RESUMO
BACKGROUND: Gaultheria trichophylla (Royle) is used as food and for treating many ailments in folk medicine especially against inflammation. The purpose of this in vitro study was to evaluate the ability of extracts of G. trichophylla as anti-oxidant and anti-inflammatory agent and for its mineral contents. METHODS: Powdered plant material (100 g) was extracted with 100 ml each of methanol, chloroform, and n-hexane using soxhlet extractor. Antioxidant activity of methanol extract was assessed by DPPH radical scavenging and FRAP assays. Determination of enzyme inhibition activity was determined using 5-LOX inhibitory activity. Total phenolic and flavonoids contents were measured by Folin-Chicalteu and colorimeteric methods respectively. Minerals and heavy metals contents were determined using Atomic absorption spectrophotometer. Qualitative HPLC analysis were performed using some standard phenolic compounds. RESULTS: The highest phenolic (17.5 ± 2.5 mg GA equivalent/g) and flavonoids (41.3 ± 0.1 mg QE equivalent/g) concentrations were found in methanol extract, which also showed more scavenging activity of 1, 1-diphenyl-2-picrylhydrazyl and ferrous reducing power with IC50 = 81.2 ± 0.2 and IC50 = 11.2 ± 0.1 µg/ml, respectively. The methanol and chloroform extracts showed best inhibition of 5-lipoxygenase enzyme with 90.5 ± 0.7% and 66.9 ± 0.1% at 0.5 mg/ml, respectively. G. trichophylla extract was also evaluated for mineral contents (K, Na, Ca, Mg, Fe, and Cu), and for chemical profiling of heavy metals (Cr, Pb, Cd, Co, Zn, Ni and Hg). CONCLUSION: Our current findings suggest that this plant is good source of minerals and concentration of all heavy metals were within permissible limits. The results revealed that this ignored plant has great pharmaceutical and nutraceutical potential.
Assuntos
Antioxidantes/análise , Gaultheria/química , Inibidores de Lipoxigenase/análise , Minerais/análise , Flavonoides/análise , Lipoxigenases/análise , Metais Pesados/análise , Oxirredução , Fenóis/análise , Proteínas de Plantas/análise , Glycine max/enzimologiaRESUMO
Copao (Eulychnia acida Phil., Cactaceae) is an endemic species occurring in northern Chile. The edible fruits of this plant are valued for its acidic and refreshing taste. Phenolic-enriched extracts from copao fruit pulp and epicarp, collected in the Elqui and Limari river valleys, were assessed by its in vitro ability to inhibit the pro-inflammatory enzymes lipoxygenase (LOX) and cyclooxygenases (COX-1 and COX-2). At 100 µg/mL, pulp extracts showed better effect towards LOX than epicarp extract, while COX-2 inhibition was observed for both epicarp and pulp samples. In general, the extracts were inactive towards COX-1. A positive correlation was observed between the anti-inflammatory activity and the main phenolic compounds found in this fruit. Copao fruits from the Limari valley, a main place of collection and commercialization, showed major activity, adding evidence on the possible health-beneficial effects of this native Chilean fruit.
Assuntos
Anti-Inflamatórios/farmacologia , Cactaceae/química , Frutas/química , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/análise , Antioxidantes/análise , Antioxidantes/farmacologia , Chile , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/análise , Inibidores de Ciclo-Oxigenase/farmacologia , Humanos , Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/análise , Inibidores de Lipoxigenase/farmacologia , Fenóis/análise , Fenóis/farmacologia , Extratos Vegetais/análise , Proteínas Recombinantes/metabolismo , OvinosRESUMO
ER-34122, a poorly water-soluble dual 5-lipoxygenase/cyclooxygenase inhibitor, exists as a crystalline form. According to an Oak Ridge thermal ellipsoid plot drawing, carbonyl oxygen O (5) makes an intermolecular hydrogen bond with the hydrogen bonded to N (3) in the crystal structure. The FTIR and the solid-state ¹³C NMR spectra suggest that the network is spread out in the amorphous state and the hydrogen bonding gets weaker than that in the crystalline phase, because the carbonyl signals significantly shift in both spectra. When amorphous ER-34122 was heated, crystallization occurred at around 140°C. Similar crystallization happened in the solid dispersion; however, the degree of crystallization was much lower than that observed in the pure amorphous material. Also, the DSC thermogram of the solid dispersion did not show any exothermic peaks implying crystallization. The heat of fusion (ΔHf) determined in the pure amorphous material was nearly equal to that for the crystalline form, whereas the ΔHf value obtained in the solid dispersion was less than a third of them. These data prove that crystallization of the amorphous form is dramatically restrained in the solid dispersion system. The carbonyl wavenumber shifts in the FTIR spectra indicate that the average hydrogen bond in the solid dispersion is lower than that in the pure amorphous material. Therefore, HPMC will suppress formation of the intermolecular network observed in ER-34122 crystal and preserve the amorphous state, which is thermodynamically less stable, in the solid dispersed system.
Assuntos
Benzamidas/química , Inibidores de Ciclo-Oxigenase/química , Excipientes/química , Inibidores de Lipoxigenase/química , Metilcelulose/análogos & derivados , Pirazóis/química , Tensoativos/química , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/metabolismo , Benzamidas/análise , Varredura Diferencial de Calorimetria , Cristalografia por Raios X , Inibidores de Ciclo-Oxigenase/análise , Estabilidade de Medicamentos , Emulsões , Temperatura Alta , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Derivados da Hipromelose , Inibidores de Lipoxigenase/análise , Espectroscopia de Ressonância Magnética , Metilcelulose/química , Conformação Molecular , Difração de Pó , Pirazóis/análise , Solubilidade , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Setileuton [4-(4-fluorophenyl)-7-[({5-[(1S)-1-hydroxy-1-(trifluoromethyl)propyl]-1,3,4-oxadiazol-2-yl}amino)methyl]-2H-1-benzopyran-2-one] is a selective inhibitor of the 5-lipoxygenase enzyme, which is under investigation for the treatment of asthma and atherosclerosis. During the development of setileuton, a metabolite (M5) was identified in incubations with rat, dog, and human liver microsomes that represented the addition of 18 Da to the 1,3,4-oxadiazole portion of the molecule. Based on mass spectral data, a ring opened structure was proposed and confirmed through comparison with a synthetic standard. The metabolic ring opening was examined in vitro in rat liver microsomes and was determined to be mediated by cytochrome P450s (P450s). Upon examination of the specific P450s involved using cDNA-expressed rat P450s, it was shown that CYP1A2 likely was the major isoform contributing to the formation of M5. Studies using stable labeled molecular oxygen and water demonstrated that the oxygen was incorporated from molecular oxygen, rather than water, and confirmed that the metabolic formation was oxidative. An alternative, comparatively slow pathway of chemical hydrolysis also was identified and described. Three potential mechanisms for the two-step metabolic ring opening of the 1,3,4-oxadizole are proposed.
Assuntos
Cumarínicos/química , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/metabolismo , Microssomos Hepáticos/enzimologia , Oxidiazóis/química , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Asma/tratamento farmacológico , Aterosclerose/tratamento farmacológico , Cumarínicos/análise , Cumarínicos/farmacologia , Sistema Enzimático do Citocromo P-450/química , Inibidores de Lipoxigenase/análise , Inibidores de Lipoxigenase/farmacologia , Masculino , Microssomos Hepáticos/metabolismo , Oxidiazóis/análise , Oxidiazóis/metabolismo , Oxidiazóis/farmacologia , RatosRESUMO
Eucalyptus bridgesiana, Cymbopogon martinii, Thymus vulgaris, Lindernia anagallis, and Pelargonium fragrans are five species of herbs used in Asia. Their essential oils were analyzed by GC-MS, and a total of 36 components were detected. The results of our study indicated that, except for the essential oil of P. fragrans, all of the essential oils demonstrated obvious antimicrobial activity against a broad range of microorganisms. The C. martinii essential oil, which is rich in geraniol, was the most effective antimicrobial additive. All of the essential oils demonstrated antioxidant activities on 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, ß-carotene/linoleic acid assay, and nitric oxide radical scavenging assay. Furthermore, the T. vulgaris essential oil, which possesses plentiful thymol, exhibited the highest antioxidant activity. For P. acnes-induced secretion of pro-inflammatory cytokines, the essential oils of P. aeruginosa, C. martinii, and T. vulgaris reduced the TNF-α, IL-1ß, and IL-8 secretion levels of THP-1 cells.
Assuntos
Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Sequestradores de Radicais Livres/farmacologia , Inibidores de Lipoxigenase/farmacologia , Magnoliopsida/química , Óleos Voláteis/farmacologia , Anti-Infecciosos/análise , Anti-Inflamatórios/análise , Araquidonato 5-Lipoxigenase/metabolismo , Bactérias/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/metabolismo , Sequestradores de Radicais Livres/análise , Fungos/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Concentração Inibidora 50 , Inibidores de Lipoxigenase/análise , Testes de Sensibilidade Microbiana , Óleos Voláteis/análiseRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Zi-shen pill (ZSP), a traditional Chinese medicine, is widely used for the treatment of benign prostatic hyperplasia (BPH) and has remarkable curative effect. AIM OF THE STUDY: To screen the potential 5-Lipoxygenase(5-LOX) inhibitors from ZSP extract. MATERIALS AND METHODS: A new approach based on affinity ultrafiltration-ultra performance liquid chromatography-mass spectrometry(UPLC-MS) was established and validated. Zileuton and glipizide were chosen as positive and negative control drug, respectively. For better screening result, the concentration of 5-LOX enzyme, incubation temperature and time, pH and ion strength were optimized. In addition, 5-LOX inhibitory assay in vitro and molecular docking technique were used for further verification. RESULTS: 20 compounds were characterized in the ultrafiltrate by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and 16 ligands showed binding ability to 5-LOX. Among them, six ligands were deduced as high-potential 5-LOX inhibitors with their high specific binding values (>2.0). The inhibitory activities of anemarrhenasaponin I, timosaponin AI, nyasol and demethyleneberberine were confirmed by the 5-LOX inhibitory assay for validating the reliability of affinity ultrafiltration approach and the computer-simulated molecular docking technique further clarified the possible mechanism of action between the active compounds and the 5-LOX active sites.
Assuntos
Inibidores de Lipoxigenase/análise , Araquidonato 5-Lipoxigenase/química , Cromatografia Líquida de Alta Pressão , Ligantes , Simulação de Acoplamento Molecular , Compostos Fitoquímicos/análise , Espectrometria de Massas por Ionização por Electrospray , UltrafiltraçãoRESUMO
Lipoxygenase (LOX) are enzymes implicated in a broad range of inflammatory diseases, cancer, asthma and atherosclerosis. These diverse biological properties lead to the interesting target for the inhibition of this metabolic pathway of LOX. The drugs available in the market against LOX reported to have various side effects. To develop potent and selective therapeutic agents against LOX, it is essential to have the knowledge of its active site. Due to the lack of structural data of human LOX, researchers are using soybean LOX (sLOX) because of their availability and similarities in the active site structure. Based on the crystal structure of sLOX-3 and its complex with known inhibitors, we have designed a tripeptide, FWY which strongly inhibits sLOX-3 activity. The inhibition by peptide has been tested with purified sLOX-3 and with LOX present in blood serum of breast cancer patients in the presence of substrate linoleic acid and arachidonic acid respectively. The dissociation constant (K(D)) of the peptide with sLOX-3 as determined by Surface Plasmon Resonance (SPR) was 3.59x10(-9) M. The kinetic constant (K(i)) and IC(50), as determined biochemical methods were 7.41x10(-8) M and 0.15x10(-6) M respectively.
Assuntos
Desenho de Fármacos , Inibidores de Lipoxigenase/síntese química , Lipoxigenase/química , Lipoxigenase/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/enzimologia , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Cinética , Lipoxigenase/sangue , Lipoxigenase/isolamento & purificação , Inibidores de Lipoxigenase/análise , Inibidores de Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/farmacologia , Ligação Proteica , Glycine max/enzimologia , Glycine max/metabolismoRESUMO
Developments of novel inhibitors to prevent the function of 5-lipoxygenase (5-LOX) proteins that are responsible for a variety of inflammatory and allergic disease are a major challenge in the scientific community. In this study, robust QSAR classification models for predicting 5-LOX activity were developed using machine learning algorithms. The Support Vector Machines (SVM), Logistic Regression, k-Nearest Neighbour (NN) and Decision Trees were adopted to improve the prediction ability of the classification models. The most informative molecular descriptors that contribute to the prediction of 5-LOX activity are screened from e-Dragon, Ochem, PowerMV and Combined databases using Filter-based feature selection methods such as Correlation Feature Selection (CFS) and Information Gain (IG). Performances of the models were measured by 5-fold cross-validation and external test sets prediction. Evaluation of performance of feature selection revealed that the CFS method outperforms the IG method for all descriptor databases except for PowerMV database. The best ensemble classification model was obtained with the IG filtered 'PowerMV' descriptor database using kNN (k = 5) algorithm which displayed an overall accuracy of 76.6% for the training set and 77.9% for the test set. Finally, we employed this model as a virtual screening tool for identifying potential 5-LOX inhibitors from the e-Drug3D drug database and found 43 potential hit candidates. This top screened hits containing one known 5-LOX inhibitors zileuton as well as novel scaffolds. These compounds further screened by applying molecular docking simulation and identified four potential hits such as Belinostat, Masoprocol, Mefloquine and Sitagliptin having a comparable binding affinity to zileuton.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores de Lipoxigenase/análise , Inibidores de Lipoxigenase/química , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Humanos , Inibidores de Lipoxigenase/farmacologia , Modelos LogísticosRESUMO
This work is part of the search in native food matrices from arid regions of Argentina of interest to improve human health. Prosopis species are ethnic food resources in South America capable of growing in arid and semi-arid environments. This work was focused to determine the nutritional and phytochemical composition of Prosopis nigra fine flour and to evaluate its biological properties. Flour showed a high level of sucrose (30.35 g/100 g flour), fiber (6.34 g/100 g flour), polyphenols (0.45 g GAE/100 g flour), and minerals (potassium, calcium, and magnesium). Apigenin C glycosides and phenylpropanoid acids were identified in free and bound phenolic enriched extracts, respectively. Polyphenols (especially free polyphenols) were able to inhibit enzymes associated with the metabolic syndrome, including α-amylase (IC50 30.1 µg GAE/mL), α-glucosidase (IC50 22.5 µg GAE/mL), while bound phenolics may control lipase activity (IC50 33.5 µg GAE/mL) and exhibit antioxidant activity by different action mechanisms (SC50 between 16 and 93 µg GAE/mL). Both extracts were more effective to inhibit cyclooxygenase-2 than phospholipase A2 and lipoxygenase, proinflammatory enzymes. Polyphenolic extracts did not show any mutagenic effect. Our studies add value to this non-conventional flour as a promising food resource that could be used as a functional food or functional ingredient in formulations to reduce the risk of the development of obesity. These studies revalue our native resources by promoting their conservation, their use and their propagation. PRACTICAL APPLICATION: Pods of P. nigra are traditional food resources in South America. The non-conventional flour obtained from them is a food that inhibits enzymes linked to carbohydrates metabolism and lipids metabolism, show antioxidant activity and anti-inflamatory activity, principally on COX-2. This natural product is a promising resource that could be used as a functional food or as functional ingredient in food formulations for reduce the risk of the development of obesity. Our studies are relevant to stimulate a sustainable management of this specie and for its development as potential new crops.
Assuntos
Farinha/análise , Síndrome Metabólica/dietoterapia , Estresse Oxidativo , Compostos Fitoquímicos/farmacologia , Prosopis/química , Alcaloides/análise , Aminoácidos/análise , Antocianinas/análise , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Ácido Ascórbico/análise , Proteínas Sanguíneas/análise , Inibidores de Ciclo-Oxigenase 2/análise , Gorduras na Dieta/análise , Fibras na Dieta/análise , Proteínas Alimentares/análise , Inibidores de Glicosídeo Hidrolases/análise , Inflamação , Inibidores de Lipoxigenase/análise , Valor Nutritivo , Polifenóis/análise , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Acanthus mollis is a plant native to the Mediterranean region, traditionally used as diuretic, anti-inflammatory and soothing of the mucous membranes of the digestive and urinary tract and externally as healing of wounds and burns, also demonstrating analgesic and anti-inflammatory activities. However, studies focused on its phytochemical composition as well as scientific proof of Acanthus mollis efficacy are scarce. AIM OF THE STUDY: The proposed work aims to perform a phytochemical characterization and evaluation of the therapeutic potential of Acanthus mollis, based on biological properties that support its traditional uses. MATERIAL AND METHODS: In this study, an 96% ethanol extract from Acanthus mollis leaves was obtained and its phytochemical composition evaluated using High Performance Liquid Chromatography with Photodiode Array Detector coupled to Electrospray Ionization Mass Spectrometry (HPLC-PDA-ESI/MSn). The chemical structure of the compound isolated was elucidated using 1H and 13C Nuclear Magnetic Resonance (NMR), 1H-correlation spectroscopy (1H-COSY), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC). The quantification of the constituents was performed using two external standards (2,4-dihydroxy-1,4-benzoxazin-3-one and verbascoside). The antioxidant activity was determined by the 2,2-diphenyl-1-pycrylhydrazyl (DPPH) assay. Anti-inflammatory activity was determined measuring the inhibition of nitric oxide production by RAW 264.7 macrophages stimulated with the TLR4 agonist lipopolysaccharide (LPS) and through lipoxygenase (LOX) inhibition assay. The cytotoxicity was screened on two lines (RAW 264.7 and HaCaT) using the resazurin assay. RESULTS: Compounds such as verbascoside and its derivatives, as well as benzoxazinoids were found as the main constituents. A percentage of 5.58% was verified for the 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) derivatives. DIBOA was the main compound of the extract. Significant concentrations were also found for phenylpropanoids, which constitute about 4.39% of the total compounds identified. This extract showed antioxidant capacity against DPPH (IC50 = 40.00⯱â¯1.59⯵g/mL) and superoxide anion (IC50 = 29.42⯱â¯1.99⯵g/mL). It also evidenced anti-inflammatory potential in RAW 264.7 macrophages, presenting capacity for nitric oxide reduction (IC50 = 28.01⯵g/mL). Moreover, in vitro studies have shown that this extract was able to inhibit the lipoxygenase, with an IC50 of 104.39⯱â¯4.95⯵g/mL. Importantly, all effective concentrations were devoid of cytotoxicity in keratinocytes, thus highlighting the safety of the extract for the treatment of skin inflammatory related diseases. Concerning macrophages it was also possible to disclose concentrations showing anti-inflammatory activity and without cytotoxicity (up to 30⯵g/mL). The benzoxazinoid DIBOA demonstrated a considerable anti-inflammatory activity suggesting its important contribution to this activity. CONCLUSIONS: These results corroborate the anti-inflammatory properties traditionally attributed to this plant. Among the compounds identified in this study, benzoxazinoids exhibited a significant anti-inflammatory activity that was never previously described. Ethanol seems to be a good option for the extraction of these bioactive compounds, since relevant antioxidant/anti-radical and anti-inflammatory activities were found for this extract.
Assuntos
Acanthaceae , Anti-Inflamatórios/farmacologia , Benzoxazinas/farmacologia , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/análise , Antioxidantes/análise , Antioxidantes/farmacologia , Benzoxazinas/análise , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Inibidores de Lipoxigenase/análise , Inibidores de Lipoxigenase/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/análise , Folhas de Planta , Células RAW 264.7RESUMO
BACKGROUND: Coffee is important source of natural antioxidants in the diet, such as phenolic compounds, alkaloids, mainly caffeine, diterpenes (cafestol and kahweol) and Maillard reaction products formed during roasting. METHODS: In aqueous and methanolic extracts of coffee (Coffea arabica L.) roasted using traditional techniques from Brazil (B), Colombia (C), Ethiopia (E), Kenya (K) and coffee roasted using an industrial technique from Brazil (T), the phenolic and caffeine content as well as antioxidant properties were determined. RESULTS: Comparing the results from water and methanolic extracts it should be noted that the highest amount of phenolics was determined for a methanolic extract of coffee roasted using the industrial technique (650.96 mg GAE/g DW) and a water extract of Kenya coffee (461.63 mg GAE/g DW). Caffeine content was on average two times higher in all methanolic extracts than in water extracts. The radical scavenging activity of aqueous extracts was found to be higher than methanolic extracts. The highest antioxidant scavenging activity was determined for C (EC50 = 1.16 mg DW/ml) and E (EC50 = 1.3 mg DW/ml) water extracts. Compared to water extracts methanolic extracts showed significantly higher reducing power, ability to chelate Fe2+, inhibition of linoleic acid peroxidation and inhibition of lipoxygenase. CONCLUSIONS: This study demonstrated that the methanolic extracts obtained from different types of coffee exhibit potential anti-inflammatory and antioxidant properties. The highest antioxidant activity was shown by traditionally roasted coffees from Colombia and Ethiopia.
Assuntos
Antioxidantes/farmacologia , Coffea/química , Extratos Vegetais/farmacologia , Alcaloides/análise , Alcaloides/farmacologia , Anti-Inflamatórios/análise , Anti-Inflamatórios/farmacologia , Antioxidantes/análise , Brasil , Café/química , Colômbia , Diterpenos/análise , Diterpenos/farmacologia , Etiópia , Quênia , Ácido Linoleico/química , Peroxidação de Lipídeos/efeitos dos fármacos , Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/análise , Inibidores de Lipoxigenase/farmacologia , Metanol/química , Fenóis/análise , Fenóis/farmacologia , Extratos Vegetais/análise , Água/químicaRESUMO
In this study, a 15-lipoxygenase-modified carbon paste electrode (15-LOX-MCPE) was developed in connection with the help of voltammetry, which can be used as an assay system for screening drugs with inhibiting lipoxygenase (LOX) activity. The influence of different experimental conditions (LOX loading of carbon paste, pH, type of buffer system etc.) was investigated in order to optimise the biosensing device. The best composition of the biosensor is 30% paraffin oil, 68% graphite powder and 2% LOX. The optimised voltammetric measurement medium is Sörensen/NaOH (0.1M, pH 9.0) using linoleic acid as a substrate. Under these conditions the hydroperoxy linoleic acid is formed, which can be oxidised at a potential of +0.9 V versus Ag/AgCl/3M KCl. The applicability of the LOX biosensor as assay of lipoxygenase inhibitors was successfully tested with nordihydroguaiaretic acid, zileuton and fenleuton, which are well known inhibitors of LOX.
Assuntos
Técnicas Biossensoriais/instrumentação , Carbono/química , Eletroquímica/instrumentação , Eletrodos , Inibidores de Lipoxigenase , Inibidores de Lipoxigenase/análise , Araquidonato 15-Lipoxigenase/análise , Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Enzimas Imobilizadas/química , Desenho de Equipamento , Análise de Falha de Equipamento , Inibidores de Lipoxigenase/química , PomadasRESUMO
Aloe vera is a rich source of many natural-health-promoting substances. The results of contemporary research on animal models indicate that the extracts have an antiinflammatory property. In this work the results of some in vitro experiments are shown: determination of the inhibitory effect of the Aloe vera extracts on the activity of partially purified lipoxygenase from the rat lung cytosol fraction, and quantitative determination of the trace elements presented in the extract (Mn, Fe, Cu, Zn) carried out by using the x-ray fluorescence analysis. The findings could explain the inhibitory effect (antilipoxygenase activity) of the Aloe vera extract in the acute inflammation process, expecially in the topical application for healing of minor burns and skin ulcers.
Assuntos
Aloe/química , Inibidores de Lipoxigenase/análise , Extratos Vegetais/química , Oligoelementos/análise , Animais , Técnicas In Vitro , Pulmão/enzimologia , RatosRESUMO
The proinflammatory leukotrienes (LT) play important roles in host defense and disease states. However, no endogenous mechanisms to downregulate 5-lipoxygenase (5-LO), the enzyme catalyzing LT synthesis, have been described. We observed that the cytosolic fraction of rat alveolar macrophages (AMs) and peritoneal macrophages (PMs), and of peripheral blood monocytes (PBMs) contain substantial amounts of 5-LO protein, but little detectable 5-LO activity. We therefore examined these mononuclear phagocyte (MNP) cytosolic fractions for inhibitory activity against 5-LO. MNP cytosol dose-dependently reduced the 5-LO activity in neutrophil (PMN) cytosol and AM membrane. Furthermore, MNP cytosol dose-dependently prolonged the lag phase of soybean lipoxygenase (LO) without affecting the rate of product formation. This effect was overcome by subsequent addition of 13(S)-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HpOD), suggesting that the active factor scavenges hydroperoxides. Inactivation by boiling and roteinase K suggest that is a protein. We speculate that this cytosolic factor(s) may serve as an endogenous means for the down-regulation of 5-LO in macrophages.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Citosol/química , Homeostase , Inibidores de Lipoxigenase/análise , Fagócitos/enzimologia , Animais , Células Cultivadas , Citosol/enzimologia , Peróxido de Hidrogênio/farmacologia , Immunoblotting , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/ultraestrutura , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/ultraestrutura , Monócitos/enzimologia , Monócitos/ultraestrutura , Neutrófilos/enzimologia , Fagócitos/ultraestrutura , Ratos , Glycine max/enzimologiaRESUMO
A mandarin-type citrus fruit, ponkan (Citrus reticulata), was processed by in-line, chopper pulper, and hand-press extractions to investigate the effect of extraction method on the concentrations of bioactive compounds in processed juice. Concentrations of polymethoxylated flavones (tangeretin, nobiletin, and sinensetin) and beta-cryptoxanthin in juice, and inhibitory activities against arachidonate cyclooxygenase and lipoxygenases of the juice extract were analyzed. The juice processed by hand-press extraction contained the largest amounts of nobiletin (3.56 mg/100 mL), tangeretin (4.10 mg/100 mL), and sinensetin (0.13 mg/100 mL). Concentrations of beta-cryptoxanthin were 0.66, 0.59, 0.55, and 0.50 mg/100 mL in chopper pulper, in-line (5/64 in.), in-line (8/64 in.) and hand-press juices, respectively. Both extracts of in-line juices showed greater inhibitory activity toward platelet 12-lipoxygenase than the others. The inhibitory effect of hand-press juice extract on platelet cyclooxygenase activity was remarkable among juice extracts. All juice extracts effectively inhibited polymorphonuclear 5-lipoxygenase activity at nearly the same rate.
Assuntos
Bebidas/análise , Citrus/química , Manipulação de Alimentos/métodos , Frutas/química , Extratos Vegetais/química , beta Caroteno/análogos & derivados , Criptoxantinas , Inibidores de Ciclo-Oxigenase/análise , Flavonoides/análise , Inibidores de Lipoxigenase/análise , Xantofilas , beta Caroteno/análiseRESUMO
1-Benzo[b]thien-2-ylethanone (2-acetylbenzothiophene, 2-ABT) and related impurities were determined using a reverse-phase high performance liquid chromatography system and UV detection at 254 nm. Separation was achieved isocratically on a 4.6 mm x 25 cm, 5 microns Zorbax Rx-C8 column using an eluent which is 0.2% perchloric acid/THF in a ratio of 60:40 (v/v). The chromatographic system resolved 2-ABT and known impurities in less than 45 min with near baseline resolution. Known impurities were quantitated versus 2-ABT with corrections made for differences in detector response at the specified wavelength. Linearity for 2-ABT was demonstrated with a correlation coefficient > 0.9999. Assay precision (RSD values) for impurities at 0.5% ranged from +/- 1.8% to +/- 14%, while precision (RSD values) for the 2-ABT determination ranged from +/- 0.81% to +/- 1.1%. A variety of different chromatographic columns and conditions are discussed for the application.
Assuntos
Hidroxiureia/análogos & derivados , Inibidores de Lipoxigenase/análise , Cromatografia Líquida de Alta Pressão/métodos , Hidroxiureia/análise , Hidroxiureia/química , Inibidores de Lipoxigenase/químicaRESUMO
A high performance liquid chromatography assay utilizing an automated sample preparation procedure for the determination of a novel leukotriene biosynthesis inhibitor, (MK-591), in human plasma has been developed. After aliquoting plasma samples and adding internal standard manually, the BenchMate Workstation executed protein precipitation and solid-phase extraction. Following evaporation to dryness, the residue was reconstituted and chromatographed isocratically on a cyano-phase analytical column. MK-591 and the internal standard were separated from each other and from endogenous plasma substances and detected with an absorbance detector. The assay has been validated in the concentration range 10-1000 ng ml-1 and has the sensitivity and specificity necessary to quantify plasma concentrations from several clinical studies.
Assuntos
Indóis/análise , Inibidores de Lipoxigenase/análise , Quinolinas/análise , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Indóis/sangue , Quinolinas/sangue , Sensibilidade e EspecificidadeRESUMO
The activities and side effects of 5-lipoxygenase (5-LO) inhibitors can be predicted by identifying their redox mechanisms. In this study, we developed a fluorescence-based method to measure the redox potential of 5-LO inhibitors and compared it to the conventional, absorbance-based method. After the pseudo-peroxidase reaction, the amount of remaining lipid peroxide was quantified using the H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) fluorescence dye. Our method showed large signal windows and provided comparable redox potential values. Importantly, the redox mechanisms of known inhibitors were accurately measured with the fluorescence assay, whereas the conventional, absorbance-based method showed contradictory results. Our findings suggest that our developed method is a better alternative for classifying the redox potential of 5-LO inhibitors, and the fluorescence assay can be effectively used to study the mechanisms of action that are related to redox cycling.
Assuntos
Araquidonato 5-Lipoxigenase/química , Bioensaio/métodos , Corantes Fluorescentes/análise , Inibidores de Lipoxigenase/análise , Inibidores de Lipoxigenase/química , Relação Dose-Resposta a Droga , Fluoresceínas/análise , Fluorescência , Humanos , Peróxidos Lipídicos/análise , OxirreduçãoRESUMO
"Água-mel" is a honey-based product produced in Portugal for ancient times. Several attributes have been reported to "água-mel" particularly in the alleviation of simple symptoms of upper respiratory tract. Samples of "água-mel" from diverse beekeepers from different regions of Portugal were studied in what concerns antimicrobial, antioxidant and antiviral properties. The amounts of phenol and brown pigment were also evaluated and correlated with the antioxidant activities. A great variability on the levels of these compounds was found among samples which were responsible for the variability detected also on the antioxidant activities, independent on the method used. Generally, antioxidant activity correlated better with brown pigments' amount than with phenols' content. The antimicrobial activity found for "água-mel" samples confirm the virtues reported by popular findings. In addition, this work also reveals the antiviral properties of "água-mel" evidenced by a decrease on the infectivity of the Qß bacteriophage.
Assuntos
Anti-Infecciosos/farmacologia , Antioxidantes/farmacologia , Antivirais/farmacologia , Mel/análise , Benzotiazóis/análise , Inibidores de Lipoxigenase/análise , Reação de Maillard , Óxido Nítrico/análise , Polímeros/análise , Polifenóis/análise , Portugal , Ácidos Sulfônicos/análiseRESUMO
Two faba bean (Vicia faba L.) subspecies major and minor and lentil seeds grown in Algeria were separated into cotyledons and hulls. These fractions, together with their corresponding whole seeds, were extracted with two solvents, aqueous (70%) acetone and (80%) ethanol, and evaluated for antioxidant activity in relation to their phenolic contents. Acetone selectively extracted tannins from faba beans. The hulls always exhibited high antioxidant activity, measured using the reducing power (RP), antiradical activity (DPPH) or oxygen radical absorbance capacity (ORAC) assays. Aqueous ethanol (80%) extract of lentil hulls exhibited high antioxidant and anti-inflammatory activities preferentially inhibiting 15-LOX (IC(50), 55 µg/ml), with moderate COX-1 (IC(50), 66 µg/ml) and COX-2 (IC(50), 119 µg/ml) inhibitory effects on the COX pathway, whereas faba bean hull extracts exerted relatively mild LOX inhibitory activity.