Artificial intelligence-driven identification and mechanistic exploration of synergistic anti-breast cancer compound combinations from Prunella vulgaris L.-Taraxacum mongolicum Hand.-Mazz. herb pair

Introduction: Hand.-Mazz. (TH) herb pair, which is commonly used in traditional Chinese medicine (TCM), has been applied for the treatment of breast cancer. Although its efficacy is validated, the synergistic anti-breast cancer compound combinations within this herb pair and their underlying mechanisms of action remain unclear. Methods: cellular assays to identify the most effective superior extracts. These superior extracts were subjected to liquid chromatography-mass spectrometry (LC-MS) analysis to identify their constituent compounds. A deep learning-based prediction model, DeepMDS, was applied to predict synergistic anti-breast cancer multi-compound combinations. These predicted combinations were experimentally validated for their anti-breast cancer effects at actual content ratios found in the extracts. Preliminary bioinformatics analyses were conducted to explore the mechanisms of action of these superior combinations. We also compared the anti-breast cancer effects of superior extracts from different geographical origins and analyzed the contents of compounds to assess their representation of the anti-tumor effect of the corresponding TCM. Results: The results revealed that LC-MS analysis identified 27 and 21 compounds in the superior extracts (50% ethanol extracts) of PVL and TH, respectively. Based on these compounds, DeepMDS model predicted synergistic anti-breast cancer compound combinations such as F973 (caffeic acid, rosmarinic acid, p-coumaric acid, and esculetin), T271 (chlorogenic acid, cichoric acid, and caffeic acid), and T1685 (chlorogenic acid, rosmarinic acid, and scopoletin) from single PVL, single TH and PVL-TH herb pair, respectively. These combinations, at their actual concentrations in extracts, demonstrated superior anti-breast cancer activity compared to the corresponding extracts. The bioinformatics analysis revealed that these compounds could regulate tumor-related pathways synergistically, inhibiting tumor cell growth, inducing cell apoptosis, and blocking cell cycle progression. Furthermore, the concentration ratio and total content of compounds in F973 and T271 were closely associated with their anti-breast cancer effects in extracts from various geographical origins. The compound combination T1685 could represent the synergistic anti-breast cancer effects of the PVL-TH pair. Discussion: This study provides insights into exploring the representative synergistic anti-breast cancer compound combinations within the complex TCM.