**2. Results**

#### *2.1. NTK Inhibits Paw Edema Induced by Di*ff*erent Inflammatory Agents*

Because edema is one of the first inflammatory cardinal signs in acute responses, we investigated the e ffects of NTK on paw edema induced by di fferent triggering agents. Initially, we analyzed the effect of a single oral pre-treatment with varying doses of NTK (10, 100, and 300 mg/kg) at di fferent time points after challenge with carrageenan (Figure 2a) or dextran (Figure 2c). These treatments caused significant inhibition of the edema caused by both agents at all evaluated time-points. In addition, as demonstrated by the analysis of the area under the curve (AUC) (Figure 2b,d), no significant di fference among the doses was observed. Therefore, the lower dose (10 mg/kg) was used throughout the experiments.

**Figure 2.** *Cont.*

**Figure 2.** Time-point analysis of treatment with different NTK doses on paw edema formation induced by carrageenan (**a**) or dextran (**c**). These data are also represented as area under the curve (AUC) ((**b**,**d**), respectively). a4 = *p* < 0.0001 vs. saline; b3 = *p* < 0.001 vs. NTK 100 mg/kg group; c4 = *p* < 0.0001 vs. NTK 10 mg/kg group. Statistical significance was determined with two-way ANOVA (**<sup>a</sup>**,**<sup>c</sup>**) and post hoc Tukey test.

Histamine and arachidonic acid (AA)-derived lipid mediators are crucially involved in edema formation during inflammatory conditions [16]. Therefore, we investigated the participation of these mediators on NKT-mediated edema inhibition. As shown in Figure 2a, a single oral pre-treatment with NTK (10 mg/kg) inhibited paw edema formation at 30 and 60 min after challenge in comparison with vehicle pre-treatment. Promethazine (PTZ) (6 mg/kg), a histamine receptor antagonist, caused similar inhibition of the edema, suggesting that NTK-mediated antiedematogenic effects may involve, at least partially, inhibition of histamine vascular actions. Accordingly, the most significant antiedematogenic effect of NTK was observed in AA-stimulated mice (Figure 3b). Moreover, indomethacin (IND), an NSAID used as a positive control, caused comparable inhibition at all evaluated time-points, suggesting that the antiedematogenic effects of NTK might involve arachidonic acid metabolism inhibition.

**Figure 3.** Potential mechanisms associated with NTK-mediated paw edema inhibition. The antiedematogenic effect is expressed as a percentage of edema induced by histamine (**a**) or arachidonic acid (**b**). a4 = *p* < 0.0001 vs. saline; a3 = *p* < 0.001 vs. saline and a2 = *p* < 0.01 vs. saline. Statistical significance was determined with two-way ANOVA and post hoc Tukey test.

#### *2.2. The E*ff*ects of NTK on Carrageenan-Induced Peritonitis*

The intraperitoneal administration of carrageenan (1%) induced an intense influx of leukocytes associated with elevated myeloperoxidase (MPO) and albumin levels into the peritoneal cavity of mice 4 h after challenge (Figure 4a–c), indicating the development a peritoneal inflammation associated with leukocyte migration and activation, and increased vascular permeability. The animals treated with NTK (10 mg/kg) or indomethacin (25 mg/kg) presented a significant reduction in all these inflammatory parameters, demonstrating that the treatments exerted anti-inflammatory effects in mice.

**Figure 4.** The effects of NTK on carrageenan-induced peritonitis. (**a**) The number of total leukocytes; (**b**) concentrations of myeloperoxidase (MPO), and (**c**) concentrations of albumin in the peritoneal fluid of mice. a4 = *p* < 0.0001 vs. saline and a3 = *p* < 0.001 vs. saline. Statistical significance was determined with one-way ANOVA and post hoc Tukey test.

#### *2.3. NTK Inhibits Leukocyte Recruitment and Cytokine Production on Carrageenan-Induced Pleurisy*

Interleukin (IL)-1β and Tumor Necrosis Factor (TNF)-α have critical roles in the development of acute inflammation [17]. The group of mice challenged with carrageenan and pre-treated with NTK (10 mg/kg) presented a significantly reduced number of total leukocytes associated with lower concentrations of IL1-β and TNF-α in the pleural lavages in comparison with untreated animals (Figure 5a–c), indicating that inhibition of cytokine production is potentially related with NTK anti-inflammatory mechanisms.

**Figure 5.** The effects of NTK on carrageenan-induced pleurisy. (**a**) The number of total leukocytes; (**b**) concentrations of Interleukin (IL)-1β, and (**c**) concentrations of Tumor Necrosis Factor (TNF)-α in the pleural lavages of mice. a4 = *p* < 0.0001 vs. saline and a2 = *p* < 0.01 vs. saline. Statistical significance was determined with one-way ANOVA and post hoc Tukey test.

#### *2.4. Investigation of NTK-Mediated COX-2 and H1 Inhibition in Silico*

In the present study, the docking procedure was validated by removing and repositioning each ligand into the binding site. The root-mean-square deviation (RMSD) resulting from the X-ray crystallography structures found conformations of 0.87Å and 0.32Å (for COX-2 and histamine, respectively), demonstrating convergence in the calculated complex formation and attesting the performance of the docking protocol. The results of docking analysis determine how closely the lowest energy pose (binding conformation) from the COX-2 enzyme and the H1 receptor.

The docked ligands showed score energies of −8.6 kcal/mol to diclofenac and −8.0 kcal/mol for nootkatone in the COX-2 binding site. Considering the H1 receptor, the score energies were −11.5 kcal/mol for doxepin and −8.1 kcal/mol for nootkatone. Of note, these data were useful to analyze the hydrophobic and polar interactions in the binding site of the complexes. The best conformation of nootkatone into COX-2 enzyme and H1 receptor binding sites indicate a favorable interaction with both proteins (Figure 6a,b), as well as their respective control ligands diclofenac and doxepin, suggesting a link between COX-2 and H1 inhibition and the NTK-mediated anti-inflammatory effects (Figure 7a–d).

**Figure 6.** The binding poses of best stability between NTK and diclofenac into the cyclooxygenase (COX)-2 enzyme binding site (**a**) and between NTK and doxepin into the binding site of the H1 receptor (**b**).

**Figure 7.** Maps of amino acid residues within the binding pocket of COX-2 (**<sup>a</sup>**,**b**) and the H1 receptor (**<sup>c</sup>**,**d**). The interactions of NKT (**<sup>a</sup>**,**<sup>c</sup>**), Diclofenac (**b**), and Doxepin (**d**) with these amino acids is illustrated.

#### *2.5. The E*ff*ects of NTK on Cotton Pellet-Induced Granuloma*

The cotton pellet-induced granuloma model was used to evaluate the effects of NTK on chronic inflammation. Administration of NTK (10 mg/kg) to mice decreased both granuloma weight (Figure 8a) and concentration of proteins in the homogenates (Figure 8b) in comparison with untreated mice, indicating that the treatment inhibited, at least partially, the inflammatory response in this model.

**Figure 8.** The effects of NTK treatment on cotton pellet-induced granuloma in mice. (**a**) Final weight of the granuloma. (**b**) Protein concentration in the homogenates. a4 = *p* < 0.0001 vs. saline and a1 = *p* < 0.05 vs. saline. Statistical significance was determined with T test.
