*4.3. Evaluation of the Anti-Inflammatory Activity*

The effects of NTK on acute inflammation were evaluated using the following mice models: carrageenan-induced peritonitis, carrageenan-induced pleurisy, and paw edema induced by either carrageenan, dextran, histamine, or arachidonic acid (AA). In all these protocols, the mice received a single oral treatment one hour before the challenge. Each challenge was performed through a single administration of an inflammatory agen<sup>t</sup> (carrageenan, histamine, dextran, or AA) to the mice, triggering an acute inflammatory response, which was monitored immediately after the challenge at specific time-points, according to standard protocols. The paw edema models were used to evaluate the local inflammatory response, while pleurisy and peritonitis were used to analyze systemic inflammatory parameters. In the chronic inflammation protocol, the animals were treated daily for ten consecutive days, after surgically receiving a persistent stimulus (cotton pellets). This protocol induces the formation of granuloma, which is considered as a parameter of chronic inflammation.

#### 4.3.1. Evaluation of the Antiedematogenic Activity

The animals (*n* = 6 per group) were randomly assigned into groups and orally treated with NTK (10, 100, or 300 mg/kg), vehicle (0.9% saline, negative control) or control drugs (promethazine, 10 mg/kg or indomethacin, 25 mg/kg) 1 h before challenge with 20 μL of carrageenan, dextran, histamine, or AA at 1% (*w*/*v*). Each inflammatory agen<sup>t</sup> used in the challenge was administered in the hind right paw and an equal volume of saline was administered in the hind left paw. The volume of both paws was measured at different time-points according to the type of challenge, using a plethysmometer and the results were expressed as a percentage of edema, considering the difference between the hind and left paw of the untreated group as 100% [19,21,22].
