Bioactivities and Chemotaxonomy of Four Heracleum Species: A Comparative Study Across Plant Parts

<b>Background/Objectives</b>: This study investigates the phytochemical profile, essential oil composition, and bioactivities-including antioxidant, antimicrobial, antibio-film, and anti-quorum sensing (QS) activities-of four <i>Heracleum</i> L. species (<i>H. crenatifolium</i> Boiss, <i>H. paphlagonicum</i> Czeczott, <i>H. sphondylium</i> subsp. <i>montanum</i> Schleich. ex Gaudin, and <i>H. pastinacifolium</i> subsp. <i>incanum</i> (Boiss. & A.Huet) P.H.Davis). <b>Methods:</b> Total phenolic and flavonoid contents were quantified using the Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. Essential oils were extracted by hydrodistillation and analyzed via Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS), while Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) evaluated chemical variability among the species. Antioxidant activities were assessed using DPPH and ABTS free radical scavenging assays. Antimicrobial activity was assessed using the broth microdilution method to determine Minimum Inhibitory Concentration (MIC) values, while antibiofilm activity was evaluated using an in vitro microplate-based biofilm model against <i>Pseudomonas aeruginosa</i> PAO1. Anti-QS activity was analyzed using a disc diffusion assay with <i>Chromobacterium violaceum</i> ATCC 12472 as the reporter strain. <b>Results:</b> It was observed that the amounts of total phenolic compounds and total flavonoids were higher in root extracts than in aerial parts extracts for the four species in this study (<i>H. sphondylium</i> subsp. <i>montanum</i> excluding phenolic content). In the analysis of essential oil, it was determined that the major component in the roots was mostly myristicin, and in the fruits it was mostly octyl acetate. Phenolic and flavonoid contents were positively correlated with antioxidant activity. Methanol and <i>n</i>-hexane extracts of <i>H. pastinacifolium</i> (aerial parts) and <i>n</i>-hexane extracts of <i>H. paphlagonicum</i> (root) exhibited notable antimicrobial activity, primarily against Gram-positive bacteria, but none of the extracts showed activity against <i>Klebsiella pneumoniae</i> ATCC 13383 or <i>P. aeruginosa</i> ATCC 27853. Among methanol extracts, <i>H. pastinacifolium</i> (aerial parts) exhibited the highest antibiofilm activity (73.2%), while <i>H. paphlagonicum</i> (aerial parts) showed the highest activity among <i>n</i>-hexane extracts (75.5%). All <i>n</i>-hexane extracts exhibited anti-QS activity, whereas the methanol extracts showed no activity. <b>Conclusions:</b> These findings underscore the chemical diversity and bioactive potential of <i>Heracleum</i> species, contributing to the chemotaxonomic understanding of the genus and supporting their potential applications in medicine and industry. To our knowledge, this is the first study that reveals the antibiofilm and anti-QS properties of these <i>Heracleum</i> species.