Biological evaluation of biomaterials, alkaloids from Pilocarpus microphyllus, in human neutrophils: toxicity and anti-inflammatory activity

Abstract

Event Abstract Back to Event Biological evaluation of biomaterials, alkaloids from Pilocarpus microphyllus, in human neutrophils: toxicity and anti-inflammatory activity Talita Magalhaes Rocha1, 2, Luzia Kalyne Almeida Moreira Leal1, 2, David Fernandes Lima3, José Roberto Leite4, Glauce Socorro Barros Viana1 and Cristina Dislich Ropke5 1 Federal University of Ceará, Department of Physiology and Pharmacology, Brazil 2 Federal University of Ceará, Department of Pharmacy, Brazil 3 Federal University of Vale of the São Francisco, Brazil 4 Federal University of Piauí, Brazil 5 Phytobios, Brazil Introduction: Pilocarpus microphyllus, popularly known as jaborandi, is a small tree extensively used by Brazilian industries and rich in imidazole alkaloids, such as pilocarpine, epiisopilosine (EPIL) and epiisopiloturine (EPIT). Objective: The aim of the present study was to evaluate the toxicity, anti-inflammatory and antioxidant activities of the EPIL and EPIT in human neutrophils. Methods: The EPIL (≥ 90% Purity) and EPIT (99,7% purity) were obtained from a residual product obtained during the isolation process of pilocarpine from plant leaves. Polymorphonuclear cells - PMNs (2.5 x 106cells/mL), predominantly neutrophils (90%) with cell viability of 95% (Tripan blue assay) were isolated from human blood residual product[1]. The toxicity of alkaloids (10, 50, 100 μg/mL) was evaluated by MTT test (620nm)[2] and lactate dehydrogenase (LDH) activity (340 nm)[3]. The potential of alkaloids in modulating pro-inflammatory mechanisms of human neutrophils was evaluated by two assays, degranulation and burst respiratory assays. The cells were incubated with alkaloids (1, 10, 25, 50, 100 μg/mL), indomethacin (INDO-standard drug, 36µg/mL), DMSO 1% (vehicle/control) or HBSS (sham), and then the cells were activated by addition of PMA (0,1 µM) with consequent release of myeloperoxidase (MPO) which was measured at 450nm[4],[5]. The antioxidant activity was determined by chemiluminescence (QL) assay[6]. The PMNs (5x106 cells/mL) were incubated (37° C) with EPIT or EPIL (1 - 100 μg/mL), HBSS (sham), quercetin (Querc, 25μg/mL, standard drug) or DMSO 1% (vehicle/control), plus probe luminol (lum, 280 μM). After stimulation with PMA (0,1 μM), the production of QL for 20 min was registered. The results were expressed as a mean ± standard error, and analyzed by ANOVA (followed by Tukey test, p < 0.05). Results and Discussion: The addition of EPIT or EPIL up to the highest concentration (100 µg/mL) in human neutrophils suspension did not reduce significantly the cells viability evaluated by LDH activity or MTT test. The maximal reduction of neutrophil degranulation induced by EPIT was approximately 35%, whereas EPIL inhibited by 68 %, an effect comparable to INDO, standard drug (inhibition: 62 %). Both alkaloids reduced ROS production measured as QL by lum in human neutrophils. However, EPIL (inhibition: 74 %) again was much more potent than EPIT (inhibition: 33 %), showing an effect similar to standard drug quercetin (75 %). Conclusions: The alkaloids from Pilocarpus microphyllus modulated pro-inflammatory mechanisms of human neutrophils through the MPO-H2O2-HOCl system and this effect does not seem to be related to a cytotoxic action. EPIL showed a higher anti-inflammatory effect than EPIT, and additional studies are needed to establish the precise mechanism of action of this molecule. CNPq; CAPES