Melatonin is a neurohormone that has been reported to protect against oxidative stress in vivo. This antioxidant effect is a result of several mechanisms, including free radicals scavenging and up regulation of endogenous antioxidant defenses. Furthermore, the antioxidant effect of melatonin may be modulated by its capability to alter the membrane fluidity. There are a limited number of studies in the literature about the effect of melatonin on membrane dynamics, and these studies are not in agreement with each other. In this work, the antioxidant potential of the neurohormone against hydroxyl radical (^·OH) and peroxynitrite (ONOO^-) was evaluated considering the central focus its interaction with the phosphatidylcholine liposomes. Melatonin changes in membranes phase transitions and molecular mobility were investigated through AFM and NMR. AFM technique through topographical images allows to observe the melatonin influence on supported membrane phase transition. Through NMR technique it is possible to evaluate the melatonin effect on the motional freedom of the phosphatidylcholine structure.

Egg phosphatidylcholine (EPC) and dimyristoyl phosphatidylcholine (DMPC) liposomes were used as membrane models, both prepared by dialysis method. Melatonin was incorporated into the vesicles during this process. Incorporated-melatonin efflux from the liposomes as a function of time was obtained in presence of Triton X-100 and monitored at 285nm. ^·OH was produced from the H₂O₂ + FeCl₃ + ascorbic acid system. ONOO^- from the H₂O₂/HCl+ NaNO₂ + NaOH system. Lipid peroxidation levels were detected by the TBARS method. For AFM experiments, DMPC supported layers were obtained by vesicle fusion method. The measurements were performed in liquid contact mode (HEPES-NaCl buffer, pH 7.4), in a temperature range of 16-30 °C. 81 MHz ³¹P-NMR experiments were performed at 298 K, using phosphoric acid in D₂0 as an external standard. Proton spin-lattice relaxation times (T₁) were carried out in a low field NMR spectrometer, at 27^oC, using a t range varying from 500 a 30 e⁶ ms, with 5 s of recycle delay.

The antioxidant potential of EPC liposomes-incorporated melatonin against ^·OH and ONOO^- increases about 40% and 16%, respectively, when compared with the membrane protection induced by melatonin only added in the lipid peroxidation reaction medium. At 28 °C, DMPC membranes are predominantly in liquid crystalline state, as shown in height histograms obtained from AFM images. After interacting with melatonin, an inversion of DMPC phase predominance to gel state is observed, at the same temperature. The fluidity-reorientation at polar head level of EPC liposomes was investigated in the presence of melatonin, by ³¹P-NMR and the results suggest that melatonin induces the decrease of the motional freedom related to EPC polar head groups. Changes in the proton T₁ were observed in EPC liposomes-incorporated melatonin. A decrease of about 18% in T₁ was observed after incorporation of melatonin in the liposomes, suggesting an increase of the lipid mobility.

The neurohormone can retard the gel-liquid crystalline transition process of DMPC membranes. Melatonin can increase the lipid mobility of EPC liposome, as observed by the proton T₁ studies, despite of the restriction observed in the motional freedom of EPC liposomes head polar group, by ³¹P-NMR. Considering the proton predominance in the phosphatidylcholine fatty acids, the results suggest a strongest interaction of melatonin with the liposome hydrophobic core. The fluidizing effect in this region can be associated with the antioxidant efficiency of the neurohormone against the reactive species tested.