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

摘要

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 (a 90% Purity) and EPIT (99,7% purity) were obtained from a residual product obtained during the isolation process of pilocarpine from plant leaves. Polymorphonudear cells - PMNs (2.5 × 10~6cells/ml_), predominantly neutrophils (90%) with cell viability of 95% (Tripan blue assay) were isolated from human blood residual product.The toxicity of alkaloids (10,50,100 μg/mL) was evaluated by MTT test (620nm)l2! and lactate dehydrogenase (LDH) activity (340 nm). The potential of alkaloids in modulating pro-inflammatory mechanisms of human neutrophils was evaluated by two assays, degradation and burst respiratory assays. The cells were incubated with alkaloids (1,10,25,50,100 μg/mL), indomethacin (INDO-standard drug, 36μg/mL), DMS01% (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. The antioxidant activity was determined by chemiluminescence (QL) assay.The PMNs (5×10~6 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 degradation 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-H_2O_2-HOCI 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.