Nanoemulsões contendo óleo de Pelargonium graveolens : estudos de estabilidade e atividade antibiofilme em material médico hospitalar

As pesquisas por substâncias de origem natural, como óleos essenciais, têm aumentado progressivamente, devido principalmente à resistência dos microrganismos aos fármacos comumente comercializados. A finalidade de aumentar o arsenal terapêutico deve-se ao índice cada vez mais elevado de infecções microbianas, principalmente no ambiente hospitalar. Neste contexto, o presente trabalho utilizou o óleo de Pelargonium graveolens, conhecido popularmente como óleo de gerânio (GO) com o objetivo de verificar suas possíveis atividades antimicrobianas, antioxidantes, anti-inflamatórias e antibiofilme. Sabe-se que entre seus constituintes majoritários encontram-se o citronelol e o geraniol e os mesmos são responsáveis por algumas destas atividades citadas. A utilização do óleo puro não é muito vantajosa, pois a concentração dos constituintes pode ser afetada pela volatilização. A estratégia utilizada neste trabalho foi o desenvolvimento de sistemas nanoestruturados, como as nanocápsulas e nanoemulsões, a fim de aumentar e melhorar as atividades do óleo de gerânio. Após a realização dos diferentes testes, verificouse que o óleo e as nanoestruras possuem atividade antifúngica e quando testados em material médico, demonstraram atividade antibiofilme. A atividade antioxidante demonstrou ser maior no óleo livre, entretanto quando as amostras foram utilizadas em macrófagos, as nanoemulsões demonstraram ter uma melhor atividade anti-inflamatória. O presente trabalho revela a importância do desenvolvimento de nanoestruturas contendo óleo de gerânio como uma alternativa no combate a formação de biofilmes em dispositivos médicos hospitalares.Research by substances of natural origin, essential oils, have steadily increased, mainly due to the resistance of microorganisms to commonly marketed drugs. The purpose of increasing the therapeutic arsenal due to the increasingly high rate of microbial infections, particularly in the hospital environment. In this context, this study used the oil Pelargonium graveolens, popularly known as geranium oil (GO) in order to verify their possible antimicrobial activity, antioxidant, anti-inflammatory and antibiofilm. It is known that among its major constituents are citronellol and geraniol and are responsible for some of these cited activities. The use of pure oil is not very advantageous, because the concentrations of constituents may be affected by volatilization. The strategy used in this study was the development of nanostructured systems, such as nanocapsules and nanoemulsions in order to increase and improve the activities of geranium oil. After performing various tests, it was found that oil and nanoestruras have antifungal activity when assayed in medical and material shown antibiofilm activity. The antioxidant activity was shown to be higher in oil free, but when the samples were used in macrophages, nanoemulsions shown to have better anti-inflammatory activity. This study reveals the importance of developing nanostructures containing geranium oil as an alternative to combat the formation of biofilms in hospital medical devices

ANTIMICROBIAL PEPTIDE P34 INFLUENCES GENE EXPRESSION OF LISTERIA MONOCYTOGENES GROWING IN SOFT CHEESE

Objective: To evaluate whether antimicrobial substances produced by autochthonous lactic acid bacteria (LAB) from Minas Frescal cheese are able to enhance the activity of bacteriocin P34 against Listeria monocytogenes and investigate the influence of P34 in specific gene expression of this bacterium after the inoculation in Minas Frescal cheese.Methods: Bacillus sp. P34 and L. monocytogenes ATCC 7644 were used in this study. The antimicrobial peptide P34 was purified and applied (0, 800 or 6400 AU/ml) to cheese surface before inoculation with L. monocytogenes. Antimicrobial activity and synergism were detected using the agar diffusion technique. Expression levels of D-Alanine-D-alanyl carrier protein ligase (dltA), Putative phospholipid lysinylation (Imo 1695) and EIIABMan of mannose-specific PTS (mptA) mRNAs in bacteriocin-treated L. monocytogenes growing in Minas Frescal cheese were determined using real-time PCR.Results: The peptide P34 showed increased antilisterial activity when combined with culture supernatants of some selected LAB isolated from Minas Frescal cheese. The addition of peptide P34 to cheese caused a decrease of up to 3 log cycles in viable counts of artificially inoculated L. monocytogenes. The influence of peptide P34 on the expression of genes associated with components of the cell surface of L. monocytogenes was investigated by real-time PCR. A significant increase in the expression of the genes dltA, Imo 1695 and mptA was observed after 96 h in the presence of peptide P34.Conclusion: These results suggest that the peptide P34 influences the expression of genes involved in D-alanylation of teichoic acids and lipoteichoic acids and lysination of the cell membrane of phospholipids

INFLUENCE OF PEPTIDE P34 ON GENE EXPRESSION OF LISTERIA MONOCYTOGENES AND LISTERIA SEELEGERI

Objective: Investigate the influence of the antimicrobial peptides P34 and nisin on the expression of genes associated with components of the cell surface of Listeria monocytogenes and Listeria seeligeri.Methods: Antimicrobial activity was determined by addition of peptide P34 and nisin (12.5 µg/ml) onto Brain Heart Infusion agar (BHI) plates previously inoculated with indicator strains (L. monocytogenes ATCC 7644 or L. seeligeri AC 82/4) after incubation for 24 h at 37 °C or 240 h at 4 °C. Ribonucleic acid (RNA) was directly extracted from bacterial colonies at the border of the inhibition zones, and the expression levels of genes D-alanine-D-alanyl carrier protein ligase (dltA), putative phospholipid lysinylation (Imo 1695) and EIIABMan of mannose-specific PTS (mptA) were determined using real-time PCR.Results: A non-significant increase in the levels of transcription of genes dltA, Imo1695 and mptA was observed for L. monocytogenes treated with peptide P34 or nisin. Both peptides caused a similar decrease in dltA gene expression in L. seeligeri. The expression of gene Imo1695 significantly decreased (about 2000-fold) after treatment with the peptide P34 at 37 °C, while at 4 °C a reduction of 12-fold and 5-fold was detected for P34 and nisin, respectively. A significant decrease in mptA gene expression was observed by exposition to peptide P34 (31.872-fold) and nisin (16.047-fold) for 24 h at 37 °C.Conclusion: The results suggest that both peptide P34 and nisin influence the expression of genes related with the cell-surface/cell-membrane structure of L. seeligeri and in lesser extent L. monocytogenes

ANTIMYCOBACTERIAL, ANTIMICROBIAL AND ANTIFUNGAL ACTIVITIES OF GERANIUM OIL-LOADED NANO CAPSULES

Objective: The aim of this study was to perform the first ever investigation of the effect of activities in the nano capsules containing Geranium oil (NC1) against different species of pathogens such as Mycobacterium genus (both fast growing and slow growing), bacterial, and yeasts.Methods: The GO was analyzed by GC and GC/MS. Nano capsule suspensions (NC) were prepared by interfacial deposition of a preformed polymer method and the MICs were determined for the antimycobacterial, antimicrobial, and antifungal activities.Results: GO-loaded nano capsules (NC1) presented nano metric mean diameters (188 nm), polydispersity indices below 0.149, pH (5.5), and zeta potentials (about-10.8 mV). The MICs were determined for the antimycobacterial, antimicrobial, and antifungal activities. The NC1 was effective to Mycobacterium smegmatis (149.7 µg ml-1), M. abscessos (35.9 µg ml-1), M. massiliense (35.9 µg ml-1), M. avium (71.8 µg ml-1), Enterococcus faecalis, Streptococcus sp. (149.7 µg ml-1) and Listeria monocytogenes (35.9 µg ml-1). The NC1 was able to significantly reduce the number of cells of C. albicans (by approximately 5 log), 4 log the number of cells of C. dublinensis, C. glabrata, and C. krusei, and 2 log the number of cells of C. parapsilosis compared to the control group.Conclusion: Our study showed that the geranium oil-loaded nano capsules have antimycobacterial activities similar to free oil. The GO was effective in inhibiting the formation of germ tubes of Candida albicans, yet the nano capsule containing GO failed to inhibit the formation of this important virulence factor.Â

DEVELOPMENT OF NANOEMULSION CONTAINING PELARGONIUM GRAVEOLENS OIL: CHARACTERIZATION AND STABILITY STUDY

Objective: To develop, characterize and evaluate the stability of nanoemulsions containing geranium oil (NEG) at different temperatures (4 °C, 25 °C and 45 °C) for 90 d.Methods: The quantification of oil in the nanostructure was performed by gas chromatography–mass spectrometry (GC-MS). The NEG was prepared in Ultra-Turrex and characterized by determining the particle size, polydispersity indices and pH. The thermo gravimetric analysis (TGA), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM) to evaluate the thermal stability of the compounds, the thermal events and morphological analyses of NEG, respectively.Results: The results allow us to suggest that the use the ultra-turrax method is a strategy good to NEG preparation. The stability of the NEG was strongly influenced by storage temperature, with droplet size increasing rapidly at higher temperatures (45 °C), which was attributed to coalescence near the phase inversion temperature. The NEG submitted the low temperatures (4±2 °C) remained with the same particle size value (164 nm). However, the citronellol and geraniol showed a significant reduction throughout the test even in these conditions of temperature. Thermogram of NEG shows the crystallization peak at the cooling cycle in-20.1 °C and a melting was observed at 1.5 °C. TEM images indicated that NEG was spherical and nanometric.Conclusion: The proposed Ultra-Turrax method is simple which prevents volatilization of GO for the production of NEG. The formulations presented good physicochemical characteristics and stability for 90 d was only achieved in 4 °C

Resveratrol and resveratrol-hydroxypropyl-β-cyclodextrin complex recovered the changes of creatine kinase and Na+, K+-ATPase activities found in the spleen from streptozotocin-induced diabetic rats

Type 1 diabetes (T1D) is the result of the selective destruction of the pancreatic β-cells by T cells of the immune system. Although spleen is a secondary lymphoid organ, it is also involved in the T1D pathogenesis. However, the alterations in a variety of cellular processes of this disease need to be further understood. We aimed to analyze the benefits of resveratrol, and its complexed form on diabetic complications in the spleen of rats. To this end, we investigated important enzymes of phosphoryl transfer network, and Na+, K+-ATPase activity. Wistar rats were divided into non-diabetic groups: Control, Ethanol, Resveratrol, Hydroxypropyl-β-cyclodextrin, Resveratrol-hydroxypropyl-β-cyclodextrin, and diabetic groups with the same treatments. Diabetes was induced by a single dose of 60 mg/kg of streptozocin intraperitoneally, and treatments by intragastric gavage once daily for 60 days. Hyperglycemia reduced creatine kinase activity, which was reversed by the administration of resveratrol. Na+, K+-ATPase activity was greatly affected, but it was reversed by resveratrol and resveratrol-hydroxypropyl-β-cyclodextrin. This suggest an energetic imbalance in the spleen of diabetic rats, and in case this also occurs in the diabetic patients, it is possible that resveratrol supplementation could be beneficial to the better functioning of the spleen in diabetic patients

Production, characterization and application of proteases produced by strains of Bacillus sp

Algumas enzimas são encontradas facilmente em diferentes microrganismos do ambiente e requerem um destaque especial devido a seu grande valor econômico. As proteases apresentam grande diversidade bioquímica, sendo facilmente manipuláveis, favorecendo dessa maneira suas aplicações biotecnológicas. Três linhagens bacterianas de Bacillus sp. foram isoladas com o objetivo de produzir protease e determinar as condições ótimas de temperatura, pH, e efeito de substratos e substâncias químicas sobre a atividade da enzima, assim como verificar a atividade depilatória e a utilização como agentes de modificação de proteína. As linhagens de Bacillus sp. apresentaram halos de atividade proteolítica em placas de Ágar Leite em diferentes pHs. O padrão proteolítico das enzimas em extrato bruto utilizando inibidores de protease indica que são serina-proteases, com alta especificidade pelo substrato azocaseína, pH e temperatura ótimos de 9,0 e 37ºC, respectivamente. As enzimas mantiveram-se estáveis à 37ºC por 30 minutos, entretanto quando submetidas a uma temperatura de 55ºC, perderam 50% da atividade inicial nos primeiros 20 minutos. A atividade enzimática foi parcialmente inibida por PMSF e benzamidina e totalmente por HgCl2. A adição de detergentes como o SDS ou o Triton X100 ocasionou um leve aumento na atividade das enzimas. Os sobrenadantes das linhagens de Bacillus sp. apresentaram enzimas com a capacidade de promover depilação de peles bovinas. Exames histológicos demonstraram que o tratamento com as enzimas não acarretou danos ao colágeno. Esses resultados indicam que as proteases produzidas pelos isolados apresentaram potencial para aplicação em processos envolvendo hidrólise de queratina, além da utilização como agente de modificação de proteínas.Some enzymes are easily found in different microorganisms of the environment and they require a special prominence due its great economic value. Proteases present great biochemistry diversity, being easily manipulated allowing in this way, their biotechnological applications. Three Bacillus sp. strains were isolated intending to produce protease and determine optimal conditions of temperature, pH, and effect of chemical substances on the activity of the enzyme, as well as to verify the depilatory activity and utilization as agents protein modification. Bacillus sp. strains showed proteoyitic activity in milk plates in different pH.The proteolytic characteristic of enzymes in crude extract using protease inhibitors indicates that they are serine-proteases, with high specificity to azocasein, with optimal pH and temperature at 9,0 and 37oC, respectively. The enzymes have been kept stable at 37oC for 30 minutes. Nevertheless, when they were subjected to 55oC they lost 50% of their initial activity in the first 20 minutes. The enzymatic activity was partially inhibited by PMSF and benzamidine and totally by HgCl2. The addition of detergents, as SDS and Triton X100, caused a light increase on enzymes activity. The supernatants of Bacillus sp. strains showed enzymes with capacity to depilate bovine skin. Histological evaluation showed that the treatment with enzymes do not promote damage to collagen. These results indicate that proteases produced by isolated present a powerful application in processes involving keratin hydrolysis, as well as the use as an agent that modifies proteins

Production, characterization and application of proteases produced by strains of Bacillus sp

Algumas enzimas são encontradas facilmente em diferentes microrganismos do ambiente e requerem um destaque especial devido a seu grande valor econômico. As proteases apresentam grande diversidade bioquímica, sendo facilmente manipuláveis, favorecendo dessa maneira suas aplicações biotecnológicas. Três linhagens bacterianas de Bacillus sp. foram isoladas com o objetivo de produzir protease e determinar as condições ótimas de temperatura, pH, e efeito de substratos e substâncias químicas sobre a atividade da enzima, assim como verificar a atividade depilatória e a utilização como agentes de modificação de proteína. As linhagens de Bacillus sp. apresentaram halos de atividade proteolítica em placas de Ágar Leite em diferentes pHs. O padrão proteolítico das enzimas em extrato bruto utilizando inibidores de protease indica que são serina-proteases, com alta especificidade pelo substrato azocaseína, pH e temperatura ótimos de 9,0 e 37ºC, respectivamente. As enzimas mantiveram-se estáveis à 37ºC por 30 minutos, entretanto quando submetidas a uma temperatura de 55ºC, perderam 50% da atividade inicial nos primeiros 20 minutos. A atividade enzimática foi parcialmente inibida por PMSF e benzamidina e totalmente por HgCl2. A adição de detergentes como o SDS ou o Triton X100 ocasionou um leve aumento na atividade das enzimas. Os sobrenadantes das linhagens de Bacillus sp. apresentaram enzimas com a capacidade de promover depilação de peles bovinas. Exames histológicos demonstraram que o tratamento com as enzimas não acarretou danos ao colágeno. Esses resultados indicam que as proteases produzidas pelos isolados apresentaram potencial para aplicação em processos envolvendo hidrólise de queratina, além da utilização como agente de modificação de proteínas.Some enzymes are easily found in different microorganisms of the environment and they require a special prominence due its great economic value. Proteases present great biochemistry diversity, being easily manipulated allowing in this way, their biotechnological applications. Three Bacillus sp. strains were isolated intending to produce protease and determine optimal conditions of temperature, pH, and effect of chemical substances on the activity of the enzyme, as well as to verify the depilatory activity and utilization as agents protein modification. Bacillus sp. strains showed proteoyitic activity in milk plates in different pH.The proteolytic characteristic of enzymes in crude extract using protease inhibitors indicates that they are serine-proteases, with high specificity to azocasein, with optimal pH and temperature at 9,0 and 37oC, respectively. The enzymes have been kept stable at 37oC for 30 minutes. Nevertheless, when they were subjected to 55oC they lost 50% of their initial activity in the first 20 minutes. The enzymatic activity was partially inhibited by PMSF and benzamidine and totally by HgCl2. The addition of detergents, as SDS and Triton X100, caused a light increase on enzymes activity. The supernatants of Bacillus sp. strains showed enzymes with capacity to depilate bovine skin. Histological evaluation showed that the treatment with enzymes do not promote damage to collagen. These results indicate that proteases produced by isolated present a powerful application in processes involving keratin hydrolysis, as well as the use as an agent that modifies proteins

Trypanocidal activity of the compounds present in Aniba canelilla oil against Trypanosoma evansi and its effects on viability of lymphocytes

Aniba canelilla (H.B.K.) Mez, popularly known as “casca-preciosa” (precious bark), is a plant of the Lauraceae family, widely distributed in the Amazon region. Its major constituent is 1-nitro-2-phenylethane, a rare molecule in plants which is responsible for this plant's cinnamon scent. The present study aimed to report the chemical characterization of the oil extracted from Aniba canelilla using gas-chromatography/mass spectrometry and to assess its in vitro trypanocidal activity against Trypanosoma evansi, a prevalent haemoflagellate parasite that affects a broad range of mammal species in Africa, Asia and South America. The oil presented 1-nitro-2-phenylethane (83.68%) and methyleugenol (14.83%) as the two major components. The essential oil as well as both major compounds were shown to exert trypanocidal effect. Methyleugenol was slightly more active than 1-nitro-2-phenylethane. In vitro studies showed that the oil extracted from the stems of A. canelilla may be regarded as a potential natural treatment for trypanosomosis, once proven their in vivo action, may be an interesting alternative in the treatment of infected animals with T. evansi. © 2016 Elsevier Lt

Resveratrol and resveratrol-hydroxypropyl-β-cyclodextrin complex recovered the changes of creatine kinase and Na+, K+-ATPase activities found in the spleen from streptozotocin-induced diabetic rats

Type 1 diabetes (T1D) is the result of the selective destruction of the pancreatic β-cells by T cells of the immune system. Although spleen is a secondary lymphoid organ, it is also involved in the T1D pathogenesis. However, the alterations in a variety of cellular processes of this disease need to be further understood. We aimed to analyze the benefits of resveratrol, and its complexed form on diabetic complications in the spleen of rats. To this end, we investigated important enzymes of phosphoryl transfer network, and Na+, K+-ATPase activity. Wistar rats were divided into non-diabetic groups: Control, Ethanol, Resveratrol, Hydroxypropyl-β-cyclodextrin, Resveratrol-hydroxypropyl-β-cyclodextrin, and diabetic groups with the same treatments. Diabetes was induced by a single dose of 60 mg/kg of streptozocin intraperitoneally, and treatments by intragastric gavage once daily for 60 days. Hyperglycemia reduced creatine kinase activity, which was reversed by the administration of resveratrol. Na+, K+-ATPase activity was greatly affected, but it was reversed by resveratrol and resveratrol-hydroxypropyl-β-cyclodextrin. This suggest an energetic imbalance in the spleen of diabetic rats, and in case this also occurs in the diabetic patients, it is possible that resveratrol supplementation could be beneficial to the better functioning of the spleen in diabetic patients