Preliminary study on in vitro effects of selected plant compounds on sea bass (D. labrax) head kidney leukocytes

In accordance with the aims of the Horizon2020 ParaFishControl project, the present research focuses on the development of novel prophylactic measures suitable to promote fish immune responses and limit parasite infections in sea bass (D. labrax). Plant derived compounds could be used in in vivo functional trials, however, in vitro surveys on substances activity using fish primary cell cultures or cell lines have to be performed preliminarily. In fact, this approach fulfils the 3Rs responsibilities and allows clarification on compounds basic mechanisms of action in fish. In this study, we investigated the effects of 16 plant derived candidates provided by ZF-Screens (2I,4I - dihydroxychalcone; 7 – hydroxyflavone; artemisinin; camphor (1R); diallyl sulfide; esculetin; eucalyptol; garlicin 80%; harmalol hydrochloride dihydrate; palmatine chloride; piperine; resveratrol; rosmarinic acid; sclareolide; tomatine and umbelliferone) on leukocytes from sea bass head kidney (HK) in terms of respiratory burst modulation. Cells purified by Histopaque® gradient (1119, 1077) were tested for their viability (>98%) by Trypan blue exclusion method, counted, suspended in isosmotic HBSS (5×106/ml) and incubated in triplicate microtitre wells with graded concentrations of substances in the presence of phorbol myristate acetate (PMA, 1.25 µg/ml). Reactive oxygen species (ROS) production was measured immediately by a luminol-enhanced chemiluminescence assay and expressed as kinetic and cumulative response (RLU). Results indicate that five compounds show a dose-dependent antioxidant activity by reducing the ROS release promoted by PMA stimulation. The percentages [average ± standard error] of ROS reduction were (at the substance concentration of 1.56, 3.13 and 6.25 µg/ml respectively): 41.7±6.40%, 79.7±2.81% and 99.0±2.18% for resveratrol; 50.2±4.31 %, 66.0±2.82% and 84.2±3.03% for harmalol hydrochloride dihydrate; 66.2± 2.91%, 74.5±3.21% and 80.9±2.13% for 2I,4I – dihydroxychalcone; 42.1±3.53%, 55.3±4.40% and 58.4±4.03% for rosmarinic acid; 36.1±5.00%, 42.3±3.99% and 47.2±4.69% for esculetin. Further studies will be performed to determine whether these compounds can modulate other key immune parameters involved in the effective host response against parasites, or if they can act as parasiticides. Some of these compounds could be considered as candidates for future inclusion in functional feeds due to their potential antioxidant activities in order to replace synthetic additives.