In Vitro Free Radical Scavenging Properties and Anti-Inflammatory Activity of Ilex paraguariensis (Maté) and the Ability of Its Major Chemical Markers to Inhibit the Production of Proinflammatory Mediators.

Ilex paraguariensis A. St. Hil. (Aquifoliaceae), popularly known as "yerba mate," has great economic and social significance for the population of Southern Latin America. This study was conducted (1) to investigate the phytochemical composition of four different standardized extracts, (2) to investigate its free radical scavenging properties, and (3) to investigate the anti-inflammatory action of I. paraguariensis and its major chemical markers. The chemical profile was achieved by Folin-Ciocalteu, by LC/DAD, and by LC/MS assays, while the antioxidant and anti-inflammatory properties were investigated, respectively, by DPPH assay and by inhibition of nitric oxide (Griess reaction) and TNF-α (ELISA). Our results demonstrated that the IA (aqueous infusion extract) showed higher amounts of total phenolic contents (266.62 ± 10.85 mg CAE·g-1 DE), the highest amounts of all six chemical markers (theobromine, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, caffeine, and rutin), and stronger antioxidant activity (EC50 = 54.4 ± 5.14 µg · mL-1). The IA extract also showed the lowest inhibition of NOx secretion (50.10 ± 8.97%) as well as inhibition of TNF-α (83.33 ± 4.01%). Regarding the chemical markers, all compounds showed strong inhibition of NOx secretion, especially theobromine, which was 200x more potent than dexamethasone. Furthermore, TNF-α secretion was also significantly decreased by THEO at 0.033 µM (22.15 ± 6.49%), NCA at 1.97 µM (27.46 ± 3.98%), CCA at 0.35 µM (39.76 ± 5.73%), CGA at 0.56 µM (23.58 ± 5.79%), CAF at 0.52 µM (26.45 ± 5.34%), and RUT at 0.16 µM (40.18 ± 3.70%). Our results suggest that I. paraguariensis and its major chemical markers have strong free radical scavenging properties as well as showed important anti-inflammatory activity and that these compounds in a plant extract may work based on several different mechanisms synergistically, resulting in moderating the immune system.

In vitro cytotoxicity of co-exposure to superparamagnetic iron oxide and solid lipid nanoparticles.

Increased production and use of different types of nanoparticles (NPs) in the last decades has led to increased environmental release of these NPs with potential detrimental effects on both the environment and public health. Information is scarce in the literature on the cytotoxic effect of co-exposure to many NPs as this concern is relatively recent. Thus, in this study, we hypothesized scenarios of cell's co-exposure to two kinds of NPs, solid lipid nanoparticles (SLNs) and superparamagnetic iron oxide nanoparticles (SPIONs), to assess the potential cytotoxicity of exposure to NPs combination. Cytotoxicity of SPIONs, SLNs, and their 1:1 mixture (MIX) in six tumor and six non-tumor cell lines was investigated. The mechanisms underlining the induced cytotoxicity were studied through cell cycle analysis, detection of reactive oxygen species (ROS), and alterations in mitochondrial membrane potential (ΔΨM). Double staining with acridine orange and ethidium bromide was also used to confirm cell morphology alterations. The results showed that SPIONs induced low cytotoxicity compared to SLNs. However, the mixture of SPIONs and SLNs showed synergistic, antagonistic, and additive effects based on distinct tests such as viability assay, ROS generation, ΔΨM, and DNA damage, depending on the cell line. Apoptosis triggered by ROS and disturbances in ΔΨM are the most probable related mechanisms of action. As was postulated, there is possible cytotoxic interaction between the two kinds of NPs.

Vasorelaxant effect of standardized extract of Cecropia glaziovii Snethl encapsulated in PLGA microparticles: In vitro activity, formulation development and release studies.

In this study, the standardized dry extract of C. glaziovii (SDE) provided a significant vasorelaxant effect after contractions induced by phenylephrine in rat aortic rings in an endothelium-dependent manner, confirming that endothelial factors are needed to stimulate this response. A vasorelaxation close to that of acetylcholine was achieved, justifying the development of new formulations for this plant material. In this context, microparticles were selected to encapsulate SDE and the double emulsion technique was considered because of the hydrophilic nature of plant material. Two experimental designs were applied. Firstly, the effect of formulation parameters on particle size, size distribution and encapsulation efficiency (EE) was evaluated. As low EE was achieved, the effect of the osmotic pressure of the external phase was evaluated in a second experimental design. The presence of the osmotic agent (NaCl) impacted positively on the EE and slower in vitro release profile was obtained, which is desired in controlled release systems. The formation of denser and less porous particle surface, which was identified by SEM analysis, contributes to explain these findings. Microstructures showed to be a promising delivery system for the C. glaziovii SDE considering that a sustained release was achieved.

Association of PLGA Microspheres to Carrier Pellets by Fluid Bed Coating: A Novel Approach towards Improving the Flowability of Microparticles.

Micro- and nanoparticles have been vastly studied due to their biopharmaceutical advantages. However, these particles generally display very weak packing and poor mechanical properties. Hereby, a new methodology is proposed to associate poorly flowing particles to macrostructures targeting the improvement of flowability and redispersibility of the particles. Cecropia glaziovii-loaded PLGA microspheres (4.59 ± 0.04 µm) were associated with carrier pellets by film coating in a top-spray fluid bed equipment. Optimal conditions were determined employing a IV-Optimal factorial design and RGB image analysis as 1% (w/v) Kollicoat® Protect as coating polymer (2:1 weight ratio of coating suspension to carrier pellets), containing 5 mg/mL microspheres (loading of 28.07 ± 1.01 mg/g). The method led to an improvement of the overall flowability. No relevant molecular interactions between PLGA microspheres and polymers were found. Microspheres detached rapidly from the surface of the pellets, without agglomeration, when exposed to hydrodynamic forces. In vitro release profiles, prior to and after fluid bed coating, showed no relevant changes in drug release rate and extent. The methodology developed is suitable for further applications when an improvement on the flow properties and redispersibility of the product is desired. We showed an easy-to-implement methodology that can be executed without significant increase in costs.

Development and effect of storage on the stability of enriched flavonoid fraction of Cecropia glaziovii-loaded PLGA nanoparticles.

Studies employing Cecropia glaziovii Snethl leaves have shown great potential in regard to their antiviral activity, mainly related to the phenolic compounds present in this species. The main goal of this work is to combine the therapeutic potential of this species with new technological strategies targeted at the development of an herbal nanoparticulate system for the preparation of a phytotherapeutic formulation. Poly (lactic-co-glycolic acid) nanoparticles containing the enriched flavonoid fraction of Cecropia glaziovii Snethl were developed through a study for the choice of preparation technique, amount of drug and surfactants used. These nanostructured systems were characterized by particle size, polydispersity, zeta potential, encapsulation efficiency and drug-loading capacity. A stability study of the formulations was conducted at room temperature over a period of 60 days. The optimal formulation that best fit the characteristics of the encapsulated material was determined. Sorbitan monooleate and the poloxamer 188 resulted in better colloidal stability, added to the organic and aqueous phases, respectively. These findings suggest that in the field of nanoparticles stability, it is important to evaluate the composition of the nanoparticulate system. This work highlights the importance of the optimization process, searching for a good formulation with suitable structural stabilization.

Manufacture and characterization of chitosan/PLGA nanoparticles nanocomposite buccal films.

Oral bioavailability of C-glycosyl flavonoid enriched fraction of Cecropia glaziovii (EFF-Cg) is limited due to its chemical complexity. The purpose of this study is the prospective evaluation of chitosan buccal films impregnated with EFF-Cg-loaded nanospheres as a drug delivery system for labial herpes treatment or for buccal administration. EFF-Cg-loaded PLGA nanospheres were prepared by double emulsion solvent evaporation technique. Nanoparticles were embedded into buccoadhesive chitosan films in different concentrations in order to obtain nanocomposite films. Films were characterized in term of morphology, mechanical properties and water absorption test. Furthermore a cytotoxicity assay was analyzed to evaluate the biocompatibility of systems. The results obtained from these analyses revealed that nanocomposite films present transparent appearance in all composition and Scanning Electron Microscopy (SEM) images show a continuous and compact section structure. Compared to the control film, mechanical responses of nanocomposites presented lower tensile strength values and no significant effect on the elongation at break. Dynamic Mechanical Analysis (DMA) tests indicated that increasing of NP concentration caused decreased stiffness and an increased of glass transition temperature values. Direct cytotoxicity test shows that nanoparticles and chitosan films not induce cytotoxic effect. Given the promising results, the study concludes that the developed buccal film impregnated with EFF-Cg-loaded nanospheres could be a promising approach for effective delivery of EFF-Cg.

LC/QTOF profile and preliminary stability studies of an enriched flavonoid fraction of Cecropia pachystachya Trécul leaves with potential antidepressant-like activity.

There is increasing interest in natural antioxidants that are candidates for the prevention of brain damage occurring in major depressive disorders. Cecropia pachystachya is a tropical tree species of Central and South America and a rich source of polyphenols, particularly flavonoids. The aim of this study was to characterize the flavonoid profile of an enriched flavonoid fraction of C. pachystachya (EFF-Cp) and evaluate the antidepressant-like effects of its acute administration in behavior, cytokine levels, oxidative stress and energy metabolism parameters. The EFF-Cp chemical characterization was performed by HPLC/DAD and LC/QTOF. The antidepressant-like effects were performed by the forced swimming test, splash test and open field test. EFF-Cp revealed 15 flavonoids, including seven new glycosyl flavonoids for C. pachystachya. Quantitatively, EFF-Cp showed isoorientin (43.46 mg/g), orientin (23.42 mg/g) and isovitexin (17.45 mg/g) as major C-glycosyl flavonoids. In addition, EFF-Cp at doses 50 and 100 mg/kg reduced the immobility time in the forced swimming test, without changing the locomotor activity and grooming time. In addition, EFF-Cp was able to prevent the oxidative damage in some brain areas. In conclusion, the results of this study suggest that EFF-Cp exerts antidepressant-like effects with its antioxidant properties.

In vitro antiherpes effect of C-glycosyl flavonoid enriched fraction of Cecropia glaziovii encapsulated in PLGA nanoparticles.

In this work is reported a novel and promising approach for the preparation of C-glycosylflavonoid enriched fraction of Cecropia glaziovii (EFF-Cg) loaded PLGA nanoparticles (NP) with antiherpes properties. The purpose of this study was to evaluate and to compare the effect of two nonionic surfactants (poloxamer 188 (PLU) and polyvinyl alcohol (PVA)), and also an emulsion stabilized by solid particles of cellulose nanocrystal (CNC) in place of surfactants. The characterization of these nanoparticles was in terms of size, polydispersity index, zeta potential, morphology, thermogravimetric analysis (TGA), loading capacity and percent yield. Since TGA analysis revealed thermo stability especially for NP-PLU, this formulation was selected for the evaluation of drug release profile, cytotoxicity and antiherpes activity. The drug delivery profile demonstrated a sustained release through the polymer structure and a significant reduction of the polymer molecular weight at 21-day period. The cytotoxicity of these nanoparticles was determined on Vero cells, and the selected formulation did not exhibit cytotoxicity even at the highest tested concentration. The results demonstrated a potential antiherpetic effect of the EFF-Cg loaded NP at 48h of testing. In summary, EFF-Cg loaded NP exhibited a promising system for the effective drug delivery in the treatment of herpes infections.

Application of Response Surface Methodology for the Technological Improvement of Solid Lipid Nanoparticles.

Solid lipid nanoparticles (SLN) are colloidal particles consisting of a matrix composed of solid (at room and body temperatures) lipids dispersed in aqueous emulsifier solution. During manufacture, their physicochemical properties may be affected by several formulation parameters, such as type and concentration of lipid, proportion of emulsifiers and amount of solvent. Thus, the aim of this work was to study the influence of these variables on the preparation of SLN. A D-optimal Response Surface Methodology design was used to establish a mathematical model for the optimization of SLN. A total of 30 SLN formulations were prepared using the ultrasound method, and then characterized on the basis of their physicochemical properties, including particle size, polydispersity index (PI) and Zeta Potential (s). Particle sizes ranged between 107 and 240 nm. All SLN formulations showed negative sigma and PI values below 0.28. Prediction of the optimal conditions was performed using the desirability function targeting the reduction of all responses. The optimized SLN formulation showed similar theoretical and experimental values, confirming the sturdiness and predictive ability of the mathematical model for SLN optimization.

Antidepressant-like effects of aqueous extract from Cecropia pachystachya leaves in a mouse model of chronic unpredictable stress.

Chronic stressful stimuli influence disease susceptibility to depression, cardiovascular, metabolic and neurodegenerative disorders. The present work investigated antidepressant and antioxidant properties of the aqueous extract from Cecropia pachystachya in a mouse model of chronic unpredictable stress (CUS). Our results indicated that acute administration of the aqueous extract (AE) from C. pachystachya (200 and 400mg/kg, p.o.) produced an antidepressant-like effect in the forced swimming test (FST). The chronic treatment with C. pachystachya extract (200mg/kg, p.o., for 14 days) prevented the depressant-like effect but not the anxiogenic effect induced by CUS. In addition to the behavioral modifications, the 14 days of CUS increased lipid peroxidation in the hippocampus (HP) and prefrontal cortex (PFC), decreased total thiol content and glutathione peroxidase activity in the HP. C. pachystachya AE administration during CUS protocol was able to prevent the oxidative damage induced by stress. However, no changes were observed in the activity of the antioxidant enzymes superoxide dismutase and catalase in the above cited brain areas after the stress protocol and treatment. Our results suggest that C. pachystachya prevented both depressive behavior and oxidative damage induced by CUS, supporting its neuroprotective potential against behavioral and biochemical dysfunctions induced by chronic stress.