RESUMO
8-Hydroxy-2'-deoxyguanosine (8OHdG) is regarded as an important biomarker of oxidative DNA damage and it may be estimated by using different techniques in various biological matrices, most notably DNA and urine. In the case of DNA, artifactual oxidation may take place during the isolation of DNA, its hydrolysis and possible derivatization (as for GC-MS), invalidating the measurement of 8OHdG. Therefore, the direct analysis of 8OHdG excreted into urine was preferred. Interferences from the urine matrix were excluded by applying LC-APCI-MS/MS in the multiple reaction monitoring (MRM) mode. The abundant fragment ion at m/z 168 arising from 8OHdG was monitored in the urine sample of volunteers supplemented with tomato concentrate for different times. The procedure allowed the detection of levels of 8OHdG as low as 1 ng/ml in urine sample.
Assuntos
Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , 8-Hidroxi-2'-Desoxiguanosina , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Desoxiguanosina/química , Humanos , Espectrometria de Massas/métodosRESUMO
BACKGROUND/AIMS: Besides antioxidant vitamins and minerals, fruits and vegetables contain flavonoids and related phenolics. The biological activities of these polyphenols have become well known in recent years evidencing their beneficial effects on human health. In this context, the characterization of the flavonoids present in tomatoes is of great interest. Thus the polyphenol pattern (including flavonols, flavanones and cinnamate derivatives), lycopene and beta-carotene concentrations and the total antioxidant activity (TAA) of the phenolic fraction from different tomato lines and cultivars have been determined. METHODS: The characterization was obtained by means of spectrophotometry and HPLC analyses. RESULTS: Mean values for single flavonoids were 0.68 +/- 0.16 for naringenin, 0.74 +/- 0.12 for rutin and 0.32 +/- 0.06 for a rutin-pentoside. Mean total polyphenol content was 13.15 +/- 1.15 mg/100 g and mean TAA value was 1.3 +/- 0.10 mmol/g. The obtained TAA values resulted in good accordance with the total polyphenol content (R(2) = 0.7928). The main phenolic acids were chlorogenic (mean +/- SE 0.20 +/- 0.03) and caffeic acid (mean +/- SE 0.03 +/- 0.01). Mean levels of lycopene and beta-carotene were 5.38 +/- 0.90 and 1.18 +/- 0.40 mg/100 g, respectively. CONCLUSIONS: Almost all the lines characterised by low carotenoid content produce high levels of polyphenols, and consequently have the most powerful antioxidant potential.
Assuntos
Antioxidantes/análise , Fenóis/análise , Polímeros/análise , Solanum lycopersicum/química , Solanum lycopersicum/genética , Antioxidantes/metabolismo , Carotenoides/análise , Cromatografia Líquida de Alta Pressão/métodos , Flavonoides/análise , Flavonóis , Licopeno , Espectrofotometria/métodos , beta Caroteno/análise , beta Caroteno/metabolismoRESUMO
Propolis is a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants, and it is composed of 50% resin (composed of flavonoids and related phenolic acids), 30% wax, 10% essential oils, 5% pollen and 5% various organic compounds. Propolis cannot be used as raw material, and it must be purified by extraction with solvents. This process should remove the inert material and preserve the polyphenolic fraction, which is considered to contribute more to the observed healing effects than the other propolis constituents. Therefore, the assay of propolis polyphenols is of interest, and this paper describes the results obtained in the analysis of propolis by means of a gradient HPLC or mass spectrometry. HPLC in the gradient mode and coupled with photodiode array detection remains the method of choice for the assay of most relevant components of propolis. Direct analysis by APCI-IT-MS represents a valuable alternative to obtain typical fingerprints of propolis and a reliable identification of a large number of propolis components.
Assuntos
Flavonoides , Fitoterapia , Própole/química , Química Farmacêutica , Cromatografia Líquida de Alta Pressão/normas , Humanos , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Fenóis/química , Fenóis/uso terapêutico , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Polímeros/química , Polímeros/uso terapêutico , Polifenóis , Própole/uso terapêutico , Controle de QualidadeRESUMO
The aim of this study was to evaluate the bioavailability of caffeic acid and the modification of plasma antioxidant status following red wine intake. Five healthy male volunteers consumed 100, 200, and 300 mL of red wine corresponding to approximately 0.9, 1.8, and 2.7 mg of caffeic acid, respectively. Plasma samples collected at different times (0-300 min) were evaluated for their content of caffeic acid and their total antioxidant status. Both these parameters, i.e., plasma concentration of caffeic acid and antioxidant potential, were dose-dependent and the C(max) was reached at about 60 min after red wine intake. The results indicate that caffeic acid is bioavailable and it may be correlated with the antioxidant potential of plasma.
Assuntos
Consumo de Bebidas Alcoólicas/sangue , Antioxidantes/metabolismo , Ácidos Cafeicos/sangue , Adulto , Área Sob a Curva , Disponibilidade Biológica , Ácidos Cafeicos/farmacocinética , Cromatografia Líquida de Alta Pressão , Radicais Livres/sangue , Humanos , Peróxidos Lipídicos/sangue , Masculino , Fatores de TempoRESUMO
Flowers and fruits of St. John's wort collected in different Italian regions were evaluated for their naphtodianthrone (hypericin and pseudohypericin), phloroglucinol (hyperforin and adhyperforin) and flavonol (rutin, quercetin, quercitrin, isoquercitrin and hyperoside) content. The quantitative evaluation was performed by HPLC-DAD. The crude drug collected at the fruit ripening period had the highest content in phloroglucinols and the lowest level of both naphtodianthrones and flavonols. Phloroglucinols peaked in the samples collected in Puglia followed by Lombardia and Veneto, while hypericins and flavonols were highest in the samples harvested in Sardegna and Trentino.
Assuntos
Hypericum/química , Perileno/análogos & derivados , Antracenos , Compostos Bicíclicos com Pontes , Calibragem , Cromatografia Líquida de Alta Pressão , Frutas/química , Itália , Perileno/análise , Floroglucinol/análogos & derivados , Padrões de Referência , Rutina/análise , Espectrofotometria Ultravioleta , Terpenos/análiseRESUMO
Liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-ITMS) was applied to evaluate the levels of ginkgolides A and B and bilobalide in plasma of volunteers after administration of Ginkgo biloba extracts in free (Ginkgoselect) or phospholipid complex (Ginkgoselect Phytosome) forms, providing 9.6 mg of total terpene lactones. The maximum plasma concentrations, C(max), of total ginkgolides A, B and bilobalide were 85.0 and 181.8 microg/mL for Ginkgoselect and Ginkgoselect Phytosome, respectively. The C(max) values were reached at 120 min for the free form and at 180--240 min for the phospholipid complex form. In both cases, the mean elimination half-life of each terpene lactone was in the range 120--180 min. Due to its sensitivity (about 1 ng/mL) and specificity, LC/APCI-ITMS proved to be a very powerful tool for pharmacokinetic studies of these phytochemicals.
Assuntos
Cromatografia Líquida/métodos , Ciclopentanos/sangue , Diterpenos , Furanos/sangue , Ginkgo biloba , Lactonas/sangue , Espectrometria de Massas/métodos , Extratos Vegetais/farmacocinética , Plantas Medicinais , Adulto , Área Sob a Curva , Privação de Alimentos , Ginkgolídeos , Meia-Vida , Humanos , MasculinoRESUMO
Flavonoids are phenolic substances isolated from a wide range of vascular plants, with over 8000 individual compounds known. They act in plants as antioxidants, antimicrobials, photoreceptors, visual attractors, feeding repellants, and for light screening. Many studies have suggested that flavonoids exhibit biological activities, including antiallergenic, antiviral, antiinflammatory, and vasodilating actions. However, most interest has been devoted to the antioxidant activity of flavonoids, which is due to their ability to reduce free radical formation and to scavenge free radicals. The capacity of flavonoids to act as antioxidants in vitro has been the subject of several studies in the past years, and important structure-activity relationships of the antioxidant activity have been established. The antioxidant efficacy of flavonoids in vivo is less documented, presumably because of the limited knowledge on their uptake in humans. Most ingested flavonoids are extensively degraded to various phenolic acids, some of which still possess a radical-scavenging ability. Both the absorbed flavonoids and their metabolites may display an in vivo antioxidant activity, which is evidenced experimentally by the increase of the plasma antioxidant status, the sparing effect on vitamin E of erythrocyte membranes and low-density lipoproteins, and the preservation of erythrocyte membrane polyunsaturated fatty acids. This review presents the current knowledge on structural aspects and in vitro antioxidant capacity of most common flavonoids as well as in vivo antioxidant activity and effects on endogenous antioxidants.
Assuntos
Antioxidantes/farmacologia , Flavonoides/farmacologia , Antioxidantes/química , Antioxidantes/farmacocinética , Biotransformação , Flavonoides/química , Flavonoides/farmacocinética , Humanos , Estrutura MolecularRESUMO
Extracts of selected medicinal plants were examined by electrospray mass spectrometry (ESI-MS). This technique allowed identification of the main components of each extract, thereby providing a typical finger-print of the examined plants. More specifically, anthocyanins (Vaccinium myrtillus), isoflavones (Glycine max, soybean), flavonol-glycosides and terpenes (Ginkgo biloba), triterpenes (Centella asiatica), caffeoyl-quinic acids (Cynara scolymus, artichoke), ginsenosides (Panax ginseng), catechins (Camellia sinensis, green tea) and flavones and flavanones (Propolis) were detected rapidly at levels in the range of 0.1-1 microg/ml, using 0.2-1 mg/ml of each medicinal plant extract.
Assuntos
Extratos Vegetais/química , Plantas Medicinais/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Eight commercial Italian vini novelli (red wines prepared by carbonic maceration and supposed to be consumed within three months from their wine-making) were evaluated for their total antioxidant activity. The wines had an average total phenol content (1605.4 +/- 337.4 mg/L gallic acid equivalents) lower than that of wines prepared by traditional maceration and consumable after aging (2057. 3 +/- 524.0 mg/L gallic acid equivalents). The average flavanol content (424.7 +/- 121.3 mg/L catechin equivalents) and the total antioxidant activity (16.8 +/- 3.8 mmol/L Trolox equivalents) of vini novelli were higher than the corresponding values (382.7 +/- 174.5 mg/L catechin equivalents and 12.3 +/- 3.3 mmol/L Trolox equivalents) found for aged wines. Three couples of experimental wines were prepared from the same grapes by traditional or carbonic maceration. These wines showed a different phenolic pattern, anthocyanins being more abundant in vini novelli. However, the average total antioxidant activities of the wines were similar, suggesting that aging (and not the wine-making technique) is the main factor influencing the antioxidant activity of red wines.
Assuntos
Antioxidantes/análise , Flavonoides/análise , Fenóis/análise , Polímeros/análise , Vinho/análise , Flavonóis , HumanosRESUMO
Oligonucleotides have become widely used tools in molecular biology and molecular diagnostics. Their parallel synthesis in large numbers and the increasing interest in microarray technology has raised the requirement for fast and informative analytical tools for their quality control. A direct injection electrospray ionization mass spectrometry (ESI-MS) technique based on the use of aqueous 2-propanol as running eluent, and spermidine (or triethylamine) as DNA modifiers, has been applied to analyze a large set of samples (about 200 synthetic oligonucleotides) ranging from 5 to 15 kDa (17-51mers) with good results in terms of sensitivity, suppression of sodium adduct formation, and speed of analysis. Copyright 2000 John Wiley & Sons, Ltd.
RESUMO
Hypericum perforatum L. (St. John's Wort) is a widely distributed herbaceous perennial plant which has been well known as a medicinal plant since antiquity. In recent years, H. perforatum has received increasing attention for the treatment of depression and other neuralgic disorders. The main constituents of H. perforatum extract include flavonoids, naphthodianthrones, phloroglucinols, essential oils and xanthones. The present work reports the analysis of naphthodianthrones and phloroglucinols in H. perforatum extracts by means of high performance liquid chromatography (HPLC) coupled simultaneously to a diode array detector (DAD) and electrospray mass spectrometry (ESI-MS). Hypericin, pseudohypericin, hyperforin and adhyperforin were separated and identified on the base of their on-line UV and mass spectra. Quantitative analysis of hypericin derivatives in different extracts of H. perforatum using DAD and MS detectors was performed. In addition, direct infusion ESI-MS of H. perforatum extracts was applied to obtain rapid mass fingerprints of constituents present in the sample.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Hypericum/química , Espectrometria de Massas/métodos , Extratos Vegetais/química , Plantas MedicinaisRESUMO
Standardized extracts of Ginkgo biloba leaves are mainly used in the treatment of peripheral and celebral circulation disorders, and also as a remedy against asthma, coughs, bladder inflammation, blenorrhagia and alcohol abuse. The leaf extracts contain biflavones, flavonol glycosides and terpene lactones. This paper reports a method based on liquid chromatography coupled with electrospray mass spectrometry for the analysis of terpenoids in G. biloba extracts. This method allows the rapid isocratic separation of underivatized ginkgolides (GA, GB, GC and GJ) and bilobalide at very low levels (10 pg on the column) and their quantitative detection by external standardization with relative standard deviations of 3 and 5% for intra- and inter-day analyses, respectively.
Assuntos
Diterpenos , Medicamentos de Ervas Chinesas/química , Ginkgo biloba/química , Plantas Medicinais , Terpenos/química , Cromatografia Líquida , Ciclopentanos/análise , Ciclopentanos/química , Ciclopentanos/normas , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/normas , Furanos/análise , Furanos/química , Furanos/normas , Ginkgolídeos , Humanos , Lactonas/análise , Lactonas/química , Lactonas/normas , Espectrometria de Massas , Extratos Vegetais/análise , Extratos Vegetais/química , Extratos Vegetais/normas , Padrões de Referência , Terpenos/análise , Terpenos/normasRESUMO
A method for oligonucleotides analysis by using capillary electrophoresis at low pH in free solution is described. It may be considered an alternative to classical analytical techniques which use basic buffers and require the presence of sieving media to separate oligonucleotides as a function of their length. On the contrary, at low pH oligo nucleotides can be separated only depending on their base composition. A large set of samples consisting of 72 synthetic oligonucleotides bearing a 5'-alkylamine moiety and designed for HLA genotyping were analysed. The quality of these synthetic oligos was easily assessed, and a single base difference in oligonucleotides of equal sequence was detected. The results suggest the application of this method to the emerging field of mutation detection and single nucleotide polymorfism analysis.
Assuntos
Eletroforese Capilar/métodos , Oligonucleotídeos/análise , Análise Mutacional de DNA/métodos , Concentração de Íons de Hidrogênio , Polimorfismo Genético , Controle de QualidadeRESUMO
Flavonoids continue to attract wide attention as possible very useful agents for combating free radical pathologies, i.e. the pathological states associated with free radical overproduction. Commonly used methods for the analysis of plant flavonoids include high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). On the other hand, the soft-ionization approach based on electrospray ionization (ESI-MS) permits highly selective analysis of complex matrices. In this work, we examined firstly the ESI-MS behaviour of representative aglycones and glycosides of flavonols, flavones and isoflavones with the aim of suggesting a possible relationship between structure and mass spectra. Using HPLC coupled to a diode array detector (DAD) for on-line UV spectra acquisition, and in parallel to ESI-MS for mass spectra (LC/DAD-ESI-MS), we have developed methodology to observe flavonols directly in tomato puree extract. In this way, it has been possible to detect intact flavonol glycosides in tomato extracts and to characterize a flavonol trisaccharide. For the first time, using LC/ESI-MS, it has been possible to detect intact flavonol glycosides in plasma of healthy volunteers and to provide further evidence on the absorption of flavonoid glycosides after consumption of common vegetables like tomatoes.
Assuntos
Flavonoides/sangue , Glicosídeos/sangue , Solanum lycopersicum/química , Calibragem , Cromatografia Líquida de Alta Pressão , Flavonoides/química , Glicosídeos/química , Humanos , Espectrometria de Massas , Rutina/análise , Espectrofotometria UltravioletaRESUMO
Surface layers (S-layers) are regularly ordered protein subunits found as the outermost cell envelope component of many bacteria. Most S-layers are composed of a single protein or glycoprotein species with a molecular weight varying between 40 and 200 kDa. Clostridium difficile is the most common cause of antibiotic associated diarrhea (AAD) and pseudomembranous colitis (PMC) in humans. Detection of the S-layer in some C. difficile strains, and preliminary characterization of two glycoproteins, P36 and P47, involved in the composition of the S-layer of one of these strains (C. difficile C253), led us to investigate the most appropriate conditions for purification and chemical characterization of these proteins. This work describes the results obtained when liquid chromatography (LC) coupled to mass spectrometry (MS) using electrospray ionization was applied to the analysis of C. difficile S-layer proteins (SLPs). In this way the molecular weights of the two SLP components, P36 and P47, were detected to be 34,258 +/- 2 and 39,545 +/- 3 Da, respectively. These data deviate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results by 1.85 and 7.5 kDa. To confirm the LC-MS results, an alternative molecular weight analysis was performed: the two S-layer proteins were isolated by semipreparative high performance liquid chromatography (HPLC), concentrated, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). The two SLP subunits were digested with protease V8, and the peptide maps were determined by LC-MS using a C18 column. Finally, preliminary results about peptide glycosylation were obtained.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Clostridioides difficile/química , Parede Celular/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/química , Hidrólise , Espectrometria de Massas , Peso Molecular , Fragmentos de Peptídeos/química , Mapeamento de PeptídeosRESUMO
Flavonoids are described to exert a large array of biological activities, which are mostly ascribed to their radical-scavenging, metal chelating and enzyme modulation ability. Most of these evidences have been obtained by in vitro studies on individual compounds and at doses largely exceeding those dietary. Little is known about a possible relationship between rate and extent of the absorption and modifications of plasma antioxidants. To elucidate this aspect, human volunteers were supplemented with single doses of green tea catechins in free (Greenselect) or phospholipid complex form (Greenselect Phytosome) equivalent to 400 mg epigallocatechingallate (EGCg). EGCg was chosen as biomarker for green tea catechin absorption, and its time course plasma concentration was correlated to the subsequent percent variations of plasma ascorbate, total glutathione, alpha-tocopherol, beta-carotene and Total Radical Antioxidant Parameter (TRAP). Green tea catechins were absorbed more extensively when administered as phospholipid complex rather than as free catechins. Single dose intake of both forms of catechins produced a transient decrease (10-20%) of plasma ascorbate and total glutathione and an increase of plasma TRAP (16-19%). These variations were consistent with the plasmatic levels of EGCg, ascorbate and total glutathione.
Assuntos
Antioxidantes/metabolismo , Catequina/análogos & derivados , Catequina/farmacocinética , Adulto , Ácido Ascórbico/sangue , Biomarcadores/sangue , Catequina/administração & dosagem , Catequina/sangue , Suplementos Nutricionais , Glutationa/sangue , Humanos , Masculino , Fosfolipídeos/metabolismo , Chá/metabolismo , Fatores de Tempo , Vitamina E/sangue , beta Caroteno/sangueRESUMO
Green tea contains relatively large amounts of catechins, that have been recognized to be efficient free-radical scavengers. In spite of a largely described antioxidant effect, the metabolic fate of catechins in humans has been scarcely studied. An infusion of green tea (about 400 mg of catechins) was given to healthy volunteers; plasma and urine samples were collected for 5 h and 2 days, respectively. Epigallocatechin gallate and epicatechin gallate were detected in plasma samples, reaching the maximum concentration (2 microM) at 2 h. Urine samples collected at 6-48 h contained detectable amounts of final catechin metabolites, including 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). The total content of these metabolites averaged 60 mg. The levels of free plasma catechins account only partly for the increased (approximately +20%) total radical-trapping antioxidant parameter (TRAP) detected after green tea intake. Catechin conjugates (glucuronide and sulphate) and metabolites may add further contribution and explain the measured TRAP increase.