In vivo anti-inflammatory assays

In vivo experiments were performed with male SPF (specific-pathogen free) C57BL/6JUnib mice, purchased from CEMIB / UNICAMP (Multidisciplinary Center for Biological Research, SP, Brazil), weighing between 22 and 25 g. All animals were housed in vivarium under humidity (40–60%) and temperature (22 ± 2 °C) control in 12 h light-dark cycle, with access to food and water ad libitum. For the experiment, all animals were deprived of food for 8 h before oral administration. This study complied with the National Council for Animal Experimentation Control guidelines for the care and use of animals in scientific experimentation (https://www.mctic.gov.br/mctic/opencms/institucional/concea/paginas/legislacao.html), according to the Brazilian Law 11,794, of October 8, 2008. All animals were euthanized by deepening anesthesia with Ketamine and Xylazine (300 mg/kg and 30 mg/kg, respectively) and, after, submitted to cervical dislocation. The study protocol was previously approved by the Institutional Ethics Committee on Animal Research at the University of Campinas (CEUA/UNICAMP, Protocol Number 4371–1, approved on 09/23/2016).

Mice received orally (via gavage) single doses of Ese (3 or 10 mg/kg) or F3 (3 or 10 mg/kg). The negative control group received oral administration of 0.9% saline (vehicle) and 2 mg/kg dexamethasone. After 1 h, all animals received an inflammatory challenge by intraperitoneal (i.p.) injection of the flogistic agent carrageenan (500 μg/cavity) for 4 h, except the vehicle group. Next, the animals were sacrificed, their peritoneal cavities were washed and recovered to count for the total number of leukocytes and neutrophils. The results were expressed as number of neutrophils per cavity. TNF-α and CXCL2/MIP-2 levels were determined using an ELISA microplate reader. The results were expressed as pg/mL.