Efeitos clínicos e microbiológicos do óleo de copaíba (copaifera officinalis) sobre bactérias formadoras de placa dental em cães / Clinical and microbiological effects of copaiba oil (copaifera officinalis) on dental plaque forming bacteria in dogs

O potencial de uso do óleo de copaíba (Copaifera officinalis) na prevenção da doença periodontal, eliminando seu agente etiológico, foi avaliado em 18 cães sem raça definida, distribuídos homogeneamente em três grupos: teste, (contendo óleo de copaíba) controle positivo e controle negativo. Os tratamentos ocorreram três vezes ao dia, durante oito dias. Ao nono dia, os animais receberam aplicação tópica de fucsina básica 0,5 por cento para evidenciação do biofilme. Mudanças na halitose e gengivite foram avaliadas diariamente por inspeção visual. Adicionalmente, foram realizados testes laboratoriais de inibição de aderência de Streptococcus mutans e ensaio antimicrobiano de difusão em ágar, sobre bactérias formadoras de placa dental. Os resultados da placa evidenciada apontaram áreas de cobertura microbiana nos dentes de 53,4±8,8 por cento, 28,5±5,4 por cento, e 22,3±5,3 por cento para os grupos negativo, positivo e teste, respectivamente, indicando diferença entre o controle negativo e os demais grupos (P<0,05). Quanto à melhora nos aspectos clínicos, halitose e gengivite, o grupo teste respondeu melhor quando comparado ao grupo controle negativo (P<0,05). A análise dos ensaios de difusão e inibição de aderência mostrou superioridade do grupo da copaíba (teste) em relação aos outros grupos (P<0,05). Os resultados sugerem o uso do óleo de copaíba na prevenção da doença periodontal e como um possível substituto da clorexidina na terapia antimicrobiana oral.

The copaiba oil (Copaifera officinalis) potential was evaluated in preventing periodontal disease and reducing its etiology. For that 18 mongrel dogs were homogeneously distributed in three groups: test (copaiba oil), positive control (chlorexidine) and negative control. The treatments were carried out three times a day, during eight days. On the 9th day, the animals were tested with a 0.5 percent basic solution of fuchsin for the detection of biofilm. Changes in halitosis and gingivitis were daily observed. In addition, the following laboratory tests were done: inhibition of the adherence of Streptococcus mutans, and plaque forming bacteria antimicrobial assays by the agar diffusion method. The results of the fuchsin test showed that dental plaque reached areas of 53.4±8.8 percent, 28.5±5.4 percent, and 22.3±5.3 percent in the negative control, positive control, and test groups, respectively, showing differences between dogs from the negative control group and dogs from the other two groups (P<0.05). Furthermore, halitosis and gingivitis decreased in the copaiba group animals when compared with the negative group (P<0.05). The results of the attachment inhibition and agar diffusion tests showed that copaiba induced better effects against the microorganisms as compared to the results of the other groups (P<0.05). These findings suggest that copaiba oil may effectively replace chlorexidine for oral antimicrobial therapy and prevention of periodontal disease.

Anti-inflammatory and cicatrizing activities of a carbohydrate fraction isolated from sugary kefir.

Kefir is an association of microrganisms generally grown in milk, with known probiotic activities identified from its soured suspensions. Aqueous media are also able to grow kefir, but little is known about the probiotic properties of its fermented products. This work aimed to evaluate some probiotic properties of a carbohydrate fraction isolated from sugary kefir (sugary kefir carbohydrate [SKC]). Anti-inflammatory activity of the isolated fraction of carbohydrate was tested both in vitro (cellular respirometry and macrophage culture) and in vivo (50% effective dose, rat paw edema, vascular permeability, and cicatrizing test). The results indicated no significant difference for oxygen uptake or macrophage culture between control and test groups. Rat paw edema, however, showed a significant inhibitory activity by 30 +/- 4% and 54 +/- 8% (P < .001) for carrageenan and dextran, respectively. In the cicatrizing test, animals treated with SKC cream also presented less trauma after treatments as compared to the negative control group (P < .05). The overall data suggested the SKC as a natural product that could be used as a constituent of an anti-inflammatory compound.

Anti-inflammatory properties of kefir and its polysaccharide extract.

Kefir is a fermented beverage originating form the Caucasian regions composed of a number of bacteria and yeasts living together in polysaccharide grains secreted by them. Kefir can be considered a probiotic source as it presents anti-bacterial, anti-mycotic, anti-neoplasic and immunomodulatory properties. Aiming to appraise a possible anti-inflammatory effect of kefir we conducted cotton-induced granuloma and paw oedema assays in rats, the latter using carrageenan, dextran and histamine as stimuli. Kefir samples were thawed and continuously cultured during 15 days both in a molasses solution (50 g/l) and in cow's milk. A polysaccharide extract isolated from the grains (kefiran) was also tested in cotton-pellet experiments. The results showed significant inhibition in the formation of granuloma tissue for all the test groups, as compared to the blank group. Kefir suspensions in molasses presented an inhibition of 41 +/- 3% for the inflammatory process, fermented milk prepared from kefir showed 44 +/- 6% inhibition and kefiran extract 34 +/- 15%. Rat paw oedema also showed significant decreases with the mediators. Dextran-induced oedema was completely inhibited at 1 h after input, with a 76% inhibition after 2 h. Carrageenan stimulus was inhibited 62% after the 3rd hour, and histamine by 52% after the 2nd hour. These results points to the existence of anti-inflammatory prebiotic compounds present in symbiotic cultures of kefir growing in both aqueous and milky suspensions.

Study of anti-inflammatory activity of Tibetan mushroom, a symbiotic culture of bacteria and fungi encapsulated into a polysaccharide matrix.

Tibetan mushroom (TM) is a fermented beverage composed by a dozen of bacteria and yeasts living together into polysaccharide grains secreted by them. TM is similar to kefir, a probiotic beverage originated in the Caucasian mountains exhibiting some anti-bacterial, anti-mycotic, anti-neoplastic and immunomodulatory effects. Aiming to evaluate a plausible anti-inflammatory property of TM we conducted cotton-induced granuloma and paw edema assays in rats, the latter using carrageenin, dextran and histamine as stimuli. TM samples were thawed and continuously cultured during 15 days into molasses solutions (50 g/l). The experiments used TM suspensions after 24h fermentation and TM grains mechanically disintegrated. The results showed a significant inhibition on the formation of granuloma tissue for the test group as compared to the negative control group. TM suspensions presented an inhibition of 43% for the inflammatory process. Rat paw edema also showed significant decreases with the mediators. The edema induced by carrageenin was inhibited 62% at the 3rd hour. The edema dextran-induced was completely inhibited at 1h and antagonized the histamine edema 52% at 1h.

Structural stabilization displayed in the soluble phase of cocrystallization of flavianic acid with trypsin in acid media.

Coprecipitation and cocrystallization of proteins with synthetic dyes are known to involve reversible denaturation processes which can offer specificity towards a target protein. Although the knowledge of conformational equilibrium and how to control it are central into the basic molecular dynamics of protein precipitation, the exact molecular mechanism of the precipitation remains unknown. Aiming at understanding the events that take place before the coprecipitation step of generic dye-protein systems, we investigated the binding of flavianic acid to bovine trypsin, using approaches of visible and second-derivative ultraviolet spectroscopy, viscosimetry, densimetry and circular dichroism. The results suggest a restricted transconformation of the macromolecule linked to dye binding at a stoichiometry of 1:1. An increase on the protein secondary structures occurred together with an electro-constriction effect on trypsin, a borderline event to the coprecipitation process, suggesting a stabilized structure for trypsin as a ligand-induced molten globule-like state.

A time-dependent, two-step binding mode of the nitro dye flavianic acid to trypsin in acid media

Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule

A time-dependent, two-step binding mode of the nitro dye flavianic acid to trypsin in acid media.

Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule.

Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine ß-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using a-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of ß-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 µM; kinetic parameters for the substrate hydrolysis were: Km = 32 µM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme

Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0.

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine beta-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using alpha-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of beta-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 microM; kinetic parameters for the substrate hydrolysis were: Km = 32 microM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme.