  quercitrin  /  salvianolic acid B  /  dihydrotanshinone I  /  cryptotanshinone  /  tanshinone I  /  tanshinone II_A