Screening Natural Compounds for Analgesic Effects

In 24-well plates, 5 dpf larvae were individually exposed to four natural compounds—Eugenol 99% (CAS number: 97-53-0; Alfa Aesar, Oeiras, Portugal), Thymol 100% (CAS number: 89-83-8; Merck Millipore, Algés, Portugal), Menthol 98% (CAS number: 2216-51-5; Alfa Aesar, Oeiras, Portugal), and Carvacrol 98% (CAS number: 499-75-2; London, UK)—at three different concentrations (2, 5, and 10 mg/L), for a duration of 1 h (Figure 5) [26 (link)]. These concentrations were selected based on the substances 10-times higher anaesthetic doses [68 (link)] and prepared by diluting a previously made stock solution of 89 g/L for each substance (Menthol and Eugenol, pH 7.2 ± 0.1 and 7.1 ± 0.1, respectively) in 90% alcohol [69 (link)]. After exposure, the larvae were removed and subjected to a 1 min exposure to 0.05% AA (CAS number: 64-19-7; Fisher Scientific, Oeiras, Portugal) diluted with E3 buffer, to induce pain [26 (link)]. To ensure this method works, a positive control using paracetamol (CAS number: 103-90-2; extracted from tablets using propanone and dissolved in E3 buffer) at a concentration of 0.05 mg/L was also evaluated [27 (link),70 ].