Efeito anti-inflamatório e antinociceptivo de alcaloides imidazólicos de Pilocarpus microphyllus: estudo in vitro, in vivo e in silico

Abstract

Medicinal plants have been shown to be promising as a source of alternative therapies to modulate the inflammatory response, including inflammatory pain. Pilocarpus microphyllus, popularly known as jaborandi, is a species native to the Northeast and widely exploited by the pharmochemical industry for the extraction of pilocarpine, a cholinergic imidazole alkaloid. Its leaves also have other alkaloids, such as epiisopilosine (EPIL) and epiisopiloturine (EPIT), obtained from industrial waste. Preliminary studies determined the anti-inflammatory and antinociceptive action of epiisopiloturine in mice. On the above, the aim of the present study was to investigate the anti-inflammatory and antinociceptive potential of the alkaloids EPIT and EPIL. For this, experimental models of inflammation in human neutrophils, mechanical hyperalgesia induced by carrageenan in mice (Von Frey electronic) and in silico study were used. In assessing the anti-inflammatory potential, both EPIT as EPIL (1 - 100 μg/mL) inhibited neutrophils degranulation (5 x 106 cells/mL) measured by myeloperoxidase activity in activated neutrophils by 1 μM fMLP (CI40 = 45.2 and 62.3 μg / mL, respectively) or 0.1 μM PMA (CI40 = 14.3 and 29.5 μg / mL, respectively). This effect was related to the modulation of the oxidative metabolism, in which EPIT and EPIL inhibited up to 44 and 74%, respectively, the production of reactive oxygen species (ROS) determined by chemiluminescence - luminol probe. In addition, it was observed that EPIT and EPIL also reduced the Ca2+ influx in the cell by 46 and 55%, respectively, and inhibited the phosphorylation of the cytosolic NF-κB, thus preventing its translocation to the nucleus. It is noteworthy that the anti-inflammatory and antioxidant actions of EPIT and EPIL occurred without affecting cel viability (MTT test and lactate dehydrogenase enzyme activity). In the study of antinociceptive activity, EPIT and EPIL (0.3 and 1 mg / kg, i.p.) inhibited carrageenan-induced inflammatory hypernociception (300 μg / paw) in mice and significantly reduced MPO levels by up to 87 and 74%, respectively, an indirect measure of neutrophil migration. Pretreatment of mice with EPIT (1 mg / kg, i.p.) and EPIL (0.3 mg / kg, i.p.) reduced the expression of COX-2 in subplantar carrageenan-induced tissue in mice. EPIT and EPIL were able to establish strong interactions with amino acid residues considered essential for binding to the COX-1 and COX-2 isoforms. The results obtained suggest that the anti-inflammatory and antinociceptive activity of imidazole alkaloids, EPIT and EPIL, are related, at least in part, to a modulation in the activation and accumulation of the innate immune system cells (neutrophils) in inflammatory focus, possibly by reducing intracellular calcium influx, activation of NF-кB and COX-2 expression.