UHPLC–MS/MS-Based Identification of Active Constituents From Pinus yunnanensis Cones and the Multi-Target Mechanism Exploration Against Breast Cancer

Pinus yunnanensis cones are a rich source of bioactive compounds; yet, the systemic exposure and mechanistic basis of their anti-breast cancer activity remain unclear. Herein, we systematically identify the systemically absorbed components of P. yunnanensis cone extract and elucidate its anti-breast cancer mechanisms using an integrated analytical and computational approach. An optimized ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to overcome severe matrix interference and insufficient sensitivity, facilitating the rapid identification of active constituents in the crude extract. UHPLC–MS/MS-based analysis of rat plasma and feces revealed 16 (14 prototypes and 2 metabolites) and 12 (10 prototypes and 2 metabolites) systemically absorbed components, respectively, derived from 28 identified compounds. Subsequent network pharmacology combined with molecular docking validation was used to screen the anti-breast cancer active components, core targets, and key pathways. Integrated network pharmacology predicted coniferaldehyde, phyllanflexoid A, 3′,4′,5,5′,6,7-hexamethoxyflavone, dihydrocaffeic acid, usnic acid, and 7-oxodehydroabietic acid, as core components, targeting TP53, STAT3, PIK3R1, SRC, and PIK3CA and involving key pathways such as VEGF signaling pathway, aldosterone-regulated sodium reabsorption, prolactin signaling pathway, PD-L1/PD-1 checkpoint pathway in cancer, and ErbB signaling pathway. This study establishes a robust UHPLC–MS/MS platform for the quality control of P. yunnanensis cones and provides critical insights into their translational potential for anti-breast cancer drug development and advancing separation science applications in natural product research.