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Green Coffea arabica L. seed oil (GCO) has been used as an active cosmetic ingredient in many skin care products, due to its composition and balance of fatty acids. On the other hand, while roasted coffee oil (RCO) is mainly used for imparting aroma in the food industry, there is no data available to suggest its safety in cell-based model systems. In this context, the present study aims to evaluate the chemical composition of GCO, RCO, and their correspondent polar fractions (PFs); and assess their cytotoxicity and antioxidant potential in vitro. RCO and RCO PF exhibited significantly higher amounts of phenolic compounds, when compared to both GCO and GCO PF. In the DPPH assay, after 5 min of incubation, RCO inhibited about 80% of radicals, while GCO only achieved half of this activity. Similar results were also obtained for their PFs. Upon exposure to GCO, no cytotoxic effects were observed, in fact, there were slight increments in cell proliferation. Nevertheless, cell exposure to RCO led to significant decreases in cell viability. Increases in the concentration of coffee oil PFs were associated with correspondent relevant increased cytotoxicity. Upon hydrogen peroxide-induced oxidative stress, neither GCO nor RCO treatment were effective in protecting cells.

The species Cyathea phalerata Mart. is a tree fern, commonly known as “xaxim”, which is found in tropical and subtropical areas of Brazil. The present study investigated the mechanisms related with the vasorelaxant effects of an Ethyl Acetate Fraction (EAF) obtained from C. phalerata in rats’ thoracic aorta rings. In pre-contracted vessels, EAF (0.1–1000 μg/mL) caused a concentration-dependent relaxation. The endothelium denudation, the nitric oxide (NO) synthase and guanylyl cyclase inhibitor reduced the vasodilation, indicating the participation of NO/cGMP pathway in its effect. The relaxation of EAF was abolished in the absence of extracellular Ca2+ and was significantly decreased in the presence of Ca2+ entry blocker, suggesting that Ca2+ influx plays an important role in EAF effect and probably in eNOS activity. However, the PI3K/Akt pathway is not responsible for eNOS phosphorylation/activation. The vasodilator effect of EAF was partially inhibited by KCl 40 mM and almost totally abolished with L-NOARG + KCl 40 mM, indicating also the role of hyperpolarization in its effect. Calcium activated K+ channels are not involved in the EAF-induced hyperpolarization. The COX inhibitor, indomethacin, slightly reduced the vasodilation induced by EAF. In addition, EAF did not alter the relaxant effects of NO-donor, indicating that the relaxant activity cannot be attributed to free radical-scavenging properties. In conclusion, the present study showed that the EAF, causes an endothelium-dependent vasorelaxant effect in aorta that mainly involves the NO-cGMP pathway, hyperpolarization and prostanoids. The vasorelaxant activity of EAF can be attributed to the occurrence of polyphenol compounds., Graphical abstract Image 1, Highlights • An ethyl acetate fraction obtained from Cyathea phalerata induces vasodilatation. • EAF induced relaxation by an endothelium-dependent mechanism. • EAF increases the NO production and hyperpolarization that evoke vasodilation.

Jaboticaba is a fruit from a native tree to Brazil,Plinia peruviana. Jaboticaba peels are an important source of antioxidant molecules such as phenolic compounds. This study aimed to evaluatein vitrothe activity of a hydroalcoholic extract of jaboticaba fruit peels (HEJFP) in wound healing processes and antioxidant activity in murine fibroblasts (L929 cell line). HEJFP concentrations (0.5, 1, 5, 10, 25, 50, 100, and 200 µg/mL) were tested in MTT assay and cell proliferation was verified at 100 µg/mL after 24 h and at 25, 50, and 100 µg/mL after 48 h of extract exposure. Evaluation of antioxidant activity was performed at 0.5, 5, 25, 50, and 100 µg/mL HEJFP concentrations. Cell treatment with HEJFP at 25, 50, and 100 µg/mL for 24 h followed by H2O2exposure for 3 h showed a strong cytoprotective effect.In vitroscratch wound healing assay indicated that none of tested HEJFP concentrations (0.5, 5, 25, 50, and 100 µg/mL) were capable of increasing migration rate after 12 h of incubation. These results demonstrate a positive effect of HEJFP on the wound healing process on L929 fibroblasts cell line, probably due to the antioxidant activity exhibited by phytochemicals in the extract.

Moderate wine intake (i.e., 1-2 glasses of wine a day) is associated with a reduced risk of morbidity and mortality from cardiovascular disease. The aim of this study was to evaluate the anti-atherosclerotic effects of a nonalcoholic ethyl acetate fraction (EAF) from a South Brazilian red wine obtained from Vitis labrusca grapes. Experiments were carried out on low-density lipoprotein (LDL) receptor knockout (LDLr⁻/⁻) mice, which were subjected to a hypercholesterolemic diet and treated with doses of EAF (3, 10, and 30 mg/kg) for 12 weeks. At the end of the treatment, the level of plasma lipids, the vascular reactivity, and the atherosclerotic lesions were evaluated. Our results demonstrated that the treatment with EAF at 3 mg/kg significantly decreased total cholesterol, triglycerides, and LDL plus very low-density lipoprotein levels compared with control hypercholesterolemic mice. The treatment of mice with EAF at 3 mg/kg also preserved the vasodilatation induced by acetylcholine on isolated thoracic aorta from hypercholesterolemic LDLr⁻/⁻ mice. This result is in agreement with the degree of lipid deposit on arteries. Taken together, the results show for the first time that the lowest concentration of an EAF obtained from a red wine produced in southern Brazil significantly reduced the progression of atherosclerosis in mice.

O estudo das interações entre células e seu substrato na engenharia de tecidos é de grande importância para determinação das propriedades biológicas dos implantes. A adesão das células ao substrato influencia na morfologia, na proliferação e na viabilidade celular. Neste trabalho, foram avaliadas adesão, proliferação e viabilidade de fibroblastos de camundongo, linhagem L929, em suporte biopolimérico. Utilizaram-se membranas de celulose bacteriana como substrato para a avaliação in vitro. As células foram cultivadas sobre essas membranas em meio Dulbecco’s Modified Eagle Medium (DMEM), suplementado com 10% de Soro Bovino Fetal (SBF), a 37°C, contendo 5% CO2. Para monitorar e avaliar o comportamento dessas células em distintos tempos de cultivos nas membranas, escolheram-se diferentes parâmetros celulares. Com base nessa experiência, observaram-se diferenças morfológicas significativas nas células. Os resultados evidenciaram boa adesão, crescimento, proliferação e viabilidade das células nas membranas utilizadas.

O estudo das interações entre células e seu substrato na engenharia de tecidos é de grande importância para determinação das propriedades biológicas dos implantes. A adesão das células ao substrato influencia na morfologia, na proliferação e na viabilidade celular. Neste trabalho, foram avaliadas adesão, proliferação e viabilidade de fibroblastos de camundongo, linhagem L929, em suporte biopolimérico. Utilizaram-se membranas de celulose bacteriana como substrato para a avaliação in vitro. As células foram cultivadas sobre essas membranas em meio Dulbecco’s Modified Eagle Medium (DMEM), suplementado com 10% de Soro Bovino Fetal (SBF), a 37°C, contendo 5% CO2. Para monitorar e avaliar o comportamento dessas células em distintos tempos de cultivos nas membranas, escolheram-se diferentes parâmetros celulares. Com base nessa experiência, observaram-se diferenças morfológicas significativas nas células. Os resultados evidenciaram boa adesão, crescimento, proliferação e viabilidade das células nas membranas utilizadas.

A identificação, extração, isolamento e caracterização de princípios ativos vegetais que modulam a viabilidade celular são de grande interesse clínico e farmacológico. Testes fitoquímicos têm demonstrado a presença de vários compostos oriundos dos metabolismos primário e secundário da Aloe vera L. A aloína é uma antraquinona isolada do parênquima clorofiliano da A. vera L que, sabe-se, apresenta atividade biológica. A atividade antitumoral da aloína e do extrato do parênquima clorofiliano foi avaliada, in vitro, com a utilização de células de melanoma de camundongo da linhagem B16-F10. Nessa análise, células incubadas em atmosfera com 5% de CO2 foram tratadas em diferentes concentrações de aloína e de extrato do parênquima, a 37ºC. A aloína e o extrato alteraram a estabilidade celular, exercendo efeito modulador sobre a viabilidade das células tumorais. Os dados experimentais foram representados por meio de um modelo matemático. Neste estudo, verificou-se que o extrato do parênquima clorofiliano é 2,3 vezes mais tóxico que a aloína.

The formation of embryonic blood vessels, defined as vasculogenesis, is a complex morphogenetic process ultimately related to tubulogenesis, carried out from in situ differentiation of mesoderm-recruited or proliferated progenitor endothelial cells (angioblasts) to endothelial cells for structuring a primary vascular plexus. Subse‐ quent events involving apoptosis versus cell survival (remodeling) in the vessel network stabilizes the primordial microvasculature, which through the angiogenesis process yields new capillaries by sprouting from the preexisting ones. Methylxan‐ thinic alkaloids such as caffeine (compounds present in a number of beverages consumed worldwide) exert some well-known effects upon heart and other cardio‐ vascular structures, in part, by negatively interplaying with phosphodiesterase (PDEs) enzymes. Once caffeine as well as Ilex paraguariensis (yerba mate) infusion extract have shown to enhance the vessel formation (vasculogenesis and angiogene‐ sis), we discuss the impact afforded by I. paraguariensis constituents on the (PDEsrelated) quantities and stability of Protein kinase A (PKA) and Protein kinase C (PKC) enzymes. Besides, the text reflects on a suggested dual roles displayed by PKA and PKC enzymatic pathways in the developmental angiogenic events.

Croton celtidifolius Baill commonly known as "sangue-de-adave" is a tree found in the Atlantic Forest of south of Brazil, mainly in Santa Catarina. The bark and leaf infusions of this medicinal plant have been popularly used for the treatment of inflammatory diseases. In this study we evaluated the anti-inflammatory and antioxidant properties of crude extract (CE), aqueous fraction (AqF), ethyl acetate fraction (EAF), butanolic fraction (BuF) and catechin, gallocatechin and sub-fractions, 19SF, 35SF and 63SF that contained a mixture of proanthocyanidins and were derived from the EAF fraction. The CE, AqF, EAF, BuF, catechin and sub-fractions 35SF and 63SF reduced paw edema induced by carrageenan. The CE, fractions, sub-fractions and isolated compounds showed antioxidant properties in vitro, all were able to scavenge superoxide anions at a concentration of 100 microg ml(-1). The EAF, catechin and gallocatechin were most effective in the deoxyribose assay, IC50 0.69 (0.44-1.06), 0.20 (0.11-0.39), 0.55 (0.28-1.08) microg x ml(-1) respectively. The CE and other fractions and sub-fractions inhibited deoxyribose degradation up to 1 microg x ml(-1). In the hydrophobic system only AqF did not show lipid peroxidation inhibition. The CE, other fractions, sub-fractions and isolated compounds inhibited lipidid peroxidation only at a concentration of 100 microg x ml(-1). In summary, this study demonstrates that Croton celtidifolius bark has significant anti-inflammatory and antioxidant activity.

We investigated the antioxidant activity of phenylpropionic acids--caffeic (CAF), ferulic (FER), para-coumaric (COU) and cinnamic (CIN)--and phenolic acids and related compounds--gallic (GAL), methyl gallate (meGAL), vanillic (VAN) and gentisic (GEN)--using visible spectroscopy, inhibition of nitroblue tetrazolium (NBT) reduction, and electrochemical methods including cyclic voltammetry and potentiometry. In the spectroscopic assays, only CAF, GAL and meGAL were able to inhibit NBT reduction. The same compounds showed the lowest oxidation potentials (Epa) and the highest redox potentials deltaE) in the cyclic voltammetric and potentiometric studies, respectively. In addition, it was observed that the greater the number of hydroxyls linked to the aromatic ring, the greater was the antioxidant activity of the analysed compounds. The correlations of Spermann--used to compare the methods between themselves and the methods with the relationship structure-antioxidant activity--were r = -0.9762 for the cyclic voltammetric-potentiometric methods. r = 0.8333 for the inhibition of NBT reduction-potentiometric methods and r = -0.8095 for the inhibition of NBT reduction-cyclic voltammetric methods. The correlations for cyclic voltammetric, potentiometric and inhibition of NBT reduction methods-number of hydroxyls linked to the aromatic ring were r = -0.9636, 0.9636 and 0.9142, respectively. These findings indicate that the electrochemical methods together with spectroscopic studies are a good tool to evaluate the antioxidant activity of substances.

Electrospun materials have been widely explored for biomedical applications because of their advantageous characteristics, i.e., tridimensional nanofibrous structure with high surface-to-volume ratio, high porosity, and pore interconnectivity. Furthermore, considering the similarities between the nanofiber networks and the extracellular matrix (ECM), as well as the accepted role of changes in ECM for hernia repair, electrospun polymer fiber assemblies have emerged as potential materials for incisional hernia repair. In this work, we describe the application of electrospun non-absorbable mats based on poly(ethylene terephthalate) (PET) in the repair of abdominal defects, comparing the performance of these meshes with that of a commercial polypropylene mesh and a multifilament PET mesh. PET and PET/chitosan electrospun meshes revealed good performance during incisional hernia surgery, post-operative period, and no evidence of intestinal adhesion was found. The electrospun meshes were flexible with high suture retention, showing tensile strengths of 3 MPa and breaking strains of 8-33%. Nevertheless, a significant foreign body reaction (FBR) was observed in animals treated with the nanofibrous materials. Animals implanted with PET and PET/chitosan electrospun meshes (fiber diameter of 0.71 ± 0.28 µm and 3.01 ± 0.72 µm, respectively) showed, respectively, foreign body granuloma formation, averaging 4.2-fold and 7.4-fold greater than the control commercial mesh group (Marlex). Many foreign body giant cells (FBGC) involving nanofiber pieces were also found in the PET and PET/chitosan groups (11.9 and 19.3 times more FBGC than control, respectively). In contrast, no important FBR was observed for PET microfibers (fiber diameter = 18.9 ± 0.21 µm). Therefore, we suggest that the reduced dimension and the high surface-to-volume ratio of the electrospun fibers caused the FBR reaction, pointing out the need for further studies to elucidate the mechanisms underlying interactions between cells/tissues and nanofibrous materials in order to gain a better understanding of the implantation risks associated with nanostructured biomaterials.

Objectives In this study, we evaluated the effect of the proanthocyanidins-rich fraction (PRF) obtained from Croton celtidifolius bark in an experimental animal model of spinal cord injury and cell death induced by glutamate. Methods Experiments were conducted using adult male Wistar rats (10 weeks old and weighing 270–300g). Experimental groups were randomly allocated into the following groups: spinal cord injury (SCI) + vehicle group: rats were subjected to SCI plus intraperitoneal administration of vehicle (saline 10 ml/kg); SCI + PRF: rats were subjected to SCI plus intraperitoneal administration of PRF (10 mg/kg) at 1 and 6 h after injury and sham operated. Key findings The treatment with the proanthocyanidin-rich fraction significantly improved not only motor recovery and grip force but also H2O2 or glutamate-induced cell death and reactive oxygen species generation induced by glutamate in dorsal root ganglion cells. In this study we demonstrate that the neuroprotective effect triggered by the proanthocyanidins-rich fraction appears to be mediated in part by the inhibition of N-methyl-D-aspartate-type glutamate receptors. Conclusions Taken together, our results demonstrate that PRF treatment ameliorates spinal cord injury and glutamatergic excitotoxicity and could have a potential therapeutic use.

Objectives The present work aimed to investigate the effect of (PhSe)2 on cardiovascular age-related oxidative stress in hypercholesterolemic mice. Methods To this end, LDL receptor knockout (LDLr−/−) mice, 3 months (young adult) and 12 months (middle-aged) old, were orally treated with (PhSe)2. Key findings Hypercholesterolemia, regardless of age, impaired the mitochondrial antioxidant defence in the cardiac tissue, which was characterized by a decline in mitochondrial aortic glutathione (GSH) levels and increased reactive oxygen species production in the heart. (PhSe)2 treatment improved GSH levels, thioredoxin reductase (TRxR) and GSH reductase (GR) activity, and decreased malondialdehyde levels in the heart of young adult LDLr−/− mice. Moreover, (PhSe)2 increased GPx activity in both age groups, and GR activity in the aorta of middle-aged LDLr−/− mice. Conclusions Therefore, (PhSe)2 enhances the antioxidant defences in the cardiovascular system of LDLr−/− mice, which could explain its success as an anti-atherogenic compound.

The purpose of this study was to evaluate hybrid poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/chitosan nanofibrous mats as scaffolds for skin engineering. In vitro studies were carried out to test the potential of the scaffolds for fibroblasts adhesion, viability, and proliferation (L929 cell line). The in vivo performance was also studied in a full-thickness wound healing model. PHBV/chitosan 4:1 (w/w) exhibited a higher in vitro biocompatibility and a better ability for cell adhesion and growth, compared to PHBV/chitosan 2:3 (w/w). The in vivo assay also revealed the better performance of this scaffold, improving the wound healing process in rats.

Objectives This paper aims to evaluate the anti-tumour properties of elatol, a compound (sesquiterpene) isolated from algae Laurencia microcladia. Methods In-vitro and in-vivo anti-tumour properties of elatol were investigated using: MTT assays to assess the cytotoxic effects; flow cytometry analysis to examine the cell cycle and apoptosis; Western blot analysis for determination of the expression of cell cycle and apoptosis proteins; and study of in-vivo tumour growth in mice (C57Bl6 mice bearing B16F10 cells). Key findings Elatol exhibited a cytotoxic effect, at least in part, by inducing cell cycle arrest in the G1 and the sub-G1 phases, leading cells to apoptosis. Western blot analysis demonstrated that elatol reduced the expression of cyclin-D1, cyclin-E, cyclin-dependent kinase (cdk)2 and cdk4. A decrease in bcl-xl and an increase in bak, caspase-9 and p53 expression was also observed. In the in-vivo experiment, treatment with elatol was able to reduce tumour growth in C57Bl6 mice. Conclusions Elatol promotes a delay in the cell cycle, probably in the G1/S transition, activating the apoptotic process and this could be responsible, at least in part, for the in-vivo effects observed. Taken together, the in-vitro and in-vivo experiments suggested that elatol has anti-tumour properties. Further studies should be conducted to clarify the mechanism of action.

Novel hybrid fibrous membranes with potential application on the biomedical field were successfully developed via electrospinning of blend solutions of two biocompatible and biodegradable polymers, poly (3-hydroxybutyrate-co-hydroxyvalerate) [PHBV] and chitosan. The effect of solvent, using different proportions of trifluoracetic acid (TFA) and 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP), PHBV/chitosan ratio, and chitosan molecular weight on solution spinnability, fiber morphology and size, and in vitro degradation were investigated. Continuous nanofiber structures were obtained from pristine PHBV to a PHBV/chitosan ratio of 2:3 (w/w), at 10% (w/v) total polymer concentration. In general, main fiber average diameters increased as chitosan content and molecular weight increased and decreased with the decrease of HFIP content in the solvent. In vitro dissolution tests revealed that the hybrid fibers show much higher degradation rates than the neat PHBV.

Objectives The aim of the present study was to evaluate the possible neurobehavioural effects in rats of the proanthocyanidin-rich fraction (PRF) isolated from the bark of Croton celtidifolius (Euphorbiaceae). Methods Adult Wistar rats were treated with the PRF (0.3–30 mg/kg) and evaluated in different behavioural paradigms classically used for the screening of drugs with psychoactive effects. Key findings Acute intraperitoneal (i.p.) administration of PRF decreased spontaneous locomotor activity (open field arena and activity cage), enhanced the duration of ethyl ether-induced hypnosis, increased the latency to the first convulsion induced by pentylenetetrazole (60 mg/kg, i.p.) and attenuated apomorphine-induced (0.5 mg/kg, i.p.) stereotyped behaviour. In lower doses, PRF (0.3 or 3 mg/kg, i.p.) increased the frequency of open arm entries in the elevated plus-maze test. Conclusions The present findings suggest that the systemic administration of PRF induces a wide spectrum of behavioural alterations in rats, consistent with the putative existence of hypnosedative, anticonvulsant and anxiolytic compounds.

Roots of Wilbrandia ebracteata Cogn., Cucurbitaceae, used in folk medicine for treatment of rheumatic disease, are rich in cucurbitacins. Dihydrocucurbitacin B is the most abundant cucurbitacin while cucurbitacin B is a minor component. A reverse-phase HPLC system was developed for simultaneous quantitative assay of these cucurbitacins in the roots. The optimised experimental conditions were acetonitrile/H(2)0 40:60, flow-rate 1.2 mL/min., detection at 230 nm and isocratic elution. A variety of sample preparation modes were tested and the extraction with dichloromethane under reflux gave better results. The validation process included linearity, accuracy, repeatability and intermediate precision. The calibration curve of dihydrocucurbitacin B was linear from 40.00 to 400 μg/mL, the recovery was 95.5±3.01%, the intermediate precision was found to be 1.64% and the repeatability varied between 1.30 to 2.05%. The calibration curve of cucurbitacin B was linear from 4.00 to 240 μg/mL, intermediate precision was found to be 2.29% and repeatability varied between 1.03 to 2.95%. Analysis of the same specimen of W. ebracteata every year from 2002 to 2005 revealed a great rise on the cucurbitacin B concentration after the root was attacked by an herbivore.

A identificação, extração, isolamento e caracterização de princípios ativos vegetais que modulam a viabilidade celular são de grande interesse clínico e farmacológico. Testes fitoquímicos têm demonstrado a presença de vários compostos oriundos dos metabolismos primário e secundário da Aloe vera L. A aloína é uma antraquinona isolada do parênquima clorofiliano da A. vera L que, sabe-se, apresenta atividade biológica. A atividade antitumoral da aloína e do extrato do parênquima clorofiliano foi avaliada, in vitro, com a utilização de células de melanoma de camundongo da linhagem B16-F10. Nessa análise, células incubadas em atmosfera com 5% de CO2 foram tratadas em diferentes concentrações de aloína e de extrato do parênquima, a 37ºC. A aloína e o extrato alteraram a estabilidade celular, exercendo efeito modulador sobre a viabilidade das células tumorais. Os dados experimentais foram representados por meio de um modelo matemático. Neste estudo, verificou-se que o extrato do parênquima clorofiliano é 2,3 vezes mais tóxico que a aloína.

Proanthocyanidins are condensed tannins present in fruits, vegetables, and flowers, consumed in the human diet. These compounds are believed to decrease coronary heart disease. The present study was designed to investigate the relaxing effects of a proanthocyanidin-rich fraction (PRF) obtained from Croton celtidifolius BAILL (Euphorbiaceae) barks in rat mesenteric arterial bed (MAB) and isolated mesenteric artery (MA). In the MAB pre-contracted with phenylephrine (Phe), PRF (0.1 –100 μ g) induced a concentration-dependent relaxation of 73% (compared to the control). This effect was significantly reduced by the nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine (L-NOARG) or high K+solution and completely abolished in vessels perfused with KCl plus L-NOARG. However, the vasorelaxant effect was not altered by indomethacin, atropine, yohimbine, pyrilamine, or K+-channel blockers: BaCl2, glibenclamide, ouabain, and 4-aminopyridine. In isolated MA pre-contracted with Phe, PRF also induced a concentration-dependent relaxation (0.1 –30 μ g/mL), which was in turn inhibited by endothelial removal, guanylyl cyclase inhibitor 1H [1,2,3]oxadiazolo[4,3-alpha]quinoxalin, charybdotoxin (ChTx), and ChTx plus apamin. Moreover, the relaxant effect was not altered by HOE140 and apamin given alone. The present study demonstrates that the vasorelaxing effect of PRF is dependent upon the NO–cGMP pathway in combination with hyperpolarization due to activation of Ca2+-dependent K+channels. Keywords:: Croton celtidifolius, proanthocyanidin, vasorelaxation, nitric oxide (NO), K+channel

Croton celtidifolius Bailon, commonly known as Sangue-de-Adave or Pau-Andrade, is a tree found in the Atlantic forest of southern Brazil. It has been popularly used for the treatment of inflammatory and ulcerative disorders. Phytochemical analysis demonstrated the presence of flavonoids and proanthocyanidins in an ethyl acetate fraction (EAF) from C. celtidifolius Bailon. In this study, we have evaluated the effects of EAF and its sub-fractions (35 and 63, catechin) on inflammatory (cell migration and plasma extravasation) and oxidative (lipid peroxidation, superoxide dismutase (SOD) activity and superoxide anion production) parameters in carrageenan-induced pleurisy in rats. NO production was also measured by nitrite/nitrate levels. EAF and sub-fraction 63 (63SF) showed anti-inflammatory activity, as indicated by a reduction in plasma extravasation and cell migration (mainly polymorphonuclear leukocytes) to the pleural cavity. Furthermore, EAF treatment decreased the production of superoxide radical anion by cells isolated from the pleural cavity, while it did not affect the nitrite/nitrate levels in exudates. The results show that C. celtidifolius contains substances with antioxidant and anti-inflammatory activity that, at least in part, act by a modulation of oxidative stress by phenolic compounds.

The chemical composition of the chromatography 63 subfraction (63SF) from the ethyl acetate soluble fraction of the crude extract of Croton celtidifolius bark presented a high content of total proanthocyanidins (75.0 ± 2.3%). HPLC analysis of 63SF revealed a dimeric profile (e.g. catechin-(4α → 8)-catechin and gallocatechin-(4α → 8)-catechin) and polymeric proanthocyanidins. In pharmacological investigations, 63SF administered intraperitoneally exhibited dose-dependent antinociceptive activity against several chemical stimuli, including the intraperitoneal injection of acetic acid (ID50 (the dose of 63SF which was able to reduce the nociceptive response by 50% relative to the control value) = 0.9 (0.5–1.6)) and the intraplantar injection of capsaicin (ID50 = 13.0 (10.0–17.0)), glutamate (ID50 = 4.0 (2.0–7.0)) and formalin (ID50 first phase = 36.0 (24.0–53.0) and late phase = 11.0 (8.0–14.0)). 63SF administered orally exhibited an antinociceptive effect in the formalin test (ID50 first phase = 125.0 (89.0–177.0) and late phase = 65.0 (33.0–95.0)). In the same test, 63SF was effective when given soon after the first phase, as well as exhibiting therapeutic activity. Furthermore, 63SF was effective in models of thermal nociception including tail-flick and hot-plate tests. When the mice were treated in the neonatal period with capsaicin, the antinociceptive effect of 63SF in the first phase of the formalin test was abolished, but pretreatment with naltrexone did not change the antinociceptive effect of 63SF. Together, these results provide evidence that 63SF exerted a pronounced systemic antinociception against chemical (acetic acid, formalin, glutamate and capsaicin tests) and thermal (hot-plate and tail-flick tests) nociceptive models of pain in mice at a dose that did not interfere with the locomotor activity. The mechanism by which this sub-fraction produced antinociception remains unclear, but it is unlikely to involve the activation of the opioid system. However, unmyelinated C-fibres sensitive to treatment with capsaicin are likely to participate in antinociception caused by 63SF.

This study investigated the superoxide anion and hydroxyl radical scavenger properties, as well as the inhibition of lipid peroxidation by the crude hydroalcoholic extract (CE) and the butanolic (BF) and ethyl acetate (EAF) fractions of Cuphea carthagenensis leaves. In a enzymatic system of O 2 − production (xanthine/xanthine oxidase system) the CE, EAF and BF (0.1–100 μg ml −1 ) were effective at inhibiting both uric acid formation and NBT reduction by O 2 −1 . In the non-enzymatic system of O 2 − generation, the CE and fractions were effective only at the concentration of 100 μg ml −1 . The CE, EAF and BF were also evaluated for their ability to scavenge hydroxyl radicals and/or to chelate iron. The results showed that CE, BF and EAF from C. carthagenensis (0.1–100 μg ml −1 ) were able to inhibit deoxyribose degradation in a concentration-dependent manner. CE was more potent than the fractions. In a hydrophobic system, increasing concentrations of CE, EAF and BF (0.1–100 μg ml −1 ) caused graded inhibition of lipid peroxidation of rat liver homogenate. The EAF displayed the lowest median inhibitory concentration. The present study suggests that an extract (CE) and fractions (EAF and BF) from C. carthagenensis leaves are significant sources of phenolic compounds with antioxidant activity in vitro and may have important health effects, for example, in cardiovascular disease.

This study evaluated the vasorelaxant properties of the crude hydroalcoholic extract (CE) of Cuphea carthagenensis, as well as its butanolic (BF) and ethyl acetate (EA) fractions, in rings of rat thoracic aorta. In endothelium-intact rings contracted with phenylephrine (30-100 nM), cumulative additions of increasing concentrations of CE, BF, and EA of C. carthagenensis (0.1 microg/ml-3 mg/ml) caused graded relaxations, with BF displaying the lowest median inhibitory concentration (IC5; mean, 6.8 microg/ml; 95% confidence limits, 3.3-14.2). BF-induced relaxations of endothelium-intact rings were virtually abolished by prior incubation with the NO-synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG; 10 or 30 microM), and were markedly reduced after guanylate cyclase inhibition with either methylene blue (10 microM) or ODQ (1 microM; 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). The inhibition of BF-induced relaxation by L-NOARG was prevented to a large extent by simultaneous incubation with L-arginine (1 mM). In endothelium-denuded rings contracted with phenylephrine, CE and BF caused graded relaxations only at doses >100 microg/ml, whereas the NO-donors SNAP (S-nitroso-N-acetyl-penicillamine) and SIN-1 (3-morpholino-sydnonimine) induced full relaxation at 1 microM. BF (100 microg/ml), which caused little relaxation per se of endothelium-denuded rings, potentiated the relaxant effects of SNAP and even more so of SIN-1 (which, unlike SNAP, also releases superoxide anion O2- in addition to NO), in a manner qualitatively similar to that seen with SOD (superoxide dismutase) against SIN-1. These data indicate that the BF of C. carthagenensis induces relaxation of the rat thoracic aorta by two mechanisms: (a) an endothelium-dependent component of action, which clearly depends on the NO/cyclic guanosine monophosphate (cGMP) pathway and can be attributed, at least in part, to free radical-scavenging properties; and (b) an endothelium-independent component of action, which becomes evident at higher doses (> or = 100 microg/ml) and remains to be further characterized. These results suggest that this native South American plant might be beneficial in cardiovascular disease.

This study has evaluated the possible role played by the l-arginine-nitric oxide pathway in the vasorelaxant action of the hydroalcoholic extract from Eugenia uniflora, and fractions from the extract, in rings of rat thoracic aorta. The addition of an increasing cumulative concentration of hydroalcoholic extract from E. uniflora (1–300 μg mL−1) caused a concentration-dependent relaxation response in intact endothelium-thoracic aorta rings pre-contracted with noradrenaline (30–100 nm). The IC50 value, with its respective confidence limit, and the maximum relaxation (Rmax) were 7.02 (4.77–10.00) μg mL−1 and 83.94 ± 3.04%, respectively. The removal of the endothelium completely abolished these responses. The nitric oxide synthase inhibitors Nω-nitro-l-arginine (l-NOARG, 30 μm) and Nω-nitro-l-arginine methyl ester (l-NAME, 30 μm), inhibited the relaxation (Rmax) to −10.43 ± 7.81% and −3.69 ± 2.62%, respectively. In addition, l-arginine (1 mm), but not d-arginine (1 mm), completely reversed inhibition by l-NOARG. Methylene blue (30 μm), a soluble guanylate cyclase inhibitor, reduced the relaxation induced by the extract to 14.60 ± 7.40%. These data indicate that in the rat thoracic aorta the hydroalcoholic extract, and its fractions, from the leaves of E. uniflora have graded and endothelium-dependent vasorelaxant effects.

Cell wall polysaccharides of the red algae Laurencia microcladia were isolated by alkaline treatment (KOH 1M, 10 mg NaBH4, room temperature), yielding two sub-fractions by neutralization (AcOH, pH=5.2) and EtOH precipitation, respectively A and B. Paper electrophoresis revealed the existence of only one polysaccharide in each fraction. FT-IR spectra of both polysaccharides showed signals for COH (OH: 3400-3300 cm-, CO: 1260-1000 cm-) and CHC (1300 – 1000 cm-) typical of cell wall algal polymers. An N-glyco derivative (1640 – 1560 cm-) and a low acidic content (0.34 mol %) were structural features found, while proteins were not detected. Antimitotic activity was observed on Lytechinus variegatus (green bur) larvae, with chronic values of 38.18 mg/mL and 267.99 mg/mL for polysaccharides A and B, respectively. No bradykinin or acetylcholine antagonistic effect was observed when polysaccharides were assayed in vitro in guinea-pig ileum. Preliminary results pointed to neurotoxic effect of these macromolecules (5mg/100 mL) since asymmetry of the neural tube in chick embryo was also found.

Foram efetuados estudos químicos e farmacológicos dos extratos brutos hidroalcóolicos obtidos das folhas ou das raízes de E. argentinum ou de frações semi-purificadas desta planta. Os testes químicos revelaram a existência de alcalóides, fitoesteróides, leucoantocianidinas, glicosídeos flavonóides, cumarinas e taninos. A análise farmacológica in vivo demonstrou que o extrato das folhas é menos tóxico que o da raiz. Tanto o extrato bruto como as frações semi-purificadas obtidas das folhas reduziram a locomoção e pressão arterial de rato e potenciaram o sono barbiturico em camundongos. O extrato bruto, principalmente o da raiz, reduziu o edema induzido por carragenina na pata de rato. Testes in vitro demonstraram que o extrato bruto ou as frações semi-purificadas das folhas produziram uma ação semelhante à cocaína avaliada no canal deferente de rato. Além disso, o extrato bruto produziu relaxamento inespecífico no útero de rata e contração do íleo de cobaia. Os resultados obtidos sugerem que esta planta possui constituintes ativos dotados de distintas propriedades farmacológicas, destacando-se o efeito anti-inflamatório, além de uma ação tipo cocaína, merecendo, portanto, estudos químicos e farmacológicos complementares. Chemical and pharmacological studies were carried out using crude hydroalcholic extracts obtained from the leaves or roots of E. argentinum or the semipurified fractions of this plant. Chemical analysis detected the presence of alkaloids, phytosteroids, leucoanthocianidins, flavonoid glycosides, cumarins and tannins. Qualitatively similar pharmacological effects were observed in experiments using both extracts from the leaves or roots. Experiments in vivo revealed that the crude extract and the semipurified fractions from the leaves reduced locomotor activity, blood pressure, carragenin-induced paw edema and potentiated barbiturate-induced sleeping-time in laboratory animals. In vitro studies demonstrated that the leaf extract or the semipurified fractions induced a cocaine-like effects on the rat vas deferens, a non-specific relaxation of the rat uteri and a contraction of the guinea-pig ileum. It is concluded that E. argentinum contains active compounds responsible for the antiinflammatory effect and cocaine-like action observed in vitro, which merit emphasis in future research.

The aim of this study is to investigate the effect of Cecropia glaziovii Snethl, Urticaceae, extracts on the oral glucose tolerance curve, on glycemia in alloxan-induced diabetic rats and vasorelaxant effect after the extraction process, and to standardize the extractive solutions. The effects of the process variables and their interactions were calculated in relation to dry residue, pH, total phenolic results and chemical marker content. Furthermore, the effect of the extracts (400 mg/kg), chlorogenic (2 or 15 mg/kg) and caffeic acids (2 mg/kg) were investigated on the oral glucose tolerance curve and on glycemia in alloxan-induced diabetic rats. Oral administration of ethanol extracts 4d20 and 8d20 significantly improved glucose tolerance in the hyperglycemic rats. Chlorogenic and caffeic acids, as well as the association of the compounds were able to significantly reduce glycemia after oral gavage treatments. On the other hand, the aqueous extracts did not alter the glycemia. The aqueous extracts (8020 and 9030) and only the higher dose of chlorogenic acid presented a significant effect on serum glucose lowering in diabetic rats. Additionally, the IC50 reveals that the ethanol extracts presented more potent vasodilator effects than the aqueous extracts in aortic rings. This study shows that C. glaziovii standardized extracts exhibits antihyperglycemic action, is able to improve glucose tolerance and has a potent vascular relaxing effect. These results are probably linked to concentrations of the main phenolic compounds of the extracts. Keywords: Cecropia glaziovii, Decoction, Maceration, Total phenolic, Antihyperglycemic activity, Vasorelaxant activity

Estudos de nosso laboratório surgerem que o ácido tânico (AT) exerce diversos efeitos "in vitro" ou "in vivo", podendo ser responsável por várias ações inespecíficas freqüentemente observadas com extratos brutos de plantas medicinais. O presente trabalho amplia estes estudos em outos modelos biológicos. A administração crônica ou aguda, por vida oral (v.o.), de AT em ratos não produziu alterações gástricas ou no processo inflamatório. Em ratos não-diabéticos observou-se modificações variáveis na glicemia de acordo com a metodologia empregada. O AT, cronicamente (v.o.), não interferiu na reprodução de fêmeas, embora constatou-se redução do número de filhotes de machos tratados. O AT (3mg/kg, v.o.) provocou aumento da diurese, porém doses mais elevadas não modificaram o volume urinário. Os testes hematológicos demonstraram que o AT possui efeito anticoagulante "in vitro", sem modificar a fragilidade de hemácias ou na homeastase sanguínea "in vivo". No músculo esquelético, o AT reduziu a resposta contrátil à acetilcolina, enquanto no músculo cardíaco (átrio esquerdo) observou-se efeito inotrópico positivo ou negativo. Estes resultados estendem os estudos anteriores e reforçam a sugestão de que parte significativa dos efeitos inespecíficos observados com extratos brutos de plantas medicinais podem estar relacionados com a presença de taninos nos mesmos. Extending previous studies the present report analyses the pharmacological effects of tannic acid (TA) in several tests. TA prevented carragenin-induced edema when administered by i.p. but not p.o. route. Long term p.o. treatment with TA did not induce gastric lesions and showed inconsistent effects on glicemia in non-diabetic rats. Rats chronically exposed to TA during the fetal stage or following birth did not exhbit changes in gain of or organ weights, but adult male rats showed a lower fertility ratio. Urinary output was slightly increased by administration of a small dose of TA (3mg/kg, p.o.), but not by higher doses. The addition of TA "in vitro" to blood samples did not influence osmotic fragility but prevented clotting for at least 30 min. In isolated tissues, TA non competitively antagonized acetylcholineinduced contractions of the frog rectus-abdominal muscle and induced, depending of the dose, both positive and negative inotropic effects in the electrically-stimulated left rat atrium. These results confirm and extend our previous observations and suggest that a great number of non-specific effects detected in studies on the pharmacology of different folk medicinal plants may be related to the presence of tannins.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia, Florianópolis, 2011 A aterosclerose é uma doença progressiva caracterizada pelo acúmulo de lipídios e elementos fibrosos nas artérias de médio e grande calibre. As doenças cardiovasculares decorrentes da aterosclerose, dentre elas o infarto agudo do miocárdio e o acidente vascular cerebral, são a principal causa de mortalidade e morbidade mundial. O estresse oxidativo e a deficiência da enzima glutationa peroxidase (GPx) possuem um importante papel no desenvolvimento da aterosclerose. Neste contexto, o objetivo deste trabalho foi avaliar o efeito do disseleneto de difenila (DD), um composto orgânico de selênio, em modelos experimentais de aterosclerose, in vivo e in vitro. O tratamento com baixas doses de DD (0,1 e 1 mg/kg/dia, v.o.) por 30 dias reduziu a formação de lesões ateroscleróticas em camundongos com deleção gênica para o receptor da lipoproteína de baixa densidade (LDLr -/-) submetidos a uma dieta hipercolesterolêmica. Esta redução foi acompanhada por melhora na vasodilatação dependente do endotélio, diminuição da expressão de 3-nitrotirosina e malondialdeído (marcadores de estresse oxidativo), redução da razão colesterol não-HDL/HDL, diminuição da infiltração de células inflamatórias na lesão e dos níveis sistêmicos de MCP-1. Em cultura de células endoteliais de aorta bovina (BAECs), o pré-tratamento por 24 horas com DD (0,5 e 1 M) atenuou a apoptose induzida por LDL humana isolada associada à diminuição da formação de EROs e o consumo de GSH, e restabelecer o aumento do potencial de membrana mitocondrial. Os estudos em macrófagos murinos da linhagem J774 expostos a oxLDL (100 g/mL), demonstraram que o DD (1 M) reduziu significativamente a formação de células espumosas, a produção de EROs e de mediadores inflamatórios (MCP-1 e TNF- ). Este conjunto de resultados revela o potencial antiaterogênico do DD, tanto em modelos experimentais in vivo quanto in vitro. Os efeitos demonstrados pelo composto sugerem ser relacionados às suas propriedades antioxidante, mimética da GPx e anti-inflamatória.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia, Florianópolis, 2009.

Doutoramento em Química Esta tese centra-se no desenvolvimento de materiais biodegradáveis e nãodegradáveis produzidos por eletrofiação com aplicação na área biomédica. O poli(3-hidroxibutirato-co-3-hidroxivalerato) (PHBV), um poliéster biodegradável, foi selecionado como base dos materiais biodegradáveis, enquanto o poli(tereftalato de etileno) (PET), um polímero sintético, estável e biocompatível, foi selecionado para a produção das matrizes não degradáveis. Adicionou-se quitosana aos sistemas com o objetivo de melhorar o processo de eletrofiação e as propriedades morfológicas, físico-químicas e biológicas dos materiais resultantes. A composição química, bem como as características morfológicas e físicoquímicas dos materiais em estudo, foram manipuladas de modo a otimizar a sua performance como suportes celulares para engenharia de tecidos. Foram realizados estudos in vitro com cultura de fibroblastos L929 para avaliar o comportamento das células, i.e. viabilidade, adesão, proliferação e morte, quando cultivadas nas matrizes produzidas por eletrofiação. Adicionalmente foram realizados ensaios in vivo para investigar o potencial dos materiais em estudo na regeneração cutânea e como tela abdominal. Os principais resultados encontrados incluem: o desenvolvimento de novas matrizes híbridas (PHBV/quitosana) adequadas ao crescimento de fibroblastos e ao tratamento de lesões de pele; o desenvolvimento de um sistema de eletrofiação com duas seringas para a incorporação de compostos bioativos; diversas estratégias para manipulação das características morfológicas dos materiais de PHBV/quitosana e PET/quitosana produzidos por eletrofiação; uma melhoria do conhecimento das interações fibroblastos-suporte polimérico; a verificação de uma resposta inflamatória desencadeada pelos materiais nãodegradáveis quando utilizados no tratamento de defeitos da parede abdominal, o que sugere a necessidade de novos estudos para avaliar a segurança do uso de biomateriais produzidos por eletrofiação. This thesis focuses on the development of biodegradable and non-degradable electrospun materials with application in the biomedical field. Poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a natural biodegradable polyester, was selected as the basis of the biodegradable materials while polyethylene terephthalate (PET), a biocompatible stable synthetic polyester, was selected for the production of the non-degradable ones. Chitosan was added to both systems in order to enhance electrospinnability as well as morphological, physico-chemical, and biological features of the biomaterials. The chemical composition, morphological and some physico-chemical characteristics of these materials were manipulated toward an optimized biological performance as scaffolds for tissue engineering. In vitro cell culture studies were performed with L929 fibroblasts in order to study the cell behavior, i.e. viability, adhesion, proliferation and death, when cultured on the electrospun materials. Furthermore, in vivo assays were conducted in order to investigate the potential of the materials under study for skin and abdominal wall repair. The main achievements of this thesis include: the development of new PHBV/chitosan hybrid mats suitable for fibroblasts growth and with a good performance when used as a scaffold for skin repair; the development of a dual syringe electrospinning system for incorporation of bioactive compounds; several strategies to manipulate the morphological characteristics of electrospun materials of both PHBV/chitosan and PET/chitosan blends; an improvement of the knowledge of cell-scaffolds interactions; the detection of an important inflammatory response elicited by the non-degradable electrospun materials when used as prosthetic meshes for abdominal defect repair, suggesting the need of further studies on the safety of nanosized electrospun biomaterials.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Farmacologia. Glutamato, o principal neurotransmissor central excitatório, induz excitotoxicidade neuronal pela ativação de receptores NMDA e AMPA, promovendo o influxo de cálcio nos neurônios. Estudos recentes do nosso grupo demonstram que as frações e sub-frações isoladas da Croton celtidifolius possuem propriedades anti-inflamatórias, antioxidantes e antinociceptivas. Assim, utilizando um modelo in vitro de excitotoxicidade induzida por glutamato (Glu) e in vivo de neurotoxicidade decorrente da lesão da medula espinhal de ratos Wistar adultos, avaliou-se o possível efeito neuroprotetor da fração rica (FRP) em proantocianidinas isolada da Croton celtidifolius. Glu induziu morte de células do gânglio da raiz dorsal (GRD), de forma dependente da concentração e do tempo de exposição. O pós-tratamento com a FRP (1 - 100 µg/ml) inibiu em até 37% a morte celular induzida por Glu (1 mM). Já o co-tratamento, foi capaz de reduzir a morte celular em 27%. A exposição das células ao Glu (0,01, 0,1 e 1 mM) aumentou os níveis da enzima lactato desidrogenase de 6,5 % (células controle) para 37, 49 e 63 %, respectivamente, indicativo de alteração na permeabilidade da membrana celular. Esta alteração foi atenuada pelo tratamento das células com a FRP (100 µg/mL). Ademais, o insulto glutamatérgico induziu a geração de espécies reativas de oxigênio, a qual foi inibida pelo tratamento das células do GRD com FRP (100 µg/ml). Além disso, o efeito neuroprotetor desencadeado pela FRP parece ser mediado em parte pela inibição de receptores glutamatérgicos do tipo NMDA, mas não os metabotrópicos uma vez que a perda da viabilidade celular decorrentes da exposição das células ao agonista NMDA, mas não ao agonista metabotrópico, foi revertida pela FRP. Nos estudos in vivo, animais submetidos a lesão medular e tratados com a FRP na dose de 10 mg/kg por via intraperitoneal, durante 5 dias consecutivos, apresentaram uma melhora significante na performance locomotora e na força de preensão, quando comparado aos animais lesados tratados com veículo. Em conjunto, estes resultados fornecem a primeira evidência de que a FRP atenua a morte celular por necrose e por apoptose, bem como a produção de EROs induzida por Glu, inibe a morte celular induzida por peróxido de hidrogênio e pelo agonista NMDA. A FRP também foi capaz de promover uma melhora na atividade locomotora e na força de preensão de animais submetidos à lesão traumática da medula espinhal, reforçando a hipótese de que polifenóis podem ser ferramentas terapêuticas promissoras como agentes neuroprotetores. Glutamate, the major central excitatory neurotransmitter, induces neuronal excitotoxicity by activation of NMDA and AMPA receptors and stimulating the influx of calcium into neurons. Recently, our group showed that fractions and subfractions isolated from Croton celtidifolius have antiinflammatory, antioxidant and antinociceptive properties. Thus, using an in vitro model of excitotoxicity induced by glutamate (Glu) and in vivo neurotoxicity caused by spinal cord injury (SCI) in adult Wistar Rats, we evaluated the possible neuroprotective effect of a proanthocyanidin-rich fraction (PRF) isolated from the bark of Croton celtidifolius. Glu induced cell death of dorsal root ganglion (DRG) in a manner concentration-dependent and the exposure time. The post-treatment with FRP (from 1 to 100 ìg / mL) inhibited the cell death induced by Glu (1 mM) in 37%. Since co-treatment, was able to reduce cell death in 27%. The exposure of cells to glu (0.001, 0.1 and 1 mM) increased the levels of the enzyme lactate deydrogenase from 6.5% (control cells) to 37, 49 and 63% respectively, indicating changes in cell membrane permeability. This change was attenuated by treatment of cells with the PRF (100 ìg / mL). Moreover, the glutamatergic insult induced generation of reactive oxygen species, which was inhibited by treatment of cells with FRP (100 ìg / mL). Moreover, the neuroprotective effect triggered by FRP seems to be mediated in part by inhibition of NMDA-type glutamate receptors, but not metabotropic receptors, since the loss of cell viability after exposure of cells to NMDA agonist, but not a metabotropic agonist was reversed by FRP. The in vivo studies, animals subjected to SCI and treated with PRF at a dose of 10 mg / kg by i.p. route for 5 consecutive days showed a significant improvement in locomotor performance and grip force when compared to injured animals treated with vehicle. Taken together, these results provide the first evidence that PRF attenuated the cell death by apoptosis and the production of ROS induced by Glu, also inhibits cell death induced by hydrogen peroxide and the NMDA agonist receptors. The FRP was able to promote an improvement in locomotor activity and grip strength of animals subjected to traumatic spinal cord injury (SCI), reinforcing the hypothesis that polyphenols are promising therapeutic tools such as neuroprotective agents.

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Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia, Florianópolis, 2010 No presente estudo investigamos as possíveis propriedades psicofarmacológicas e neuroprotetoras da fração rica em proantocianidinas (FRP) obtida das cascas da Croton celtidifolius Baill (Euphorbiaceae) em ratos. Para tal finalidade, foram utilizados testes comportamentais classicamente empregados na triagem inicial de drogas com atividade sobre o sistema nervoso central e um modelo experimental da doença de Parkinson (DP). A administração intraperitoneal (i.p.) aguda da FRP (10 ou 30 mg/kg) diminuiu significativamente a atividade locomotora espontânea dos animais no campo aberto e na caixa de atividade, e prolongou a duração da hipnose induzida pelo éter etílico, sugerindo um efeito do tipo hipnosedativo. No labirinto em cruz elevado, a FRP (0,3 ou 3 mg/kg) aumentou significativamente a freqüência de entradas nos braços abertos do aparato, indicando um putativo efeito ansiolítico. Além destes efeitos, a FRP (10 ou 30 mg/kg) também aumentou a latência para a primeira convulsão induzida pelo pentilenotetrazol (60 mg/kg; i.p.), induziu ptose palpebral, hipotermia e atenuou o comportamento estereotipado induzido pela apomorfina (0,5 mg/kg; s.c.) na dose de 30 mg/kg. Estes achados sugerem que a FRP possui um amplo espectro de propriedades psicofarmacológicas como hipnosedativa, anticonvulsivante, neuroléptica e ansiolítica. No presente estudo, também foram avaliados os efeitos neuroprotetores do pré-tratamento com a FRP (10 mg/kg; i.p.), durante cinco dias, nas alterações comportamentais e neuroquímicas induzidas pela administração intranasal (i.n.) de MPTP (1 mg/narina) em ratos. A FRP foi capaz de prevenir os prejuízos cognitivos, emocionais e motores induzidos pela administração i.n. do MPTP, como também impediu a inibição da atividade do complexo-I mitocondrial e a degeneração dos neurônios dopaminérgicos do bulbo olfatório e substância negra causados pelo MPTP. Nossos resultados corroboram com outros estudos prévios acerca das propriedades neuroprotetoras dos polifenóis, sugerindo a possibilidade destes compostos serem ferramentas úteis em estratégias de neuroproteção.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-graduação em Farmacologia Uma fração rica em proantocianidinas (FRP) obtida das cascas da C. celtidifolius foi avaliada quanto ao seu potencial vasorrelaxante em preparações vasculares isoladas. Em leito arterial mesentérico, artéria mesentérica e aorta torácica isolados de ratos a FRP induz relaxamento dependente da concentração. Em vasos de pequeno calibre, este efeito foi significativamente reduzido pelo L-NOARG ou por Krebs despolarizante, e completamente abolido quando ambos são combinados. O efeito também foi reduzido na presença de ODQ e pela caribdotoxina. Em vasos de grande calibre, o L-NOARG e o ODQ também reduziram o relaxamento da fração, bem como a ausência de cálcio no e a presença de um bloqueador de influxo de cálcio (lantânio) na solução de Krebs-Henseleit. N-metilmaleimida e a neomicina também foram capazes de reduzir significativamente a vasodilatação induzida pela FRP. Em outro grupo de experimentos, a vasodilatação induzida pela FRP foi significativamente reduzida pelo Wortmannina. A FRP também foi testada quanto ao seu potencial antiaterogênico em camundongos C57/BL6 LDLr KO. Nestes experimentos, a fração não foi capaz de reduzir as concentrações de lipídios séricos (colesterol total, HDL ou VLDL mais LDL), assim como não reverteu os danos vasculares morfofuncionais provocados pela dieta, quando avaliados nos experimentos de reatividade vascular e na determinação da área de lesão aterosclerótica. Os resultados indicam que a FRP apresenta efeito vasorrelaxante dependente de endotélio em artérias de pequeno e grande calibre, que este efeito envolve a ativação da via NO/GMPc e que o mecanismo de ativação da via envolve o influxo de cálcio do meio extracelular pela ativação de proteínas sensíveis ao NEM e via ativação da PLC e por promover hiperpolarização do músculo liso vascular induzido por abertura de canais KCa2+ em vasos de pequeno calibre. A fração também promove a fosforilação da enzima eNOS, pela via PI3K/Akt, mas não apresenta atividade antiaterogênica quando avaliada no modelo de aterogênese induzida pela dieta em camundongos C57/BL6 LDLr KO.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Farmacologia Uncaria tomentosa (Willd.) DC (Rubiaceae) is an indigenous plant of the Amazon rainforest, popularly known as cat#s claw. The medicinal use of this plant by Peruvian indians has drawn scientific interest over its healing propreties, and gave rise to an increasing number of studies about its biological activities. Chemically, the substances of greatest interest present in this plant are indole and oxindole alkaloids. This study characterizes the antinociceptive activity of a standardized fraction of oxindole alkaloids from U. tomentosa (UT) in mouse models of nociception. Pretreatment of animals with UT (3-300 mg/kg, i.p.) decreased, in a dose-dependent way, nociception elicited by chemical stimuli such as the intraperitoneal injection of acetic acid and the intraplantar injection of formalin, glutamate or capsaicin. Treatment with UT (300 mg/kg) significantly increased the latency of animals to respond to thermal noxious stimuli (hot plate and tail flick tests), probably revealing central effects of the alkaloids. Moreover, treatment with UT was able to inhibit nociceptive behavior caused by intratecal injection of glutamate, NMDA, substance P and the proinflammatory cytokines TNF-á and IL-1â. The antinociceptive effect of UT (100 mg/kg, i.p.) in the formalin test was abolished by pretreatment with ketanserin, an antagonist of serotonergic 5-HT2 receptors, but not by antagonists of the opioid receptors (naltrexone), muscarinic cholinergic receptors (atropine), or adrenergic á1 and á2 receptors (prazosin and yoimbine, respectively). In a similar way, pre-treatment with L-arginine, a substrate for the nitric oxide synthase enzyme, did not affect the antinociceptive activity of UT in the formalin test. These data suggest that the antinociceptive activity of UT involves an interaction with the serotonergic and the glutamatergic systems, but not with the other systems tested here.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Farmacologia O estresse oxidativo desempenha um importante papel na patogênese de várias doenças humanas, tais como injúria por isquemia e reperfusão, aterosclerose, doenças neurodegenerativas e câncer. No sistema cardiovascular, os radicais livres estão envolvidos principalmente no desenvolvimento da hipertensão e aterosclerose. Neste contexto, o presente trabalho teve como objetivo investigar, in vitro e in vivo, a atividade antioxidante de Cyathea phalerata, além de sua atividade vasodilatadora em anéis de aorta torácica isolada de rato. A atividade antioxidante in vitro foi avaliada através da medida da capacidade seqüestradora do Extrato Bruto (EB), Precipitado (PPT), Fração Aquosa (FAQ), Fração n-Butanol (FBU) e Fração Acetato de Etila (FAE), sobre os radicais DPPH, O2"- e OH", e sobre a peroxidação lipídica. Nestes ensaios foi possível observar o relevante potencial antioxidante da FAE, principalmente a sua capacidade de captar o radical OH" e inibir a peroxidação lipídica. A avaliação in vivo do estresse oxidativo foi realizada em camundongos pré-tratados com a FAE (10, 30 ou 100 mg/kg) e posteriormente expostos ao tetracloreto de carbono (CCl4), através da medida ex vivo de fragmentação do DNA, carbonilação de proteínas e lipoperoxidação de membranas, e das defesas antioxidantes (concentração de GSH e atividades de CAT e GST). A FAE foi capaz de reduzir o dano induzido pelo agente estressor (CCl4) ao DNA, lipídeos e proteínas e aumentar a atividade das enzimas CAT e GST. O importante potencial antioxidante apresentado pela FAE de Cyathea phalerata levou ao estudo desta fração em um modelo de reatividade vascular in vitro (aorta torácica isolada de ratos). A FAE apresentou um efeito vasodilatador dependente da camada de células endoteliais, principalmente nas menores concentrações. A incubação prévia com L-NOARG e ODQ demonstrou a participação da via NO/GMPc no efeito vasodilatador desta fração. Além disso, prostaciclina produzida pelas células endoteliais também parece participar desta atividade. A FAE não alterou a contração induzida pela fenilefrina e nem o relaxamento induzido pelo SNP. O relevante potencial antioxidante e a atividade vasodilatadora de Cyathea phalerata podem estar relacionados à sua composição química, rica em flavonóides. Esta planta pode ser importante para o tratamento de patologias que envolvem estresse oxidativo, podendo diminuir os dano causados à macromoléculas (lipídeos, proteínas e DNA) e aumentar as defesas antioxidantes. Destaca-se a importância da FAE de Cyathea phalerata, rica em flavonóides, para a prevenção e tratamento de doenças cardiovasculares.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Farmacologia O objetivo geral deste estudo foi investigar a atividade de uma fração rica em compostos fenólicos - Fração Acetato de Etila (FAE), obtida a partir do extrato bruto dealcoolizado de um [vinho] tinto catarinense, sobre o sistema cardiovascular de ratos Wistar normotensos e camundongos C57/BLACK6 com ablação gênica para o receptor de LDL (LDLR KO), submetidos a uma dieta hipercolesterolêmica (DH). Foram realizados ensaios para determinar a atividade [antioxidante], experimentos de reatividade vascular, experimento crônico para verificar o efeito da FAE em camundongos LDLR KO submetidos a uma DH, dosagens bioquímicas de [colesterol] total, triglicerídeos, e lipoproteínas, cortes histológicos de arcos aórticos de camundongos LDLR KO para determinar a formação de [ateromas] e imunohistoquímica. Os resultados deste estudo demonstraram que a FAE apresenta alguns efeitos biológicos importantes: 1) atividade [antioxidante] in vitro; 2) atividade vasorelaxante sobre vasos de pequeno calibre, mais precisamente, sobre o leito arterial mesentérico de ratos; 3) efeito hipolipidemiante na menor dose utilizada (3 mg/kg); 4) Diminuição sobre a expressão dos marcadores CD40L e nitrotirosina (N-TYR). A diminuição da expressão dos referidos marcadores aterogênicos foi ao encontro de todos os demais resultados anteriores, confirmando as efetivas atividades antioxidantes, hipolipidemiantes e vasorelaxantes da FAE.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-graduação em Farmacologia O tratamento in vivo em camundongos com ligantes do receptor periférico benzodiazepínico (PBR) exerceram efeito inibitório na resposta inflamatória em dois modelos de inflamação aguda. No primeiro modelo o efeito antiinflamatório em de pré-tratamento com os ligantes PBR, PK11195 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide e Ro5-4864 7-chloro-5-(4-Chlorophenyl)-1,3-dihydro-1-methyl-2-H-1,4-benzodiazepin-2, mostraram que a formação do edema em resposta a carragenina (300 mg/pata) foi inibida por PK11195 e Ro5-4864, em diferentes intervalos de tempo. O estudo do tempo resposta mostrou que a melhor resposta com ambos ligantes foi ótima em animais injetados no tempo de 24h, por esta razão este tempo de pré-tratamento foi escolhido para estudo da dose resposta. A formação do edema de pata em resposta a carragenina (300 mg/pata) foi reduzida por PK11195 e Ro5-4864 em diferentes doses (0,00001-10mg/kg, i.p.) produzindo uma inibição dose dependente na formação do edema de pata em camundongos induzida pela carragenina. PK11195 e Ro5-4864 (0.1 mg/kg, i.p.) quando administrado 24 h antes da indução do edema, inibiram de forma significativa o edema de pata de camundongos induzido por vários mediadores inflamatórios. No segundo modelo o pré-tratamento com os ligantes (0.1 mg/kg, i.p.), 24 h antes da indução da pleurisia induzida pela carragenina, mostrou uma significante inibição no recrutamento das células totais e diferenciais, principalmente a custas de neutrófilos, inibindo também a liberação de Interleucina-13 (IL-13) e Interleucina-6 (IL-6) no líquido pleural

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Farmácia A disfunção endotelial e o envolvimento de radicais livres exercem um importante papel na patogênese da aterosclerose. A erva-mate (Ilex paraguariensis) apresenta atividade antioxidante e vasorelaxante in vitro, em leito arterial mesentérico de ratos e reduz a aterosclerose experimental em coelhos. Assim, o objetivo deste trabalho foi verificar o efeito do extrato aquoso de Ilex paraguariensis (EAI) i) na confirmação da atividade antioxidante in vitro do extrato utilizado; ii) no relaxamento in vitro de aorta torácica de ratos e; iii) na reatividade vascular ex vivo de camundongos com aterosclerose. A atividade antioxidante do EAI foi avaliada através dos ensaios de redução do NBT, degradação da desoxirribose e inibição da peroxidação lipídica. O EAI mostrou-se efetivo em todos os ensaios, de maneira dependente da sua concentração (p < 0,01). No relaxamento in vitro de aortas torácicas de ratos, previamente contraídas com fenilefrina, o EAI apresentou efeito significativo dependente da sua concentração na presença de endotélio funcional (p < 0,01). O relaxamento na ausência de endotélio íntegro ocorreu somente com as maiores concentrações de EAI avaliadas. Ensaios realizados com o inibidor das enzimas óxido nítrico sintases, L-NOARG, e com o substrato endógeno, L-arginina, sugeriram o envolvimento da via NO/GMPc no relaxamento dependente de endotélio induzido pelo EAI. O efeito do EAI na reatividade vascular ex vivo foi testado em camundongos "knockout" para o receptor de LDL (LDLR KO) administrados com 3 diferentes doses do EAI. Os animais foram distribuídos em 5 grupos (n = 7 por grupo): 1) controle (C), dieta padrão e água ad libitum; 2) hipercolesterolêmico (HC), dieta com 1,25 % de colesterol e água; 3), 4) e 5) hipercolesterolêmico-Ilex paraguariensis (HI), dieta com colesterol e EAI nas doses de 0,3, 0,9 e 2,7 g/kg/dia, respectivamente. Após 3 meses de tratamento, os camundongos do grupo HC apresentaram valores séricos de colesterol total e de LDL e VLDL significativamente elevados (p < 0,001). A administração do EAI não modificou o perfil lipídico dos animais dos grupos HI nas três doses estudadas. As artérias dos camundongos HC apresentaram estrias gordurosas e diminuição significativa na resposta de contração à fenilefrina e relaxamento à acetilcolina (p < 0,001). O tratamento com EAI reverteu a inibição da capacidade contrátil da aorta nos grupos HI 0,9 e HI 2,7 e de inibição do relaxamento no grupo HI 0,9 (p < 0,05). Em conclusão, os resultados mostraram que o EAI comercial possui atividade antioxidante e vasorelaxante in vitro, com envolvimento do NO endotelial. Além disso, a ingestão do EAI melhorou a reatividade vascular ex vivo de aortas de camundongos LDLR KO com aterosclerose, apesar da hipercolesterolemia. Tais resultados são sugestivos do potencial benéfico da erva-mate no combate à aterosclerose. Endothelial dysfunction and free radicals play an important role in the pathogenesis of atherosclerosis. "Mate" (Ilex paraguariensis) extract exhibits antioxidant activity and induce endothelium-dependent vasodilatation in mesenterical arterial bed in vitro and reduces experimental atherosclerosis in cholesterol-fed rabbits. Therefore, this study assessed the effects of a commercial Ilex paraguariensis aqueous extract (IAE) on i) the confirmation of in vitro antioxidant activity of the extract used; ii) vasodilatation of thoracic rat aortas and iii) ex vivo vascular reactivity in atherosclerotic mice. The IAE antioxidant activity was determined through the NBT reduction assay, deoxyrribose degradation and lipid peroxidation inhibition. The IAE showed to be effective in all assays, in a concentration-dependent manner (p < 0.01). In vasodilatation of endothelium-intact thoracic rat aorta rings, previously contracted with phenylephrine, the IAE caused a significant concentration-dependent effect (p < 0.01). In endothelium denuded aortic rings, the relaxation induced by the IAE occurred only at the highest tested concentrations. Pretreatment of the aortic rings with the NO synthase inhibitor, L-NOARG, and the endogenous substrate, L-arginine, confirmed the involvement of NO/GMPc pathway in the endothelium-dependent relaxation. The effect of ingesting IAE on ex vivo vascular reactivity was tested in LDL receptor knockout mice (LDLR KO) administered with 3 different doses of IAE. Animals were distributed in 5 groups (n = 7 per group): 1) control (C), normal chow and water ad libitum; 2) hypercholesterolemic (HC), cholesterol-rich diet (1.25 % of cholesterol) and water ad libitum; 3), 4), and 5) hypercholesterolemic-Ilex paraguariensis (HI), cholesterol-rich diet and IAE at the doses of 0.3; 0.9 and 2.7 g/kg/day, respectively. After a three-months treatment, HC mice showed serum total cholesterol, LDL-c plus VLDL-c values significantly elevated (p < 0.001). The IAE administration did not change the lipid profile of IAE-administered mices. HC mice aortic artery exhibited fatty streaks and showed a significant inhibition in response to phenylephrine contraction and to acethylcoline vasodilatation (p < 0.001). The IAE treatment reverted the reduction of contractile capacity in mice aorta in HI 0.9 and HI 2.7 groups and the inhibition of relaxation in HI 0.9 group (p < 0.05). In summary, the results showed that the commercial IAE have antioxidant and endothelial NO-dependent vasorelaxant properties in vitro. Furthermore, IAE ingestion improved ex vivo vascular reactivity of atherosclerotic LDLR KO mice aorta, regardless hypercholesterolaemia. Our results suggest that "mate" aqueous extract can be beneficial against atherosclerosis development.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-graduação em Farmacologia. A [SF63], obtida das cascas da [Croton Celtidifolius] administrada por via intraperitoneal, provocou antinocicepção quando avaliada nos modelos de contorção abdominal induzida pelo [Ácido acético], no teste da nocicepção induzida pela [Formalina], no teste da [Capsaicina], no teste [Glutamato], no teste da [Retirada da cauda] e no modelo da [Placa quente], em doses que não promoveram reações inespecíficas, como sedação e relaxamento muscular, avaliadas pelo teste do [Rota-rod]. Também foi observado que a [SF63] administrada por via oral apresenta atividade antinociceptiva, quando avaliada no teste da nocicepção induzida pela formalina. Os resultados sugerem que a [SF63]

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Farmacologia.