Abstract: Ophioglossaceae species are perennial herbaceous ferns with rich wild diversity, exhibiting significant edible and medicinal value. Plants of this family are commonly used in their entirety in traditional medicine for their heat-clearing and detoxifying effects, and are clinically applied in treating conditions such as lung heat cough and snakebites. Chinese Materia Medica includes several species from this family, among which Ophioglossum vulgatum and Sceptridium ternatum are the most common. Phytochemical studies have shown that both O. vulgatum and S. ternatum contain bioactive compounds such as flavonoids, terpenoids, and phenolic acids. However, the number of chemically characterized constituents in this family remains limited, which restricts further exploration of their medicinal potential. In this study, a widely targeted metabolomics approach was applied to systematically analyze the metabolic profiles of whole plants of O. vulgatum, O. reticulatum, and S. ternatum. A total of 2,651 metabolites were identified, including primary metabolites such as amino acids and their derivatives, lipids, organic acids, nucleotides and derivatives, as well as secondary metabolites such as flavonoids, alkaloids, terpenoids, phenolic acids, lignans, and coumarins. The diversity of secondary metabolites exceeded that of primary metabolites, with flavonoids being the most abundant, predominantly flavonols and flavones. Comparative metabolomic analysis revealed that the two congeneric species, O. vulgatum and O. reticulatum, exhibited closer metabolic profiles, and both contained significantly more flavonoids than S. ternatum, indicating phylogenetic consistency and interspecific specificity in metabolite composition. Among the three species, O. vulgatum showed the highest number of enriched metabolites, followed by S. ternatum, and O. reticulatum had the fewest. Furthermore, analysis of O. vulgatum samples collected at different harvesting periods indicated that samples collected in August exhibited the highest accumulation of flavonoids and terpenoids. This study systematically elucidates the diversity of metabolites in three Ophioglossaceae species, the metabolic variations among species and genera, and the influence of harvesting time on metabolite accumulation, thereby providing a scientific basis for future medicinal development and nutritional evaluation.

Key words: Ophioglossum vulgatum, Ophioglossum reticulatum, Sceptridium ternatum, metabolome, harvesting period