4.3. Formalin Test

Mice were orally administered one of six treatments (0.1 mL/10 g): (1) the vehicle, a saline solution with 0.05% Tween 80 (the control); (2) methyleugenol (1–30 mg/kg); (3) diclofenac (1–30 mg/kg); (4) ketorolac (1–30 mg/kg); (5) methyleugenol plus diclofenac; and (6) methyleugenol plus ketorolac. The 2.5% formalin test was conducted 30 min after the animals received each treatment. Formalin-induced licking behavior in mice was evaluated as previously described [30]. Briefly, each mouse was placed in an open plastic observation chamber for 30 min to become acclimated to its surroundings. Subsequently, it was removed, injected with 20 μL of 2.5% formalin into the dorsum of the right hindpaw, and returned to the chamber. The accumulated time spent licking the injected paw was taken as nociceptive behavior. Animal behavior was observed during phase I (from 1–10 min) and phase II (from 11–40 min). A brief timeline of the experimental design is given in Figure 3.

Timeline of the experimental design.

The results of this assay are expressed as the mean ± standard error of the mean (SEM) of 6–8 animals. The curve for the time course of the activity of each drug dose was constructed by plotting the licking time against the log dose. The percentage of the antinociceptive effect was calculated from the total licking time evoked during phase II, in accordance with the following equation:

The statistical differences between groups with regard to the dose-response curves were determined by one-way analysis of variance (ANOVA) followed by Dunnett’s test.

The ED₃₀ for each drug administered individual was calculated from its dose-response curve by linear regression. The isobologram was then constructed by plotting the ED₃₀ value of methyleugenol on the abscissa and the ED₃₀ value of diclofenac or ketorolac on the ordinate to obtain the theoretical additive line. The theoretical ED₃₀T value for each combination was calculated and the dose-response curve was constructed based on fractions (1/2, 1⁄4, 1⁄8m and 1⁄16) of the ED₃₀ values of the individual drugs, using a 1:1 dose ratio. Afterward, the experimental ED₃₀E value of each combination was calculated from its corresponding dose-response curve by linear regression. The difference between each ED₃₀T and the respective ED₃₀E was examined by the Student’s t-test. Interaction indices (γ) and confidence intervals for the ED₃₀ were calculated as described by Tallarida [31,32].