Comparative metabolomic characterization of secondary metabolites in Gnaphalium affine D. Don considering the influence of growth stages and cultivation systems

Gnaphalium affine has long been consumed as a food and medicinal plant due to its nutritional and bioactive properties. This study comprehensively examined variation in metabolites and antioxidant activities across growth stages and between two cultivation environments of G. affine using UPLC-QTOF/MS-based metabolomics with chemometric analysis. In total, 50 metabolites were annotated, including eight compounds isolated by successive chromatographic separations and structurally elucidated. PCA indicated that metabolite variation was associated with both growth stage and the cultivation environments examined in this study, while PLS-DA showed clearer discrimination between samples from the two cultivation environments. Phenylpropanoids and flavonoids, particularly caffeoylquinic acid derivatives, quercetin glycosides, and luteolin glycosides, were the main discriminatory metabolites. For targeted quantification, the isolated compounds were used as reference standards to develop an HPLC-DAD method, which showed excellent linearity (R² ≥ 0.999) and low detection limits (0.671–1.088 µg/mL). Total phenolic and flavonoid contents and antioxidant activities varied across growth stages and between samples from the two cultivation environments examined. Open-field samples showed increased phenolic contents and enhanced ABTS radical-scavenging activity at later growth stages under the conditions examined. These findings support G. affine as a promising functional food and nutraceutical resource for future applications.