## 4 Discussion  
_P. tridentatum_ is known as displaying anti-inflammatory activity, with fresh or shade-dried flowers of _P. tridentatum_ also being used in traditional medicine, in infusions, decoctions, and tonics [26] as anti-inflammatory [11; 12], diuretic, and depurative of the liver [8; 14; 27; 28]. Herein, we evaluated the effects of the leaves methanolic extract of P. _tridentatum_ using various in vivo tests in rodents, as well as computational models to assess the molecular interaction of its biological compounds with proteins involved in inflammatory cascades. Figure 6: Schematic representation of the interaction of isoquercetin ligand on the active site of PPARy (PDB 6 tsg) receptor in 3D (**a**) and 2D (**b**) structure; PPAR\(\alpha\) (PDB 2P54) receptor in three-dimensional (**c**) and two-dimensional (**d**) structure; CK2\(\alpha\) (PDB 6 TGU) receptor in three-dimensional (**e**) and two-dimensional (**f**) structure; 5-lipoxygenase-activating protein (PDB 2Q7M) receptor in 3D (**g**) and 2D (**h**) structure; interaction of quercetin and genistein ligand on the active site of prostaglandin H (2) synthase-1 (PDB 1Q4G) in three-dimensionsal (**i**,**m**, respectively) and two-dimensional (**j**,**n**, respectively) structure and CK2\(\alpha\) (PDB 6 TGU) receptor in three-dimensional (**k**,**o**, respectively) and two-dimensional (**l**,**p**, respectively) structure. Our data shows the topical application (100 mg/mL) and oral administration (100 and 300 mg/kg) of _P. tridentatum_ extract were able to significantly decrease ear and knee oedema, respectively, as well as mechanical hyperalgesia. Moreover, the molecular docking analysis evaluated the binding of the compounds (isoquercetin, quercetin, and genistein) with amino acid residues at the mentioned proteins active site and measured binding free energy parameter, inhibition constants, hydrogen bonding, and interactions. The free energy value can predict a compound's binding affinity and its ability to inhibit the protein. A negative free energy value indicates a stronger affinity between the ligands and receptors [24]. Based on the results in Table 2, isoquercetin, quercetin, and genistein had lower inhibition constant, negative free binding, and negative intermolecular interaction energies, indicating stronger affinity with the receptors and that they could potentially inhibit the activation of inflammatory cascade. Regarding the in vivo experiment of acute inflammation, the ear oedema induced by topical administration of croton oil mimics several characteristics observed in human skin diseases [16]. In the used model, the application of croton oil provokes an inflammatory reaction, specifically epidermal hyperplasia, increased vascularization, and increased inflammatory infiltrates [29]. After croton oil application, the ears' histopathological analysis revealed an increase in ear thickness (macroscopic and microscopic), as well, regarding the number of inflammatory cells. This effect was prevented by the topical application of _P. tridentatum_ extract, which did not affect ear macro- and microstructure when applied alone. Moreover, its effects were the same as Medrol application, a synthetic glucocorticoid used to treat local inflammation [30]. In the chronic inflammation model, the K/C-induced experimental osteoarthritis causes inflammation in the knee joint and subsequent chronic pain [20; 30]. The PAM data showed the animals developed mechanical hyperalgesia in the knee, confirming the correct induction of the model. This effect was sustained from the first week after induction and lasted until the end of the experiment. Importantly, our results showed both concentrations of the extract (100 and 300 mg/Kg) were able to decrease mechanical allodynia from the third week onwards (one week after the start of treatment). Moreover, the extract was more efficient than the commercial non-steroidal anti-inflammatory drug used as a control for drug administration (ibuprofen).