Chemotype-Resolved Cytoprotection of CAM Hydrothermal Extracts Validated in 2D/3D Fibroblast Models and Supported by...

Crassulacean Acid Metabolism (CAM) plants possess unique stress-adaptive physiologies, yet their chemotype-bioactivity relationships remain poorly understood. We established an integrated workflow combining GC-MS metabolomics, 2D/3D functional validation, and in silico profiling to evaluate hydrothermal extracts from four CAM species (BDAE [Bulbophyllum drymoglossum], CRSAE [Crassula rupestris], ECAE [Echeveria], and STAE [Hylotelephium telephium]). PCA and marker analysis resolved three distinct chemotypes: acid-rich, sugar-rich, and lipid/sterol-enriched. Using standardized noncytotoxic concentrations, we demonstrated that acid-rich extracts (BDAE/CRSAE) most consistently attenuated H₂O₂-induced oxidative stress in NIH/3T3 fibroblasts. These cytoprotective effects included significant ROS reduction and preservation of mitochondrial membrane potential. Importantly, these antioxidant properties were robustly retained in a 3D bioprinted tissue-mimetic model with high biocompatibility, as confirmed by live/dead imaging. Furthermore, in silico docking justifies prioritizing phaselic acid as a bioactive marker, showing reproducible interactions with Keap1, COX-2, and MMP-1. While acknowledging the specific analytical scope of GC-MS and the predictive nature of computational modeling, this study successfully connects chemical composition, biological activity, and mechanistic hypotheses within a robust chemotype-resolved framework. Our findings offer a strategic scientific rationale for the selective application of CAM-derived metabolites in antioxidant strategies.