Comparative HPLC fingerprinting of flavonoid profiles in Selaginella species

Abstract. Setyawan AD, Chikmawati T, Miftahudin, Lotulung PDN, Sutarno, Sugiyarto, Sunarto. 2025. Comparative HPLC fingerprinting of flavonoid profiles in Selaginella species. Asian J Nat Prod Biochem 23: 131-145. Flavonoids are among the most prominent secondary metabolites in Selaginella, yet comparative information on flavonoid composition across multiple species remains limited. Most existing phytochemical studies focus on individual species or isolated compounds, providing little insight into interspecific chemical relationships within the genus. This study applies RT-based HPLC fingerprinting to comparatively assess flavonoid profile variation among 20 samples representing 17 Selaginella species collected from diverse ecological and geographical settings in Indonesia. Methanolic extracts were analyzed under standardized chromatographic conditions, and flavonoid-related peaks were aligned using a retention time tolerance of ±0.2 min to construct a binary presence-absence fingerprint matrix comprising 11 RT-defined features. Interspecific similarity was evaluated using Sørensen and Jaccard coefficients, followed by hierarchical clustering (UPGMA) and complementary ordination to visualize chemical relationships among samples. The results reveal pronounced qualitative differences in flavonoid fingerprints among species, characterized by both shared and species-specific chromatographic features. Conspecific samples collected from different localities exhibited stable core flavonoid profiles with additional site-related variation, while interspecific comparisons showed substantial heterogeneity. Similarity patterns were not strictly associated with geographic proximity, suggesting that flavonoid composition primarily reflects intrinsic biochemical traits rather than purely environmental effects. This study provides an effective qualitative framework for interspecific discrimination and chemotaxonomic assessment in Selaginella. Although compound identity and abundance were not resolved, the approach offers a practical foundation for future targeted phytochemical, bioactivity-oriented, and integrative chemotaxonomic investigations.