4.10.3. Tail Immersion Test

The tail immersion test was performed according to a previously established method to estimate the central analgesic properties of MESF. The animals were randomly divided and treated as described for the acetic acid-induced writhing test. After drug administration, 2–3 cm of the tail from each mouse was placed in warm water at a constant temperature of 55 ± 0.5 °C, and the abrupt removal of the tail from the warm water is considered to represent a pain reaction. Afterward, the total tail immersion time and tail deflection at zero, thirty, sixty, ninety, and one hundred twenty minutes after the administration of all test samples were noted as response times; to inhibit tail tissue impairment in mice, a cutoff period of 20 s was used for the placement of the tail in warm water [55]. At the end of the experiment, the mice were euthanized using diethyl ether anesthesia. The percentage maximum possible effect (%MPE) for individual mice was measured according to the following formula: