Toxicological Aspects of the Essential Oil from Cinnamodendron dinisii.

The objective of this study was to determine cytotoxic activity, hemolytic activity, and to evaluate the ability of the essential oil from Cinnamodendron dinisii to induce DNA fragmentation of human lymphocytes. The essential oil was obtained by hydrodistillation. Cytotoxic activity was determined by the MTT method. Hemolytic activity was evaluated by spectrophotometric quantification of hemoglobin released by erythrocytes. Damage to lymphocyte DNA molecules was assessed by the Comet assay. The essential oil under study showed high cytotoxic activity on Vero cells (CC50 = 35.72 µg/mL) and induced hemolysis in both hematocrits, besides leading to the oxidation of hemoglobin released. The genotoxic activity of C. dinisii essential oil was also observed, which induced concentration-dependent DNA fragmentation of human lymphocytes and, at 50 µL/mL, it was more active than the positive control. The essential oil from C. dinisii has a toxic action, suggesting a special attention in the application of this oil to health-promoting activities; however, among its components, there are molecules with potential for future application in anticancer therapies.

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity.

Detergents are amphiphilic molecules widely used to solubilize biological membranes and/or extract their components. Nevertheless, because of the complex composition of biomembranes, their solubilization by detergents has not been systematically studied. In this review, we address the solubilization of erythrocytes, which provide a relatively simple, robust and easy to handle biomembrane, and of biomimetic models, to stress the role of the lipid composition on the solubilization process. First, results of a systematic study on the solubilization of human erythrocyte membranes by different series of non-ionic (Triton, CxEy, Brij, Renex, Tween), anionic (bile salts) and zwitterionic (ASB, CHAPS) detergents are shown. Such quantitative approach allowed us to propose Resat-the effective detergent/lipid molar ratio in the membrane for the onset of hemolysis as a new parameter to classify the solubilization efficiency of detergents. Second, detergent-resistant membranes (DRMs) obtained as a result of the partial solubilization of erythrocytes by TX-100, C12E8 and Brij detergents are examined. DRMs were characterized by their cholesterol, sphingolipid and specific proteins content, as well as lipid packing. Finally, lipid bilayers of tuned lipid composition forming liposomes were used to investigate the solubilization process of membranes of different compositions/phases induced by Triton X-100. Optical microscopy of giant unilamellar vesicles revealed that pure phospholipid membranes are fully solubilized, whereas the presence of cholesterol renders the mixture partially or even fully insoluble, depending on the composition. Additionally, Triton X-100 induced phase separation in raft-like mixtures, and selective solubilization of the fluid phase only.

Multiple stages of detergent-erythrocyte membrane interaction--a spin label study.

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate-and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5mM detergent. Between 3.2 and 10mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs; the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111).

Development and pharmacological evaluation of ropivacaine-2-hydroxypropyl-beta-cyclodextrin inclusion complex.

Ropivacaine (RVC) is an enantiomerically pure local anesthetic (LA) largely used in surgical procedures, which presents physico-chemical and therapeutic properties similar to those of bupivacaine (BPV), but associated to less systemic toxicity. This study focuses on the development and pharmacological evaluation of a RVC in 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) inclusion complex. Phase-solubility diagrams allowed the determination of the association constant between RVC and HP-beta-CD (9.46 M(-1)) and showed an increase on RVC solubility upon complexation. Release kinetics revealed a decrease on RVC release rate and reduced hemolytic effects after complexation (onset at 3.7 mM and 11.2mM for RVC and RVC HP-beta-CD, respectively) were observed. Differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and X-ray analysis (X-ray) showed the formation and the morphology of the complex. Nuclear magnetic resonance (NMR) and job-plot experiments afforded data regarding inclusion complex stoichiometry (1:1) and topology. Sciatic nerve blockade studies showed that RVC HP-beta-CD was able to reduce the latency without increasing the duration of motor blockade, but prolonging the duration and intensity of the sensory blockade (p<0.001) induced by the LA in mice. These results identify the RVC HP-beta-CD complex as an effective novel approach to enhance the pharmacological effects of RVC, presenting it as a promising new anesthetic formulation.