Obtenção de sistemas microemulsionados e estudo de simulação por dinâmica molecular de sistemas micelares objetivando a veiculação de produtos naturais bioativos

Among the new drugs launched into the market since 1980, up to 30% of them belong to the class of natural products or they have semisynthetic origin. Between 40-70% of the new chemical entities (or lead compounds) possess poor water solubility, which may impair their commercial use. An alternative for administration of poorly water-soluble drugs is their vehiculation into drug delivery systems like micelles, microemulsions, nanoparticles, liposomes, and cyclodextrin systems. In this work, microemulsion-based drug delivery systems were obtained using pharmaceutically acceptable components: a mixture Tween 80 and Span 20 in ratio 3:1 as surfactant, isopropyl mirystate or oleic acid as oil, bidistilled water, and ethanol, in some formulations, as cosurfactants. Self-Microemulsifying Drug Delivery Systems (SMEDDS) were also obtained using propylene glycol or sorbitol as cosurfactant. All formulations were characterized for rheological behavior, droplet size and electrical conductivity. The bioactive natural product trans-dehydrocrotonin, as well some extracts and fractions from Croton cajucara Benth (Euphorbiaceae), Anacardium occidentale L. (Anacardiaceae) e Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae) specimens, were satisfactorily solubilized into microemulsions formulations. Meanwhile, two other natural products from Croton cajucara, trans-crotonin and acetyl aleuritolic acid, showed poor solubility in these formulations. The evaluation of the antioxidant capacity, by DPPH method, of plant extracts loaded into microemulsions evidenced the antioxidant activity of Phyllanthus amarus and Anacardium occidentale extracts. For Phyllanthus amarus extract, the use of microemulsions duplicated its antioxidant efficiency. A hydroalcoholic extract from Croton cajucara incorporated into a SMEDDS formulation showed bacteriostatic activity against colonies of Bacillus cereus and Escherichia coli bacteria. Additionally, Molecular Dynamics simulations were performed using micellar systems, for drug delivery systems, containing sugar-based surfactants, N-dodecylamino-1-deoxylactitol and N-dodecyl-D-lactosylamine. The computational simulations indicated that micellization process for N-dodecylamino-1- deoxylactitol is more favorable than N-dodecyl-D-lactosylamine system.

Estudo da imobilização de proteases para a síntese de oligolisinas

Enzymatic synthesis of peptides using proteases has attracted a great deal of attention in recent years. One key challenge in peptide synthesis is to find supports for protease immobilization capable of working in aqueous medium at high performance, producing watersoluble oligopeptides. At present, few reports have been described using this strategy. Therefore, the aim of this thesis was to immobilize proteases applying different methods (Immobilization by covalent bound, entrapment onto polymeric gels of PVA and immobilization on glycidil metacrylate magnetic nanoparticles) in order to produce water-soluble oligopeptides derived from lysine. Three different proteases were used: trypsin, α-chymotrypsin and bromelain. According to immobilization strategies associated to the type of protease employed, trypsin-resin systems showed the best performance in terms of hydrolytic activity and oligopeptides synthesis. Hydrolytic activities of the free and immobilized enzymes were determined spectrophotometrically based on the absorbance change at 660 nm at 25 °C (Casein method). Calculations of oligolysine yield and average degree of polymerization (DPavg) were monitored by 1H-NMR analysis. Trypsin was covalently immobilized onto four different resins (Amberzyme, Eupergit C, Eupergit CM and Grace 192). Maximum yield of bound protein was 92 mg/g, 82 mg/g and 60 mg/g support for each resin respectively. The effectiveness of these systems (Trypsin-resins) was evaluated by hydrolysis of casein and synthesis of water-soluble oligolysine. Most systems were capable of catalyzing oligopeptide synthesis in aqueous medium, albeit at different efficiencies, namely: 40, 37 and 35% for Amberzyme, Eupergit C and Eupergit CM, respectively, in comparison with free enzyme. These systems produced oligomers in only 1 hour with DPavg higher than free enzyme. Among these systems, the Eupergit C-Trypsin system showed greater efficiency than others in terms of hydrolytic activity and thermal stability. However, this did not occur for oligolysine synthesis. Trypsin-Amberzyme proved to be more successful in oligopeptide synthesis, and exhibited excellent reusability, since it retained 90% of its initial hydrolytic and synthetic activity after 7 reuses. Trypsin hydrophobic interactions with Amberzyme support are responsible for protecting against strong enzyme conformational changes in the medium. In addition, the high concentration of oxirane groups on the surface promoted multi-covalent linking and, consequently, prevented the immobilized enzyme from leaching. The aforementioned results suggest that immobilized Trypsin on the supports evaluated can be efficiently used for oligopeptides synthesis in aqueous media

Obtenção de polímeros graftizados de quitosana e estudo das propriedades físico-químicas para aplicação na indústria do petróleo

Chitosan is a biopolymer derived from the shells of crustaceans, biodegradable, inexpensive and renewable with important physical and chemical properties. Moreover, the different modifications possible in its chemical structure generate new properties, making it an attractive polysaccharide owing to its range of potential applications. Polymers have been used in oil production operations. However, growing concern over environmental constraints has prompted oil industry to search for environmentally sustainable materials. As such, this study sought to obtain chitosan derivatives grafted with hydrophilic (poly(ethylene glycol), mPEG) and/or hydrophobic groups (n-dodecyl) via a simple (one-pot) method and evaluate their physicochemical properties as a function of varying pH using rheology, small-angle Xray scattering (SAXS), dynamic light scattering (DLS) and zeta potential. The chitosan derivatives were prepared using reductive alkylation under mild reaction conditions and the chemical structure of the polymers was characterized by nuclear magnetic resonance (1H NMR) and CHN elemental analysis. Considering a constant mPEG/Chitosan molar ratio on modification of chitosan, the solubility of the polymer across a wide pH range (acidic, neutral and basic) could only be improved when some of the amino groups were submitted to reacetylation using the one-pot method. Under these conditions, solubility is maintained even with the simultaneous insertion of n-dodecyl. On the other hand, the solubility of derivatives obtained only through mPEG incorporation using the traditional methodology, or with the ndodecyl group, was similar to that of its precursor. The hydrophilic group promoted decreased viscosity of the polymer solutions at 10 g/L in acid medium. However, at basic pH, both viscosity and thermal stability increased, as well as exhibited a pronounced pseudoplastic behavior, suggesting strong intermolecular associations in the alkaline medium. The SAXS results showed a polyelectrolyte behavior with the decrease in pH for the polymer systems. DLS analyses revealed that although the dilute polymer solutions at 1 g/L and pH 3 exhibited a high density of protonated amino groups along the polymer chain, the high degree of charge contributed significantly to aggregation, promoting increased particle size with the decrease in pH. Furthermore, the hydrophobic group also contributed to increasing the size of aggregates in solution at pH 3, whereas the hydrophilic group helped reduce their size across the entire pH range. Nevertheless, the nature of aggregation was dependent on the pH of the medium. Zeta potential results indicated that its values do not depend solely on the surface charge of the particle, but are also dependent on the net charge of the medium. In this study, water soluble associative polymers exhibit properties that can be of great interest in the petroleum industry

Avaliação das propriedades físico-químicas de sistemas a base de carboximetilcelulose e poli (N-isopropilacrilamida) em soluções aquosas para aplicação na indústria do petróleo

Sustainable development is a major challenge in the oil industry and has aroused growing interest in research to obtain materials from renewable sources. Carboxymethylcellulose (CMC) is a polysaccharide derived from cellulose and becomes attractive because it is water-soluble, renewable, biodegradable and inexpensive, as well as may be chemically modified to gain new properties. Among the derivatives of carboxymethylcellulose, systems have been developed to induce stimuli-responsive properties and extend the applicability of multiple-responsive materials. Although these new materials have been the subject of study, understanding of their physicochemical properties, such as viscosity, solubility and particle size as a function of pH and temperature, is still very limited. This study describes systems of physical blends and copolymers based on carboxymethylcellulose and poly (N-isopropylacrylamide) (PNIPAM), with different feed percentage compositions of the reaction (25CMC, 50CMC e 75CMC), in aqueous solution. The chemical structure of the polymers was investigated by infrared and CHN elementary analysis. The physical blends were analyzed by rheology and the copolymers by UV-visible spectroscopy, small-angle X-ray scattering (SAXS), dynamic light scattering (DLS) and zeta potential. CMC and copolymer were assessed as scale inhibitors of calcium carbonate (CaCO3) using dynamic tube blocking tests and chemical compatibility tests, as well as scanning electron microscopy (SEM). Thermothickening behavior was observed for the 50 % CMC_50 % PNIPAM and 25 % CMC_75 % PNIPAM physical blends in aqueous solution at concentrations of 6 and 2 g/L, respectively, depending on polymer concentration and composition. For the copolymers, the increase in temperature and amount of PNIPAM favored polymer-polymer interactions through hydrophobic groups, resulting in increased turbidity of polymer solutions. Particle size decreased with the rise in copolymer PNIPAM content as a function of pH (3-12), at 25 °C. Larger amounts of CMC result in a stronger effect of pH on particle size, indicating pH-responsive behavior. Thus, 25CMC was not affected by the change in pH, exhibiting similar behavior to PNIPAM. In addition, the presence of acidic or basic additives influenced particle size, which was smaller in the presence of the additives than in distilled water. The results of zeta potential also showed greater variation for polymers in distilled water than in the presence of acids and bases. The lower critical solution temperature (LCST) of PNIPAM determined by DLS corroborated the value obtained by UV-visible spectroscopy. SAXS data for PNIPAM and 50CMC indicated phase transition when the temperature increased from 32 to 34 °C. A reduction in or absence of electrostatic properties was observed as a function of increased PNIPAM in copolymer composition. Assessment of samples as scale inhibitors showed that CMC performed better than the copolymers. This was attributed to the higher charge density present in CMC. The SEM micrographs confirmed morphological changes in the CaCO3 crystals, demonstrating the scale inhibiting potential of these polymers

Differential response related to genotoxicity between eggplant (Solanum melanogena) skin aqueous extract and its main purified anthocyanin (delphinidin) in vivo

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. A number of studies have demonstrated that anthocyanins present antioxidant capacity and show inhibitory effects on the growth of some cancer cells. Thus, the goal of this study was to evaluate both the antimutagenicity/antigenotoxicity and mutagenicity/genotoxicity of aqueous extract obtained from the Solanum melanogena, a possible novel source of anthocyanin, and its main purified anthocyanin extract (delphinidin), using the single cell (comet) assay and micronucleus test. Pretreatment with higher doses of the purified anthocyanin (10 and 20 mg/kg b.w.) led to a statistically significant reduction (p < 0.05) in the frequency of micronuclei in polychromatic erythrocytes induced by cyclophosphamide. The pattern of reduction ranged from 48% to 57% independent of concentration. No apparent: genotoxicity and mutagenicity was found for either the anthocyanin or delphinidin extracts. Taken together, these results suggest that mice pre-treated with specific compounds present in anthocyanins (delphinidin) displayed a lower incidence of mutations induced by cyclophosphamide. This finding emphasizes the potential of natural colorants to prevent mutations and also the applicability of genotoxic evaluation for improving health. Furthermore, the results presented here could be an additional argument to support the use of anthocyanins in the diet. (c) 2006 Published by Elsevier Ltd.

Avaliação do Potencial para Ensilagem de Cultivares de Alfafa (Medicago sativa L.)

The objective of this trial was to study the ensilage potential of alfalfa (Medicago sativa L,.). The material used in the evaluation was harvested from an experiment in a complete randomized blocks design with three replicates, with seventeen alfalfa cultivars, Criouil, Florida 77, P555, P581, Moapa, Gilboa, 34 Linea, C/W 8754, C/W 4468, C/W 86, C/W 8746, Hunter Field, Trifecta, Sequel, CUF 101, Aurora e Siriver, during three years. The harvest used for the determinations was collected at 30(th) day of plants development and the material was submitted to two treatments: no wilting - Fresh alfalfa frozen immediately after harvest; wilting fresh alfalfa exposed to sun by three hours to get 35% DM. The dry matter content (DM), water soluble carbohydrates (WSC) and buffer capacity (BC) were evaluated in rhp forages Wilting increased the dry matter content (39,98%), however there were not differences among cultivars. The same occurred for the WSC content, with interaction among cultivars and treatments. The alfalfa cultivars, when submitted to effective wilting practice, presented appropriated characteristics of dry matter and water-soluble carbohydrates for ensilage. Wilting had no effect on alfalfa buffer capacity.

Considerations about enamel microabrasion after 18 years

Purpose: To review of the current status of enamel microabrasion method and its results 18 years after the development and application of this method. Methods: A technique performing enamel microabrasion with hydrochloric acid mixed with pumice and other techniques employing a commercially available compound of hydrochloric acid and fine-grit silicon carbide particles in a water-soluble paste have been described. Much has been learned about the application of this esthetic technique, long-term treatment results and microscopic changes to the enamel surface that has significant clinical implications. The latest treatment protocol is presented and photographic case histories document the treatment results. Clinical observations made over 18 years are discussed. Results: According to our findings, the dental enamel microabrasion technique is a highly satisfactory, safe and effective procedure.

COX23, a homologue of COX17, is required for cytochrome oxidase assembly

Deletion of reading frame YHR116W of the Saccharomyces cerevisiae nuclear genome elicits a respiratory deficiency. The encoded product, here named Cox23p, is shown to be required for the expression of cytochrome oxidase. Cox23p is homologous to Cox17p, a water-soluble copper protein previously implicated in the maturation of the Cu-A center of cytochrome oxidase. The respiratory defect of a cox23 null mutant is rescued by high concentrations of copper in the medium but only when the mutant harbors COX17 on a high copy plasmid. Overexpression of Cox17p by itself is not a sufficient condition to rescue the mutant phenotype. Cox23p, like Cox17p, is detected in the intermembrane space of mitochondria and in the postmitochondrial supernatant fraction, the latter consisting predominantly of cytosolic proteins. Because Cox23p and Cox17p are not part of a complex, the requirement of both for cytochrome oxidase assembly suggests that they function in a common pathway with Cox17p acting downstream of Cox23p.

In vitro and in vivo effects of clove on pro-inflammatory cytokines production by macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fine structure of Mytella falcata (Bivalvia) gill filaments

Bivalve filter feeders are sessile animals that live in constant contact with water and its pollutants. Their gill is an organ highly exposed to these conditions due to its large surface and its involvement in gas exchanges and feeding. The bivalve Mytella falcata is found in estuaries of Latin America, on the Atlantic as well as the Pacific Coast. It is commonly consumed, and sometimes is the only source of protein of low-income communities. In this study, gill filaments of M. falcata were characterized using histology, histochemistry and transmission electron microscopy for future comparative studies among animals exposed to environmental pollutants. Gill filaments may be divided into abfrontal, intermediate and frontal zones. Filaments are interconnected by ciliary discs. In the center of filaments, haemocytes circulate through a haemolymph vessel internally lined by an endothelium and supported by an acellular connective tissue rich in polysaccharides and collagen. The abfrontal zone contains cuboidal cells, while the intermediate zone consists of a simple squamous epithelium. The frontal zone is composed of five columnar cell types: one absorptive, mainly characterized by the presence of pinocytic vesicles in the apical region of the cell; one secretory, rarely observed and three ciliated with abundant mitochondria. All cells lining the filament exhibit numerous microvilli and seem to absorb substances from the environment. PAS staining was observed in mucous cells in the frontal and abfrontal zones. Bromophenol blue allowed the distinction of haemocytes and detection of a glycoprotein secretion in the secretory cells of the frontal region. The characteristics of M. falcata gill filaments observed in this study were very similar to those of other bivalves, especially other Mytilidae, and are suitable for histopathological studies on the effect of water-soluble pollutants. (C) 2007 Elsevier Ltd. All rights reserved.

Paracellular Absorption: A Bat Breaks the Mammal Paradigm

Bats tend to have less intestinal tissue than comparably sized nonflying mammals. The corresponding reduction in intestinal volume and hence mass of digesta carried is advantageous because the costs of flight increase with load carried and because take-off and maneuverability are diminished at heavier masses. Water soluble compounds, such as glucose and amino acids, are absorbed in the small intestine mainly via two pathways, the transporter-mediated transcellular and the passive, paracellular pathways. Using the microchiropteran bat Artibeus literatus (mean mass 80.6 +/- 3.7 g), we tested the predictions that absorption of water-soluble compounds that are not actively transported would be extensive as a compensatory mechanism for relatively less intestinal tissue, and would decline with increasing molecular mass in accord with sieve-like paracellular absorption. Using a standard pharmacokinetic technique, we fed, or injected intraperitonealy the metabolically inert carbohydrates L-rhamnose (molecular mass = 164 Da) and cellobiose (molecular mass = 342 Da) which are absorbed only by paracellular transport, and 3-O-methyl-D-glucose (3OMD-glucose) which is absorbed via both mediated (active) and paracellular transport. As predicted, the bioavailability of paracellular probes declined with increasing molecular mass (rhamnose, 90 +/- 11%; cellobiose, 10 +/- 3%, n = 8) and was significantly higher in bats than has been reported for laboratory rats and other mammals. In addition, absorption of 3OMD-glucose was high (96 +/- 11%). We estimated that the bats rely on passive, paracellular absorption for more than 70% of their total glucose absorption, much more than in non-flying mammals. Although possibly compensating for less intestinal tissue, a high intestinal permeability that permits passive absorption might be less selective than a carrier-mediated system for nutrient absorption and might permit toxins to be absorbed from plant and animal material in the intestinal lumen.

Matéria orgânica em um Latossolo submetido a diferentes sistemas de produção de milho

Com objetivo de avaliar o efeito de sistemas de produção sobre a dinâmica do carbono no solo e nas frações da matéria orgânica, durante seis anos, em Jaboticabal (SP), foram testados diferentes tratamentos, a saber: semeadura convencional de milho com pousio no inverno (C-Mi-P), plantio direto de milho e pousio no inverno (D-Mi-P), convencional de milho em rotação com soja e pousio no inverno (C-Mi-P-So), plantio direto de milho em rotação com soja e pousio no inverno (D-Mi-P-So) e plantio direto de milho com uso de Mucuna aterrina (mucuna preta), Cajanus cajan (feijão guandu) e Crotalaria juncea no inverno (D-Mi-Mu, D-Mi-Gu e D-Mi-Cr) em delineamento de blocos ao acaso e parcelas subdivididas. Após 60 dias da emergência das plântulas, coletaram-se amostras de solo a diferentes profundidades (0-0,05, 0,05-0,10 e 0,10-0,20 m). Avaliaram-se nas amostras os teores de C orgânico (CO) e C total nas frações: matérias húmicas (MH), solúvel em água (SA), ácido fúlvico (AF), ácido húmico (AH) e humina (HN) com dados expressos em base de terra seca em estufa (TSE). O uso do sistema plantio direto com pousio e o cultivo de leguminosas no inverno (mucuna preta e feijão guandu) foram os que apresentaram maiores valores de CO e C-HN na camada de 0-0,05 m. O tratamento C-Mi-P-So mostrou o maior valor de C-SA na camada de 0,05-0,10 m. Quanto aos teores de C-MH, os tratamentos C-Mi-P-So e D-Mi-P-So foram os que apresentaram os maiores valores nas camadas superficiais. O plantio direto de milho em monocultura e sucessão com leguminosas (mucuna preta e feijão guandu) parecem ter favorecido dois processos: migração de AF para as camadas mais profundas, reduzindo os valores de MH nas camadas superficiais do solo, e interconversões de AF em AH mais rápidas.

Separação e identificação de constituintes químicos polares dos galhos de Porcelia Macrocarpa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mecanismos da intoxicação do fígado de rato causada pelo gossipol

O fígado desempenha uma função central no metabolismo devido à sua interposição entre o trato digestivo e a circulação geral do organismo. Ele é também o principal órgão envolvido na biotransformação de substâncias exógenas (xenobióticos), com capacidade de converter compostos hidrofóbicos em hidrossolúveis, mais facilmente eliminados pelo organismo. O gossipol é uma substância fenólica tóxica presente na semente de algodão (Gossypium sp). Com o objetivo de estudar os mecanismos envolvidos na hepatotoxicidade do gossipol avaliou-se os seus efeitos no sistema antioxidante do fígado de ratos no que diz respeito ao estresse oxidativo e aspectos histopatológicos. Foram utilizados ratos machos da linhagem Wistar, separados em dois grupos, sendo que um recebeu óleo de canola (veículo, grupo Controle) e o outro recebeu gossipol na dosagem de 40 mg/kg de peso vivo do animal por 15 dias (grupo Tratado). O tratamento com gossipol promoveu alterações na atividade sérica das enzimas marcadoras de dano hepático e um significativo estresse oxidativo caracterizado pela diminuição nos níveis da glutationa reduzida (GSH) e consequente aumento da glutationa oxidada (GSSG), incluindo, ainda, danos à membrana plasmática e de organelas demonstrados pela peroxidação lipídica. O resultado da avaliação histopatológica demonstrou degeneração dos hepatócitos.

Potencial alelopático de forrageiras tropicais: efeitos sobre invasoras de pastagens

Extratos aquosos de sementes, parte aérea e raízes de três gramíneas e três leguminosas forrageiras foram preparados a uma concentração de 10%, com o objetivo de avaliar os efeitos potencialmente alelopáticos sobre a germinação de sementes e o alongamento da radícula das invasoras de pastagens: desmódio, guanxuma e assa-peixe. A germinação foi monitorada em períodos de dez dias, com contagens diárias e eliminação das sementes germinadas. O alongamento da radícula era medido ao final de um período de dez dias de crescimento. Os efeitos do potencial osmótico foram isolados através de cálculos. As espécies doadoras evidenciaram potencialidades alelopáticas que variou de intensidade em função da especificidade entre espécies doadoras e receptoras. B. brizantha e calopogônio foram as espécies que promoveram as reduções mais intensas sobre a germinação das sementes e o alongamento da radícula das espécies receptoras. A parte aérea das espécies doadoras constituiu-se na principal fonte de substâncias potencialmente alelopáticas, solúveis em água. Independentemente da espécie doadora, desmódio e guanxuma foram as invasoras que se mostraram mais susceptíveis aos efeitos potencialmente alelopáticos, enquanto o assapeixe foi a mais tolerante.