RESUMO
BACKGROUND: Low targeting efficacy and high toxicity continue to be challenges in Oncology. A promising strategy is the glycosylation of chemotherapeutic agents to improve their pharmacodynamics and anti-tumoral activity. Herein, we provide evidence of a novel approach using diglycosidases from fungi of the Hypocreales order to obtain novel rutinose-conjugates therapeutic agents with enhanced anti-tumoral capacity. RESULTS: Screening for diglycosidase activity in twenty-eight strains of the genetically related genera Acremonium and Sarocladium identified 6-O-α-rhamnosyl-ß-glucosidase (αRßG) of Sarocladium strictum DMic 093557 as candidate enzyme for our studies. Biochemically characterization shows that αRßG has the ability to transglycosylate bulky OH-acceptors, including bioactive compounds. Interestingly, rutinoside-derivatives of phloroglucinol (PR) resorcinol (RR) and 4-methylumbelliferone (4MUR) displayed higher growth inhibitory activity on pancreatic cancer cells than the respective aglycones without significant affecting normal pancreatic epithelial cells. PR exhibited the highest efficacy with an IC50 of 0.89 mM, followed by RR with an IC50 of 1.67 mM, and 4MUR with an IC50 of 2.4 mM, whereas the respective aglycones displayed higher IC50 values: 4.69 mM for phloroglucinol, 5.90 mM for resorcinol, and 4.8 mM for 4-methylumbelliferone. Further, glycoconjugates significantly sensitized pancreatic cancer cells to the standard of care chemotherapy agent gemcitabine. CONCLUSIONS: αRßG from S. strictum transglycosylate-based approach to synthesize rutinosides represents a suitable option to enhance the anti-proliferative effect of bioactive compounds. This finding opens up new possibilities for developing more effective therapies for pancreatic cancer and other solid malignancies.
Assuntos
Antineoplásicos , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Hypocreales/metabolismo , Rutina/farmacologia , Rutina/química , Acremonium , Gencitabina , Dissacarídeos/farmacologia , Dissacarídeos/químicaRESUMO
The fungal diglycosidase α-rhamnosyl-ß-glucosidase I (αRßG I) from Acremonium sp. DSM 24697 catalyzes the glycosylation of various OH-acceptors using the citrus flavanone hesperidin. We successfully applied a one-pot biocatalysis process to synthesize 4-methylumbellipheryl rutinoside (4-MUR) and glyceryl rutinoside using a citrus peel residue as sugar donor. This residue, which contained 3.5 % [w/w] hesperidin, is the remaining of citrus processing after producing orange juice, essential oil, and peel-juice. The low-cost compound glycerol was utilized in the synthesis of glyceryl rutinoside. We implemented a simple method for the obtention of glyceryl rutinoside with 99 % yield, and its purification involving activated charcoal, which also facilitated the recovery of the by-product hesperetin through liquid-liquid extraction. This process presents a promising alternative for biorefinery operations, highlighting the valuable role of αRßG I in valorizing glycerol and agricultural by-products. KEYPOINTS: ⢠αRßG I catalyzed the synthesis of rutinosides using a suspension of OPW as sugar donor. ⢠The glycosylation of aliphatic polyalcohols by the αRßG I resulted in products bearing a single rutinose moiety. ⢠αRßG I catalyzed the synthesis of glyceryl rutinoside with high glycosylation/hydrolysis selectivity (99 % yield).
Assuntos
Acremonium , Hesperidina , Hesperidina/química , GlicerolRESUMO
BACKGROUND & AIMS: Reaching efficacious drug delivery to target cells/tissues represents a major obstacle in the current treatment of solid malignancies including hepatocellular carcinoma (HCC). In this study, we developed a pipeline to selective add complex-sugars to the aglycone 4-methylumbelliferone (4MU) to help their bioavailability and tumour cell intake. METHODS: The therapeutic efficacy of sugar-modified rutinosyl-4-methylumbelliferone (4MUR) and 4MU were compared in vitro and in an orthotopic HCC model established in fibrotic livers. The mechanistic bases of its selective target to liver tumour cells were evaluated by the interaction with asialoglycoprotein receptor (ASGPR), the mRNA expression of hyaluronan synthases (HAS2 or HAS3) and hyaluronan deposition. RESULTS: 4MUR showed a significant antiproliferative effect on liver tumoural cells as compared to non-tumoural cells in a dose-dependent manner. Further analysis showed that 4MUR is incorporated mostly into HCC cells by interaction with ASGPR, a receptor commonly overexpressed in HCC cells. 4MUR-treatment decreased the levels of HAS2 and HAS3 and the cytoplasmic deposition of hyaluronan. Moreover, 4MUR reduced CFSC-2G activation, hence reducing the fibrosis. In vivo efficacy showed that 4MUR treatment displayed a greater tumour growth inhibition and increased survival in comparison to 4MU. 4MUR administration was associated with a significant reduction of liver fibrosis without any signs of tissue damage. Further, 60% of 4MUR treated mice did not present macroscopically tumour mass post-treatment. CONCLUSION: Our results provide evidence that 4MUR may be used as an effective HCC therapy, without damaging non-tumoural cells or other organs, most probably due to the specific targeting.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Hialuronan Sintases , Himecromona/farmacologia , Himecromona/uso terapêutico , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , CamundongosRESUMO
Diglycosidases are endo-ß-glucosidases that hydrolyze the heterosidic linkage of diglycoconjugates, thereby releasing in a single reaction the disaccharide and the aglycone. Plant diglycosidases belong to the glycoside hydrolase family 1 and are associated with defense mechanisms. Microbial diglycosidases exhibit higher diversity-they belong to the families 3, 5, and 55-and play a catabolic role. As diglycoconjugates are widespread in the environments, so are the microbial diglycosidases, which allow their utilization as nutritional source and carbon recycling. In the last 10 years, six microbial diglycosidases have been sequenced, and for two of them, the three-dimensional structure has been elucidated. This knowledge allowed the identification of their diverse phylogenetic origin, and gave insights into the understanding of the substrate specificity. Here, the last advances and the applications of microbial diglycosidases are reviewed. KEY POINTS: ⢠Substrate specificity and phylogenetic relationships of diglycosidases are reviewed. ⢠On-going and potential applications of diglycosidases are discussed.
Assuntos
Dissacarídeos , Glicosídeo Hidrolases , Tecnologia de Alimentos , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Filogenia , Especificidade por SubstratoRESUMO
Cancer constitutes the second leading cause of death globally and is considered to have been responsible for an estimated 9.6 million fatalities in 2018. Although treatments against gastrointestinal tumors have recently advanced, those interventions can only be applied to a minority of patients at the time of diagnosis. Therefore, new therapeutic options are necessary for advanced stages of the disease. Glycosylation of antitumor agents, has been found to improve pharmacokinetic parameters, reduce side effects, and expand drug half-life in comparison with the parent compounds. In addition, glycosylation of therapeutic agents has been proven to be an effective strategy for their targeting tumor tissue, thereby reducing the doses of the glycodrugs administered to patients. This review focusses on the effect of the targeting properties of glycosylated antitumor agents on gastrointestinal tumors.
RESUMO
AbstractDiglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-ß-glucosidase I and II (αRßG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-ß-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRßG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRßG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRßG I. On the other hand, αRßG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRßG I showed exclusive specificity toward 7-O-ß-rutinosyl flavonoids, whereas αRßG II hydrolyzed both 7-O-ß-rutinosyl- and 3-O-ß-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-ß-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRßG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.
Assuntos
Acremonium/enzimologia , Acremonium/genética , Flavonoides/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Peso Molecular , Filogenia , Pichia/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-ß-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-l-rhamnopyranosyl-ß-d-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ≤6.0. The structure of 4-hydroxyphenyl-ß-rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4-hydroxyphenyl-ß-rutinoside (38%, regarding hesperidin) was achieved in a 2-h process at pH 5.0 and 30 °C, with 36 mM OH-acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pKa value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.
Assuntos
Acremonium/enzimologia , Dissacarídeos/química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Acremonium/genética , Dissacarídeos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosilação , Concentração de Íons de HidrogênioRESUMO
Members of Cohnella sp. isolated from a variety of environments have been shown to be glycoside hydrolase producers. Nevertheless, most evaluations of members of this genus are limited to their taxonomic description. The strain AR92, previously identified as belonging to the genus Cohnella, formed a well-supported cluster with C. thailandensis and C. formosensis (>80% bootstrap confidence). Its growth and xylanase production were approached by using a mineral-based medium containing alkali-pretreated sugarcane bagasse as the main carbon source, which was assayed as a convenient source to produce biocatalysts potentially fitting its degradation. By means of a two-step statistical approach, the production of endoxylanase was moderately improved (20%). However, a far more significant improvement was observed (145%), by increasing the inoculum size and lowering the fermentation temperature to 25°C, which is below the optimal growth temperature of the strain AR92 (37°C). The xylanolytic preparation produced by Cohnella sp. AR92 contained mild temperature-active endoxylanase (identified as redundant GH10 family) for the main activity which resulted in xylobiose and xylo-oligosaccharides as the main products from birchwood xylan.
Assuntos
Bacillales/classificação , Bacillales/enzimologia , Endo-1,4-beta-Xilanases/metabolismo , Bacillales/citologia , Bacillales/crescimento & desenvolvimento , Carbono/metabolismo , Celulose/metabolismo , Meios de Cultura/química , DNA Ribossômico/genética , Dissacarídeos/metabolismo , Fermentação , Oligossacarídeos/metabolismo , Filogenia , Saccharum/metabolismo , Especificidade da Espécie , Temperatura , Xilanos/metabolismoRESUMO
The enzymatic deglycosylation of the plant flavonoid rutin (quercetin-3-O-(6-O-α-l-rhamnopyranosyl-ß-d-glucopyranoside) is usually assessed by means of high performance liquid chromatography (HPLC). We have developed a spectrophotometric method for the quantification of the released quercetin. After the enzymatic reaction, quercetin is extracted with ethyl acetate, and subsequently oxidized under basic conditions. The absorbance of quercetin autooxidation products at 320nm was correlated with the quercetin concentration by linear regression (molar extinction coefficient 23.2 (±0.3)×103M-1cm-1). With this method, rutin-deglycosylation activity in buckwheat flour and a commercial naringinase was measured, and showed no significant differences with the results obtained by HPLC. The convenience of this method resides on the enzymatic activity quantification using the natural substrate by UV-visible spectrometry. Moreover, the simplicity and speed of analysis allows its application for a large number of samples.
Assuntos
Flavonoides/química , Complexos Multienzimáticos/química , Rutina/química , Espectrofotometria/métodos , beta-Glucosidase/química , Cromatografia Líquida de Alta Pressão/métodos , Flavonoides/análise , Rutina/análise , Raios UltravioletaRESUMO
Tritrichomonas foetus is a flagellated protozoan that causes a sexually transmitted disease in cattle. Trichomonosis is characterized by early abortions, subfertility and a significant decrease in productivity. Vaccine preparations containing whole T. foetus can reduce the time of residence of the pathogen in the host cervix after experimental infection. Here, T. foetus vaccines prepared with different adjuvants were tested, in parallel with a commercial vaccine, for their efficacy to clear the infection. The median time for clearance of infection was 69days in non-immunized animals, 55days in animals treated with aluminum hydroxide, 41days with oil-in-water or saponin based vaccines or with a commercial vaccine and 27days in animals treated with saponin plus aluminum hydroxide. A slight increase in the risk of T. foetus clearance from the genital tract was found with the saponin based vaccine (hazard ratio, 2.52; 95% confidence interval, 1.03-6.17) or the commercial vaccine (hazard ratio, 2.61; 95% confidence interval, 1.07-6.38). A significant increase in the risk of T. foetus clearance was found with the combination of saponin plus aluminum hydroxide based vaccine (hazard ratio, 5.12; 95% confidence interval, 2.04-12.83).
Assuntos
Adjuvantes Imunológicos , Doenças dos Bovinos/prevenção & controle , Infecções Protozoárias em Animais/prevenção & controle , Vacinas Protozoárias/imunologia , Tritrichomonas foetus/imunologia , Aborto Animal/prevenção & controle , Hidróxido de Alumínio/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Colo do Útero/parasitologia , Feminino , Feto/parasitologia , Gravidez , Infecções Protozoárias em Animais/parasitologia , Vacinas Protozoárias/administração & dosagem , Saponinas , Tritrichomonas foetus/isolamento & purificação , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vagina/parasitologiaRESUMO
Bacteria represent an underexplored source of diglycosidases. Twenty-five bacterial strains from the genera Actinoplanes, Bacillus, Corynebacterium, Microbacterium, and Streptomyces were selected for their ability to grow in diglycosylated flavonoids-based media. The strains Actinoplanes missouriensis and Actinoplanes liguriae exhibited hesperidin deglycosylation activity (6-O-α-L-rhamnosyl-ß-D-glucosidase activity, EC 3.2.1.168), which was 3 to 4 orders of magnitude higher than the corresponding monoglycosidase activities. The diglycosidase production was confirmed in A. missouriensis by zymographic assays and NMR analysis of the released disaccharide, rutinose. The gene encoding the 6-O-α-L-rhamnosyl-ß-D-glucosidase was identified in the genome sequence of A. missouriensis 431(T) (GenBank accession number BAL86042.1) and functionally expressed in Escherichia coli. The recombinant protein hydrolyzed hesperidin and hesperidin methylchalcone, but not rutin, which indicates its specificity for 7-O-rutinosylated flavonoids. The protein was classified into the glycoside hydrolase family 55 (GH55) in contrast to the known eukaryotic diglycosidases, which belong to GH1 and GH5. These findings demonstrate that organisms other than plants and filamentous fungi can contribute to an expansion of the diglycosidase toolbox.
Assuntos
Proteínas de Bactérias/metabolismo , Chalconas/metabolismo , Hesperidina/análogos & derivados , Hesperidina/metabolismo , Micromonosporaceae/metabolismo , beta-Glucosidase/metabolismo , Proteínas de Bactérias/genética , Chalconas/química , Clonagem Molecular , Dissacarídeos/química , Dissacarídeos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Expressão Gênica , Glicosídeos/química , Glicosídeos/metabolismo , Hesperidina/química , Hidrólise , Micromonosporaceae/classificação , Micromonosporaceae/genética , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ramnose/química , Ramnose/metabolismo , Especificidade por Substrato , beta-Glucosidase/genéticaRESUMO
The diglycosidase, α-rhamnosyl-ß-glucosidase, from Acremonium sp. DSM24697 was immobilized by adsorption and cross-linking onto polyaniline-iron (PI) particles. The immobilization yield and the immobilization efficiency were relatively high, 31.2% and 8.9%, respectively. However, the heterogeneous preparation showed lower stability in comparison with the soluble form of the enzyme in operational conditions at 60 °C. One parameter involved in the reduced stability of the heterogeneous preparation was the protein metal-catalyzed oxidation achieved by iron traces supplied from the support. To overcome the harmful effect, iron particles were coated with polyethyleneimine (PEI; 0.84 mg/g) previously for the immobilization of the catalyst. The increased stability of the catalyst was correlated with the amount of iron released from the support. Under operational conditions, the uncoated particles lost between 76% and 52% activity after two cycles of reuse, whereas the PEI-coated preparation reduced 45-28% activity after five cycles of reuse in the range of pH 5.0-10, respectively. Hence, polymer coating of magnetic materials used as enzyme supports might be an interesting approach to improve the performance of biotransformation processes.
Assuntos
Enzimas Imobilizadas/química , Glucosidases/química , Imãs/química , Polietilenoimina/química , Acremonium/enzimologia , Adsorção , Compostos de Anilina/química , Estabilidade Enzimática , Ferro/química , TemperaturaRESUMO
Tritrichomonas foetus causes a venereal infection in cattle; the disease has mild or no clinical manifestation in bulls, while cows may present vaginitis, placentitis, pyometra and abortion in the more severe cases. T. foetus has one of the largest known genomes among trichomonads. However molecular data are fragmentary and have minimally contributed to the understanding of the biology and pathogenesis of this protozoan. In a search of new T. foetus genes, a detailed exploration was performed using recently available expressed sequences. Genes involved in the central carbon metabolism (phosphoenol pyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphate aldolase, thioredoxin peroxidase, alpha and beta chains of succinyl CoA synthetase, malate dehydrogenase, malate oxidoreductase and enolase) as well as in cell structure and motility (actin, α-tubulin and ß-tubulin) were found duplicated and, in many cases, repeatedly duplicated. Homology analysis suggested that massive expansions might have occurred in the T. foetus genome in a similar way it was also predicted for Trichomonas vaginalis, while conservation assessment showed that duplications have been acquired after differentiation of the two species. Therefore, gene duplications might be common among these parasitic protozoans.
Assuntos
Duplicação Gênica , Genoma de Protozoário/genética , Complicações Parasitárias na Gravidez/parasitologia , Infecções por Protozoários/parasitologia , Trichomonas vaginalis/genética , Tritrichomonas foetus/genética , Animais , Bovinos , Evolução Molecular , Etiquetas de Sequências Expressas , Feminino , Masculino , Gravidez , Proteínas de Protozoários/genética , Análise de Sequência de DNA/veterinária , Trichomonas vaginalis/enzimologia , Tritrichomonas foetus/enzimologiaRESUMO
The diglycosidase α-rhamnosyl-ß-glucosidase (EC 3.2.1.168) from the fungus Acremonium sp. DSM24697 was immobilized on several agarose-based supports. Covalent multipoint immobilization onto glyoxyl-activated agarose was selected as the more stable preparation at high concentration of dimethyl sulfoxide (DMSO) and high temperature. The optimal conditions for the immobilization process involved an incubation of the enzyme with agarose beads containing 220 µmol of glyoxyl groups per gram at pH 10 and 25°C for 24 h. The hydrolysis of hesperidin carried out in 10% v/v DMSO at 60°C for 2 h reached 64.6% substrate conversion and a specific productivity of 2.40 mmol h(-1) g(-1). Under these conditions, the process was performed reutilizing the catalyst for up to 18 cycles, maintaining >80% of the initial activity and a constant productivity 2.96 ± 0.42 µmol(-1) h(-1) g(-1). To the best of our knowledge, such productivity is the highest achieved for hesperetin production through an enzymatic approach.
Assuntos
Acremonium/enzimologia , Enzimas Imobilizadas/metabolismo , Glicosídeo Hidrolases/metabolismo , Hesperidina/metabolismo , Glicosídeo Hidrolases/isolamento & purificação , Microesferas , Sefarose , Temperatura , Fatores de TempoRESUMO
Tritrichomonas foetus is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to infertility and abortion. A test based on loop mediated isothermal amplification (LAMP) targeting the 5.8S rDNA subunit was designed for the specific identification of T. foetus. The LAMP assay was validated using 28 T. foetus and 35 non-T. foetus trichomonads strains. It did not exhibit cross-reaction with closely related parasites commonly found in smegma cultures like Tetratrichomonas spp. and Pentatrichomonas hominis. Bovine smegma did not show interferences for the detection of the parasite and, the sensitivity of the method (4×10(3) CFU/mL, approximately 10 cells/reaction) was slightly higher than that found for PCR amplification with TFR3 and TFR4 primers. The LAMP approach has potential applications for diagnosis and control of T. foetus and, practical use for low skill operators in rural areas.
Assuntos
Doenças dos Bovinos/parasitologia , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/veterinária , Infecções Protozoárias em Animais/parasitologia , Tritrichomonas foetus/isolamento & purificação , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/diagnóstico , DNA Ribossômico/genética , Masculino , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Protozoárias em Animais/diagnóstico , Esmegma/parasitologia , Tritrichomonas foetus/genéticaRESUMO
The release of enrofloxacin entrapped in polyvinyl alcohol (PVA) cryogel at pH 5.5 showed a first-order kinetic, releasing 69.7% of the antibiotic after 4.5 h at 37 °C. In order to slow down the fluoroquinolone release rate, high-methoxylated pectin was added into the cryogel (PVA-P). A film containing 1.0% (w/v) HM pectin and 5.0 µg/ml enrofloxacin released only 3.7% of the antibiotic after 4.5 h. Since the FTIR spectrum showed that most of the interactions between PVA-P matrix and enrofloxacin were due to polar groups (carboxylate and amine), a two-layer film system was designed to modulate the releasing rate of the drug. The top film equilibrated with 0.75 or 1.5 M NaCl release up to 41.9% and 89.0% of the enrofloxacin in 4 h, respectively. The release rate of enrofloxacin was found dependent on NaCl concentration in the upper gel layer. The two-layer cryogel system showed attractive features for transcutaneous antibiotic delivery.
Assuntos
Antibacterianos/química , Criogéis/química , Portadores de Fármacos/química , Fluoroquinolonas/química , Pectinas/química , Álcool de Polivinil/química , Preparações de Ação Retardada , Enrofloxacina , Concentração OsmolarRESUMO
A simple enzymatic-spectrophotometric method for hesperidin quantification was developed by means of a specific fungal enzyme. The method utilises the diglycosidase α-rhamnosyl-ß-glucosidase (EC 3.2.1.168) to quantitatively hydrolyse hesperidin to hesperetin, and the last is measured by its intrinsic absorbance in the UV range at 323 nm. The application of this method to quantify hesperidin in orange (Citrus sinensis) juices was shown to be reliable in comparison with the standard method for flavonoid quantification (high performance liquid chromatography, HPLC). The enzymatic method was found to have a limit of quantification of 1.8 µM (1.1 mg/L) hesperidin, similar to the limit usually achieved by HPLC. Moreover, it was feasible to be applied to raw juice, without sample extraction. This feature eliminated the sample pre-treatment, which is mandatory for HPLC, with the consequent reduction of the time required for the quantification.
Assuntos
Acremonium/enzimologia , Bebidas/análise , Citrus/química , Ensaios Enzimáticos/métodos , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Hesperidina/química , Espectrofotometria/métodosRESUMO
Transglycosylation potential of the fungal diglycosidase α-rhamnosyl-ß-glucosidase was explored. The biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the [M+Na](+) ions correlated with the theoretical monoisotopic mass (calcd [M+Na](+): 507.44 m/z; obs. [M+Na](+): 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to explore in nature the occurrence of endo-ß-glucosidases by zymographic analysis.
Assuntos
Acremonium/enzimologia , Dissacarídeos/química , Dissacarídeos/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Glucosidases/metabolismo , Glicosídeos/química , Glicosídeos/síntese química , Glicosilação , Himecromona/síntese química , Himecromona/química , Solubilidade , Solventes/química , Especificidade por Substrato , Água/químicaRESUMO
Brucella suis biovar 1 is the causative agent of brucellosis in several domestic and wild animals and it is a common agent of human brucellosis. European hares (Lepus europaeus) have been shown to be infected by B. suis biovar 1 and the transmission to other animals has been suggested. In this work, experimental rabbits (Cuniculus orictolagus) were infected with B. suis biovar 1 isolated from wild hares. Infected rabbits showed high serological response in 2 weeks after discharge and typical granulomatous lesions (2mm diameter) were found in liver, spleen and kidneys after 50 days. B. suis biovar 1 was cultured from the lesion of the organs mentioned above as well as from urine, placenta and fetuses. These data suggest that hares are a potential source for horizontal transmission of B. suis biovar 1 to other mammalians.
Assuntos
Brucella suis/patogenicidade , Brucelose/veterinária , Lebres/microbiologia , Coelhos/microbiologia , Animais , Animais Selvagens/microbiologia , Brucelose/patologia , Feminino , Rim/microbiologia , Rim/patologia , Fígado/microbiologia , Fígado/patologia , Masculino , Gravidez , Complicações Infecciosas na Gravidez/microbiologia , Complicações Infecciosas na Gravidez/veterinária , Baço/microbiologia , Baço/patologiaRESUMO
Most aroma compounds exist in vegetal tissues as disaccharide conjugates, rutinose being an abundant sugar moiety in grapes. The availability of aroma precursors would facilitate analytical analysis of plant-based foods. The diglycosidase α-rhamnosyl-ß-glucosidase from Acremonium sp. DSM 24697 efficiently transglycosylated the rutinose moiety from hesperidin to 2-phenylethanol, geraniol, and nerol in an aqueous-organic biphasic system. 2-Phenethyl rutinoside was synthesized up to millimolar level with an 80% conversion regarding the donor hesperidin. The hydrolysis of the synthesized aroma precursors was not detected in an aqueous medium. However, in the presence of ethanol as a sugar acceptor, the enzyme was able to transfer the disaccharide residue forming the alkyl-rutinoside. The aroma precursors were significantly hydrolyzed (up to 3-4% in 2 h at 30 °C), which indicated the potential use of the enzyme for biotechnological applications, for example, in aroma modulation of fermented foods.