Metabolomic Profiling of Agathosma betulina Leaves, Under Wild, Field, and Glasshouse Conditions Using UPLC-QTOF-MS and Multivariate Analysis

Agathosma betulina (P.J. Bergius) Pillans is an aromatic medicinal shrub endemic to South Africa’s Cape Floristic Region, widely used in traditional medicine to treat urinary tract infections, gastrointestinal disorders, colds, and wounds. Despite its commercial importance, limited information is available on how different growing environments influence its phytochemical composition. This study profiled the secondary metabolite composition of A. betulina leaves collected from wild, open-field cultivation, and glasshouse-grown environments using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) combined with chemometric analyses. Methanolic extracts prepared from nine samples were analysed, resulting in the confident annotation of 21 secondary metabolites (MSI Level 2), comprising phenolic acids, flavanones, flavones, and flavonol glycosides. Rutin was the predominant flavonoid, with concentrations of 194.62, 185.71, and 58.82 mg CE kg−1 DW in wild, cultivated, and glasshouse-grown plants, respectively. Neochlorogenic acid accumulated predominantly in cultivated plants (51.84 mg CE kg−1 DW) compared with wild plants (3.31 mg CE kg−1 DW), while coumaroylquinic acid (125.93 mg CE kg−1 DW) and feruloylquinic acid (30.74 mg CE kg−1 DW) were most abundant under glasshouse conditions. Tricin-7-O-glucoside (62.93 mg CE kg−1 DW) and isorhamnetin-3-O-glucoside (31.26 mg CE kg−1 DW) also accumulated significantly in cultivated plants. Partial Least Squares Discriminant Analysis (PLS-DA) score explained 92.1% of the total variation (C1 = 63.8%; C2 = 28.3%), together with hierarchical heat map clustering and VIP, clearly separating samples according to growing environment. Wild plants exhibited the greatest metabolite diversity, cultivated plants showed intermediate metabolite profiles, and glasshouse-grown plants displayed reduced flavonoid accumulation and overall chemical diversity. These findings demonstrate that growing environment strongly influences secondary metabolite accumulation in A. betulina and provide valuable information for optimizing cultivation practices, quality control, and conservation strategies for this economically important medicinal species.