RESUMO
A microwave-assisted extraction (MAE) method was developed for the extraction of bioactive inositols (D-chiro- and myo-inositols) from lettuce (Lactuca sativa) leaves as a strategy for the revalorization of these agrofood residues. Gas chromatography-mass spectrometry was selected for the simultaneous determination of inositols and sugars (glucose, fructose, and sucrose) in these samples. A Box-Behnken experimental design was used to maximize the extraction of inositols based on the results of single factor tests. Optimal conditions of the extraction process were as follows: liquid-to-solid ratio of 100:1 v/w, 40°C, 30 min extraction time, 20:80 ethanol:water (v/v), and one extraction cycle. When compared with conventional solid-liquid extraction (SLE), MAE was found to be more effective for the extraction of target bioactive carbohydrates (MAE 5.42 mg/g dry sample versus SLE 4.01 mg/g dry sample). Then, MAE methodology was applied to the extraction of inositols from L. sativa leaves of different varieties (var. longifolia, var. capitata and var. crispa). D-chiro- and myo-inositol contents varied between 0.57-7.15 and 0.83-3.48 mg/g dry sample, respectively. Interfering sugars were removed from the extracts using a biotechnological procedure based on the use of Saccharomyces cerevisiae for 24 h. The developed methodology was a good alternative to classical procedures to obtain extracts enriched in inositols from lettuce residues, which could be of interest for the agrofood industry.
Assuntos
Fracionamento Químico/métodos , Inositol/análise , Inositol/isolamento & purificação , Lactuca/química , Agricultura , Indústria Alimentícia , Cromatografia Gasosa-Espectrometria de Massas , Resíduos Industriais , Inositol/química , Micro-OndasRESUMO
SIGNIFICANCE: Mycothiol (MSH, AcCys-GlcN-Ins) is the main low-molecular weight (LMW) thiol of most Actinomycetes, including the human pathogen Mycobacterium tuberculosis that affects millions of people worldwide. Strains with decreased MSH content show increased susceptibilities to hydroperoxides and electrophilic compounds. In M. tuberculosis, MSH modulates the response to several antituberculosis drugs. Enzymatic routes involving MSH could provide clues for specific drug design. Recent Advances: Physicochemical data argue against a rapid, nonenzymatic reaction of MSH with oxidants, disulfides, or electrophiles. Moreover, exposure of the bacteria to high concentrations of two-electron oxidants resulted in protein mycothiolation. The recently described glutaredoxin-like protein mycoredoxin-1 (Mrx-1) provides a route for catalytic reduction of mycothiolated proteins, protecting critical cysteines from irreversible oxidation. The description of MSH/Mrx-1-dependent activities of peroxidases helped to explain the higher susceptibility to oxidants observed in Actinomycetes lacking MSH. Moreover, the first mycothiol-S-transferase, member of the DinB superfamily of proteins, was described. In Corynebacterium, both the MSH/Mrx-1 and the thioredoxin pathways reduce methionine sulfoxide reductase A. A novel tool for in vivo imaging of the MSH/mycothiol disulfide (MSSM) status allows following changes in the mycothiol redox state during macrophage infection and its relationship with antibiotic sensitivity. CRITICAL ISSUES: Redundancy of MSH with other LMW thiols is starting to be unraveled and could help to rationalize the differences in the reported importance of MSH synthesis observed in vitro versus in animal infection models. FUTURE DIRECTIONS: Future work should be directed to establish the structural bases of the specificity of MSH-dependent enzymes, thus facilitating drug developments. Antioxid. Redox Signal. 28, 487-504.
Assuntos
Cisteína/química , Glicopeptídeos/química , Inositol/química , Mycobacterium tuberculosis/metabolismo , Oxidantes/metabolismo , Compostos de Sulfidrila/metabolismo , Cisteína/metabolismo , Glicopeptídeos/metabolismo , Inositol/metabolismo , Mycobacterium tuberculosis/patogenicidade , Oxidantes/química , Oxirredução , Estresse Oxidativo , Peroxidases/química , Peroxidases/metabolismo , Compostos de Sulfidrila/químicaRESUMO
Understanding enzymatic reactions with atomic resolution has proven in recent years to be of tremendous interest for biochemical research, and thus, the use of QM/MM methods for the study of reaction mechanisms is experiencing a continuous growth. Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds, and are important for many biotechnological purposes, including drug targeting. Their reaction product may result with only one of the two possible stereochemical outcomes for the reacting anomeric center, and therefore, they are classified as either inverting or retaining GTs. While the inverting GT reaction mechanism has been widely studied, the retaining GT mechanism has always been controversial and several questions remain open to this day. In this work, we take advantage of our recent GPU implementation of a pure QM(DFT-PBE)/MM approach to explore the reaction and inhibition mechanism of MshA, a key retaining GT responsible for the first step of mycothiol biosynthesis, a low weight thiol compound found in pathogens like Mycobacterium tuberculosis that is essential for its survival under oxidative stress conditions. Our results show that the reaction proceeds via a front-side SNi-like concerted reaction mechanism (DNAN in IUPAC nomenclature) and has a 17.5 kcal/mol free energy barrier, which is in remarkable agreement with experimental data. Detailed analysis shows that the key reaction step is the diphosphate leaving group dissociation, leading to an oxocarbenium-ion-like transition state. In contrast, fluorinated substrate analogues increase the reaction barrier significantly, rendering the enzyme effectively inactive. Detailed analysis of the electronic structure along the reaction suggests that this particular inhibition mechanism is associated with fluorine's high electronegative nature, which hinders phosphate release and proper stabilization of the transition state.
Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Cisteína/biossíntese , Glicopeptídeos/biossíntese , Glicosiltransferases/metabolismo , Inositol/biossíntese , Metais/metabolismo , Teoria Quântica , Biocatálise , Cisteína/química , Glicopeptídeos/química , Inositol/química , Mycobacterium tuberculosis/metabolismoRESUMO
The enantioselective enzymatic desymmetrization of 4,6-di-O-benzyl-myo-inositol, a myo-inositol derivative, was effectively catalyzed by Thermomyces lanuginosus lipase (TL-IM). The product 1D-1-O-acetyl-4,6-di-O-benzyl-myo-inositol, a useful precursor to inositol phosphates, was obtained in excellent yield and enantiomeric excess. Through the investigation of the effects of solvent, biocatalyst load, and temperature, a more economical procedure resulted. The feasibility of biocatalyst reuse was also shown.
Assuntos
Ascomicetos/enzimologia , Compostos de Benzil/química , Proteínas Fúngicas/química , Inositol/análogos & derivados , Lipase/química , Biocatálise , Inositol/química , Cinética , EstereoisomerismoRESUMO
Baccharis plants have been used since ancient times in American traditional medicine. Baccharis chilco is a perennial shrub of temperate regions of South America that grows well in rainfall forests of Colombia. Neither chemical composition nor biological studies of this plant have ever been reported. Two caffeoylquinic acid (CQA) derivatives, 5-O-[(E)-caffeoyl]quinic acid (1) and 3,5-di-O-[(E)-caffeoyl]quinic acid (3), and rosmarinic acid (2) have been isolated from B. chilco growing wild in Colombia, using the on-line HPLC-DAD-DPPH radical-scavenging detection technique as guidance. In the course of the purification work, L-chiro-inositol (4) was also isolated. Structures of the four isolated compounds were determined by spectroscopic methods. Antioxidants 2 and 3 exhibited high antiradical activities evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH(.)) assay, although somewhat lower than that of the reference compound ascorbic acid. The on-line HPLC-DAD-DPPH technique allowed a rapid pinpointing of antioxidants in the studied EtOH extract, and the facile guided isolation of the target molecules.
Assuntos
Baccharis/química , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Compostos de Bifenilo/química , Cromatografia Líquida de Alta Pressão/métodos , Cinamatos/química , Cinamatos/isolamento & purificação , Cinamatos/farmacologia , Depsídeos/química , Depsídeos/isolamento & purificação , Depsídeos/farmacologia , Sequestradores de Radicais Livres/isolamento & purificação , Radicais Livres/química , Inositol/química , Inositol/isolamento & purificação , Inositol/farmacologia , Picratos/química , Extratos Vegetais/isolamento & purificação , Ácido Quínico/análogos & derivados , Ácido Quínico/química , Ácido Quínico/isolamento & purificação , Ácido Quínico/farmacologia , Ácido RosmarínicoRESUMO
In order to investigate the potential use of Boldoa purpurascens against diabetes, the antihyperglycemic effect of an ethanol extract obtained from its leaves was evaluated at doses of 50, 100 and 200 mg/kg in rats after induction of hyperglycemia by alloxan. Insulin 5 IU/kg was used as positive control and NaCl 0.9% as negative control. A similar experiment was performed with the aqueous extract used at doses of 50 and 100 mg/kg using metformin at a dose of 50mg/kg as positive control. Statistical analysis was carried using the Kruskal-Wallis test with an interval of trust of 99%. The ethanolic and aqueous extract of B. purpurascens showed a significant decrease of blood glucose levels 72 h after administration. Phytochemical analysis of the ethanol extract showed the presence of D-pinitol, a compound known for its hypoglycemic properties. In conclusion, ethanolic as well as aqueous extracts of B. purpurascens leaves show antihyperglycemic activity, possibly due to the presence of D-pinitol and flavonoids.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/farmacologia , Nyctaginaceae/química , Extratos Vegetais/farmacologia , Animais , Glicemia/análise , Inositol/análogos & derivados , Inositol/química , Inositol/farmacologia , Masculino , Metformina/uso terapêutico , Extratos Vegetais/química , Folhas de Planta/química , Ratos , Ratos WistarRESUMO
Density functional theory (DFT) calculations of (1) H NMR chemical shifts for l-quebrachitol isomers were performed using the B3LYP functional employing the 6-31G(d,p) and 6-311 + G(2d,p) basis sets. The effect of the solvent on the B3LYP-calculated NMR spectrum was accounted for using the polarizable continuum model. Comparison is made with experimental (1) H NMR spectroscopic data, which shed light on the average uncertainty present in DFT calculations of chemical shifts and showed that the best match between experimental and theoretical B3LYP (1) H NMR profiles is a good strategy to assign the molecular structure present in the sample handled in the experimental measurements. Among four plausible O-methyl-inositol isomers, the l-quebrachitol 2a structure was unambiguously assigned based only on the comparative analysis of experimental and theoretical (1) H NMR chemical shift data. The B3LYP infrared (IR) spectrum was also calculated for the four isomers and compared with the experimental data, with analysis of the theoretical IR profiles corroborating assignment of the 2a structure. Therefore, it is confirmed in this study that a combined experimental/DFT spectroscopic investigation is a powerful tool in structural/conformational analysis studies.
Assuntos
Inositol/análogos & derivados , Teoria Quântica , Configuração de Carboidratos , Inositol/química , Espectroscopia de Ressonância Magnética/normas , Prótons , Padrões de Referência , EstereoisomerismoRESUMO
A molecular modeling study was carried out to investigate the most likely enzymatic disassembly mechanism of dendrimers that were designed as potential antichagasic and antileishmanial prodrugs. The models contained myo-inositol (core), L-malic acid (spacer), and active agents such as 3-hydroxyflavone, quercetin, and hydroxymethylnitrofurazone (NFOH). A theoretical approach that considered one, two, or three branches has already been performed and reported by our research group; the work described herein focused on four (models A and B), five, or six branches, and considered their physicochemical properties, such as spatial hindrance, electrostatic potential mapping, and the lowest unoccupied molecular orbital energy (E(LUMO)). The findings suggest that the carbonyl group next to the myo-inositol is the most promising ester breaking point.
Assuntos
Dendrímeros/química , Simulação de Dinâmica Molecular , Pró-Fármacos/química , Tripanossomicidas/química , Flavonoides/química , Inositol/química , Malatos/química , Nitrofurazona/análogos & derivados , Nitrofurazona/química , Quercetina/química , Eletricidade Estática , TermodinâmicaRESUMO
Six compounds from the aerial parts of the Argentinean plant Hymenoxys robusta (Rusby) Parker were isolated and their structures elucidated using extensive spectroscopic analyses. These compounds comprise two inositol derivatives and four 3,4-seco-pseudoguaianolides, including vermeerin. Bioactivity assays of these compounds against bacterial and fungal pathogens showed that only vermeerin possessed antimicrobial activity specific against Staphylococcus aureus, and showed no toxicity when exposed to human-derived macrophages.
Assuntos
Anti-Infecciosos/farmacologia , Asteraceae/química , Inositol/farmacologia , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Aspergillus niger/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Inositol/química , Inositol/isolamento & purificação , Lactonas/química , Lactonas/isolamento & purificação , Testes de Sensibilidade Microbiana , Ressonância Magnética Nuclear Biomolecular , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Trichophyton/efeitos dos fármacosRESUMO
OBJECTIVE: The few studies applying single-voxel ¹H spectroscopy in children and adolescents with bipolar disorder (BD) have reported low N-acetyl-aspartate (NAA) levels in the dorsolateral prefrontal cortex (DLPFC), and high myo-inositol / phosphocreatine plus creatine (PCr+Cr) ratios in the anterior cingulate. The aim of this study was to evaluate NAA, glycerophosphocholine plus phosphocholine (GPC+PC) and PCr+Cr in various frontal cortical areas in children and adolescents with BD. We hypothesized that NAA levels within the prefrontal cortex are lower in BD patients than in healthy controls, indicating neurodevelopmental alterations in the former. METHOD: We studied 43 pediatric patients with DSM-IV BD (19 female, mean age 13.2 ± 2.9 years) and 38 healthy controls (19 female, mean age 13.9 ± 2.7 years). We conducted multivoxel in vivo ¹H spectroscopy measurements at 1.5 Tesla using a long echo time of 272 ms to obtain bilateral metabolite levels from the medial prefrontal cortex (MPFC), DLPFC (white and gray matter), cingulate (anterior and posterior), and occipital lobes. We used the nonparametric Mann-Whitney U test to compare neurochemical levels between groups. RESULTS: In pediatric BD patients, NAA and GPC+PC levels in the bilateral MPFC, and PCr+Cr levels in the left MPFC were lower than those seen in the controls. In the left DLPFC white matter, levels of NAA and PCr+Cr were also lower in BD patients than in controls. CONCLUSIONS: Lower NAA and PCr+Cr levels in the PFC of children and adolescents with BD may be indicative of abnormal dendritic arborization and neuropil, suggesting neurodevelopmental abnormalities.