Comparative In Vitro Bioactivity of Traditional Aqueous and Alcoholic Preparations of Arnica (Chiliadenus glutinosus): Effects on Marine Fish Pathogens, PLHC1 Cells and Gilthead Seabream (Sparus aurata) Leucocytes

Arnica (Chiliadenus glutinosus (L.) Fourr.) is an endemic plant widely used in Spanish traditional medicine as infusions and alcoholic macerates for different ailments. Despite this use, information about the biological activity of these preparations in fish-related models is scarce. In the present study, the arnica extract bioactivity assay evaluated aqueous, ethanolic, and methanolic extracts at different doses (0, 0.001, 0.01, 0.125, 0.25, 0.5, and 1 mg mL⁻¹) to compare their antioxidant activity, effects on four marine fish pathogens (Vibrio harveyi, Vibrio anguillarum, Photobacterium damselae and Tenacibaculum maritimum), cytotoxicity on the PLHC1 tumour cell line, and their impact on immunological parameters in head-kidney leucocytes (HKLs) of gilthead seabream (Sparus aurata). All extracts showed dose-dependent antioxidant activity, while bactericidal effects depended on the solvent and were mainly observed at the highest concentrations. Ethanolic and methanolic extracts displayed clear cytotoxicity, whereas the aqueous extract showed lower toxicity and was selected for further evaluation. In the carrageenan stimulation assay, selected concentrations (0, 0.25, and 0.5 mg mL⁻¹) of the aqueous extract were tested in leucocytes stimulated with λ-carrageenan (0 and 1000 µg mL⁻¹), and respiratory burst and phagocytic activity, cell morphology, and gene expression were analysed. The aqueous extract reduced respiratory burst and phagocytic capacity in activated leucocytes and was associated with morphological signs of cell activation. It also downregulated crel and casp9 expression. These results provide a comparative view of the in vitro bioactivity of different traditional preparations of arnica and show that their biological effects strongly depend on the solvent used and the concentration tested, providing initial experimental information on their cellular effects in fish.