RESUMO
Aesculetin, a coumarin compound present in the sancho tree and chicory, exhibits excellent antioxidant and anti-inflammatory activities in the vascular and immune system. In this study, a rapid and sensitive ultra-high performance liquid chromatography electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method was established and validated for the determination of aesculetin in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was performed on an Acquity UPLC HSS T3 C18 column (2.1 × 100 mm, 1.8 µm) with gradient elution at a flow rate of 0.3 ml/min, using mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Aesculetin and puerarin (internal standard) were detected by multiple reaction monitoring in negative ion mode. The method was fully validated according to the US Food and Drug Administration guidelines. The calibration curve was linear over the range of 2-1,000 ng/ml with correlation coefficient >0.9980. The carry-over, matrix effect, extraction recovery, dilution effect, intra- and inter-day precision and the accuracy were within acceptable limits. The method was then applied to a pharmacokinetic study of aesculetin in rats. After oral administration at doses of 5, 10 and 20 mg/kg, the plasma concentration reached peaks of 95.7, 219.9, 388.6 ng/ml at times of 1.22-1.78 h. The oral bioavailability was calculated as 15.6-20.3% in rat plasma. The result provided pre-clinical information for further application of aesculetin.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Umbeliferonas/sangue , Umbeliferonas/farmacocinética , Animais , Limite de Detecção , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Umbeliferonas/químicaRESUMO
Coumarin is a dietary-derived substance that is extensively metabolized by human liver to excretable 7-hydroxycoumarin. Although coumarin under daily dietary consumption is generally regarded as nontoxic, the substance is of toxicological and clinical interest because of its potential association with hepatotoxicity, which is especially evident in rats. In this study, the pharmacokinetics of coumarin were modeled after virtual oral administration in humans. The adjusted monitoring equivalents of coumarin, along with the biotransformation of coumarin to o-hydroxyphenylacetic acid (via 3,4-epoxidation) based on reported plasma concentrations from rat studies, were scaled to human coumarin equivalents using known species allometric scaling factors. Using rat and human liver preparations, data on the rapid in vitro metabolic clearance for humans (~50-fold faster than in rats) were obtained for in vitro-in vivo extrapolation. For human physiologically based pharmacokinetic (PBPK) modeling, the metabolic ratios to o-hydroxyphenylacetic acid and 7-hydroxycoumarin were set at minor (0.1) and major (0.9) levels for the total disappearance of coumarin. The resulting modeled plasma concentration curves in humans generated by simple PBPK models were consistent with reported simulated coumarin maximum concentrations. These results provide basic information to simulate plasma levels of coumarin and its primary metabolite 7-hydroxycoumarin or its secondary activated metabolite o-hydroxyphenylacetic acid (via 3,4-epoxidation) resulting from dietary foodstuff consumption. Under the current assumptions, little toxicological impact of coumarin was evident in humans, thereby indicating the usefulness of forward dosimetry using PBPK modeling for human risk assessment.
Assuntos
Cumarínicos/sangue , Cumarínicos/toxicidade , Animais , Simulação por Computador , Cumarínicos/metabolismo , Cumarínicos/farmacocinética , Conjuntos de Dados como Assunto , Humanos , Técnicas In Vitro , Fígado/metabolismo , Masculino , Modelos Biológicos , Fenilacetatos/sangue , Ratos Sprague-Dawley , Medição de Risco , Umbeliferonas/sangueRESUMO
BACKGROUND: The elucidation of the biological roles of individual active compounds in terms of their in vivo bio-distribution and bioactivity could provide crucial information to understand how natural compounds work together as treatments for diseases. PURPOSE: We examined the functional roles of Byakangelicin (Byn) to improve the brain accumulation of active compounds, e.g., umbelliferone (Umb), curcumin (Cur), and doxorubicin (Dox), and consequently to enhance their biological activities. METHODS: Active compounds were administered intravenously to mice, with or without Byn, after which organs were isolated and visualized for their ex vivo fluorescence imaging to determine the bio-distribution of each active compound in vivo. For the in vivo bioactivity, Cur, either with or without Byn, was administered to a lipopolysaccharide (LPS)-induced neuro-inflammation model for 5 days, and its anti-inflammatory effects were examined by ELISA using a brain homogenate and serum. RESULTS: We successfully demonstrated that the levels of active compounds (Umb, Cur, and Dox) in the brain, lung, and pancreas were greatly elevated by the addition of Byn via direct ex vivo fluorescence monitoring. In addition, sufficient accumulation of the active compound, Cur, greatly reduced LPS-induced neuro-inflammation in vivo. CONCLUSION: Byn could serve as a modulator to allow improved brain accumulation of diverse active compounds (Umb, Cur, and Dox) and enhanced therapeutic effects.
Assuntos
Curcumina/metabolismo , Doxorrubicina/metabolismo , Furocumarinas/farmacocinética , Inflamação Neurogênica/tratamento farmacológico , Umbeliferonas/metabolismo , Administração Intravenosa , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Curcumina/química , Modelos Animais de Doenças , Doxorrubicina/sangue , Doxorrubicina/química , Feminino , Furocumarinas/administração & dosagem , Humanos , Lipopolissacarídeos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Umbeliferonas/sangue , Umbeliferonas/químicaRESUMO
A rapid, simple and sensitive ultra-performance liquid chromatography-electrospray-ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for the simultaneous determination of aesculin, aesculetin, fraxetin, fraxin and polydatin in beagle dog plasma for the first time. Plasma samples were pretreated by protein precipitation with methanol. Chromatographic separation was performed on an Acquity UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 µm) with gradient elution at a flow rate of 0.4 mL/min, using a mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). The analytes and IS were detected by multiple reaction monitoring (MRM) via negative ion mode with ion transitions of m/z 339.1â»m/z 176.8 for aesculin, m/z 176.8â»m/z 88.9 for aesculetin, m/z 206.8â»m/z 192.1 for fraxetin, m/z 369.1â»m/z 206.9 for fraxin, m/z 389.1â»m/z 227.0 for polydatin and m/z 415.2â»m/z 295.1 for puerarin. This method was validated according to the FDA guidelines and the results met the requirements of analysis. The calibration curves of analytes were linear with correlation coefficients more than 0.9980. The intra- and inter-day precisions were less than 15% and the accuracy was within ±15%. The maximum plasma concentration (Cmax) of aesculin, aesculetin, fraxetin, fraxin and polydatin was 46.75 ± 7.46, 209.9 ± 57.65, 369.7 ± 48.87, 67.04 ± 12.09 and 47.14 ± 12.04 ng/mL, respectively. The time to reach the maximum plasma concentration (Tmax) was 1.32 ± 0.38 h for aesculin, 1.03 ± 0.27 h for aesculetin, 0.94 ± 0.23 h for fraxetin, 0.83 ± 0.18 h for fraxin and 1.15 ± 0.15 h for polydatin. The results indicated that the absorption of aesculin might be slow in beagle dog plasma. This method was successfully applied for pharmacokinetics in beagle dog plasma after oral administration of the extracts of Ledum palustre L. at a dosage of 0.27 g/kg.
Assuntos
Cumarínicos/sangue , Esculina/sangue , Glucosídeos/sangue , Ledum/química , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacocinética , Estilbenos/sangue , Umbeliferonas/sangue , Administração Oral , Animais , Calibragem , Cromatografia Líquida de Alta Pressão/métodos , Cães , Feminino , Limite de Detecção , Extratos Vegetais/sangue , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
To establish HPLC-MS/MS method for simultaneous determination of daphnetin, daphnoretin, and daphneticin in rat plasma after oral and intravenous administration of Daphne giraldii extract, and then use them in the calculation of pharmacokinetic parameters. Six sprague-dawley rats received intragastric administration of D. giraldii extract (daphnetin, daphnoretin and daphneticin were 88.40, 3.24 and 4.28 mgâ¢kg⻹, respectively). Their drug plasma concentration was determined by LC-MS/MS with schisandrin as an internal standard to draw plasma concentration-time curve. The pharmacokinetic parameters were calculated by Kinetica 4.4. The results showed that the linear range was 5-1 000 µgâ¢L⻹ for daphnetin, daphnoretin and daphneticin, and the method ological test showed conformance to the requirements.The intraday and inter-day variable coefficients (RSD) were both less than 15.0%, indicating that both of legitimate precise and accuracy were consistent with the analysis requirements of biological samples. For daphnetin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 4 h, 858.96 µgâ¢L⻹, 10 566.4 µgâ¢L⻹â¢h, 5.19 h and 9.43 h, respectively. For daphnoretin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 2.92 h, 178.00 µgâ¢L⻹, 905.89 µgâ¢L⻹â¢h, 3.50 h and 6.95 h, respectively. For daphneticin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 2 h, 36.67 µgâ¢L⻹, 355.11 µgâ¢L⻹â¢h, 4.95 h and 8.27 h, respectively. The LC-MS/MS analysis method established in this study was proved to be so accurate and sensitive that it can be applied to the pharmacokinetic study of daphnetin, daphnoretin and daphneticin.
Assuntos
Cumarínicos/sangue , Daphne/química , Extratos Vegetais/farmacocinética , Umbeliferonas/sangue , Animais , Cromatografia Líquida , Cumarínicos/farmacocinética , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Umbeliferonas/farmacocinéticaRESUMO
A sensitive and rapid ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS/MS) method was developed for the simultaneous analysis of anemoside B4, phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, toosendanin (IS1 of anemoside B4), tetrahydropalmatine (IS2 of phellodendrine, berberine, palmatine and obakunone) and scopoletin (IS3 of esculin and esculetin) and to compare the pharmacokinetics of these active ingredients in normal and ulcerative colitis rats. After methanol deproteinization, solvents were evaporated at 40°C under a gentle stream of nitrogen. Chromatography was performed using a C18 column with a gradient elution of 0.1% aqueous formic acid and acetonitrile at 0.4ml/min. Detection and measurement were performed on a 4000 QTRAP UPLC-MS/MS system from AB Sciex in the multiple reaction monitoring (MRM) mode. Phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, tetrahydropalmatine (IS2) and scopoletin (IS3) were monitored under positive ionization conditions. Anemoside B4, and toosendanin (IS1) were monitored under negative ionization conditions. The optimized mass transition ion-pairs (m/z) were 1221.1/750.7 for anemoside B4, 343.2/193.2 for phellodendrine, 337.1/321.0 for berberine, 353.0/336.9 for palmatine, 455.1/161.1 for obakunone, 341.2/179.2 for esculin, 179.1/123.0 for esculetin, 573.4/531.4 for toosendanin (IS1), 356.2/192.2 for tetrahydropalmatine (IS2) and 193.0/133.1 for scopoletin (IS3).
Assuntos
Colite Ulcerativa/sangue , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Berberina/análise , Berberina/sangue , Alcaloides de Berberina/análise , Alcaloides de Berberina/sangue , Cromatografia Líquida de Alta Pressão/métodos , Esculina/análise , Esculina/sangue , Masculino , Quinolizinas/análise , Quinolizinas/sangue , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/métodos , Umbeliferonas/análise , Umbeliferonas/sangueRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhizae uralenis (GU) is often prescribed together with Cortex daphnes (CD) in traditional Chinese medicinal practice to increase the efficacy of CD on the treatment of rheumatoid arthritis (RA), but the reasons were still unknown. In order to clarify the rationality of herbaceous compatibility between CD and GU, the comparative evaluations on pharmacokinetic behaviors of daphnetin (a predominantly active ingredient in CD) after intragastric administration of CD and CD-GU (combination of CD and GU) extract were studied. In addition, the effects of glycyrrhizin and liquiritin, active ingredients of Glycyrrhiza triterpenes and Glycyrrhiza flavones respectively, on the pharmacokinetics of daphnetin were also investigated. MATERIALS AND METHODS: Five groups of rats were orally administered with CD extract, CD-GU extract, pure daphnetin, co-administration of daphnetin and glycyrrhizin as well as co-administration of daphnetin and liquiritin at the same single dose of daphnetin (20 mg/kg). The rat plasma concentrations of daphnetin were determined by our developed UPLC-MS/MS method. The pharmacokinetics of daphnetin in above groups were investigated and compared. RESULTS: Comparing with oral administration of CD extract, AUC and Tmax of daphnetin significantly increased after giving CD-GU (p<0.05). In addition, in comparison to daphnetin alone, co-administration of daphnetin with liquiritin significantly increased the AUC and Cmax of daphnetin for ~1.5-fold, while co-administered with glycyrrhizin showed limited impact on the pharmacokinetics of daphnetin. CONCLUSIONS: In this study, it was found that liquiritin, one of the major components of GU, significantly enhanced the bioavailability of the main component daphnetin in CD. In addition, the bioavailability of daphnetin in the CD-GU prescription was also significantly higher than that in CD alone, which could be due to liquiritin. Such results explained the mechanism of the increased efficacy in treating RA with the combined use of CD and GU.
Assuntos
Daphne/química , Flavanonas/farmacocinética , Glucosídeos/farmacocinética , Glycyrrhiza uralensis/química , Extratos Vegetais/farmacocinética , Umbeliferonas/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Flavanonas/administração & dosagem , Flavanonas/sangue , Glucosídeos/administração & dosagem , Glucosídeos/sangue , Masculino , Extratos Vegetais/administração & dosagem , Extratos Vegetais/sangue , Ratos , Ratos Sprague-Dawley , Umbeliferonas/administração & dosagem , Umbeliferonas/sangueRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea laniceps Hand.-Mazz. (SL) has long been used under the herbal name Tibetan "Snow Lotus" for the treatment of rheumatoid arthritis, stomachache and dysmenorrhea in Tibetan folk medicine. Since herbal medicine (HM) is a synergistical system with multiple components, both of the metabolism and pharmacokinetic studies of HM are interdependent. This study aimed to develop an integrated strategy based on the UPLC-DAD-QTOF-MS technique for metabolism and pharmacokinetic studies of HM. MATERIAL AND METHODS: SL was used here as a test herb to verify the feasibility of the proposed strategy. SL was administered to rats, then, the blood plasma, urine and feces were analyzed to determine the metabolic profiles. Using our strategy, umbelliferone and scopoletin were evaluated to be the key bioactive components. Their pharmacokinetic parameters were measured and biotransformation pathways were elucidated. RESULTS: After oral administration of SL to rats, 17 components in blood, 10 components in urine and 2 components in feces were identified and characterized using our UPLC-DAD-QTOF-MS method. Umbelliferone, scopoletin and their metabolites were found to be the major components involved in the metabolism process. Literature reports also suggest that umbelliferone and scopoletin are responsible for the therapeutic effects of SL, thus these two components were selected as the active markers for pharmacokinetic study. In the test of validity, the established method presented good linearity with R(2)>0.99. The relative standard deviation value was below 13.9% for precision, and recovery studies for accuracy were found to be within the range 91.8-112.5%. CONCLUSION: The present strategy offers, simultaneously, precision in quantitative analysis (metabolism study) and accuracy in quantitative analysis (pharmacokinetic study) with greater efficiency and less costs, which is therefore reliably used for integrated metabolism and pharmacokinetic studies of HM.
Assuntos
Asteraceae , Extratos Vegetais/farmacocinética , Animais , Cromatografia Líquida de Alta Pressão , Fezes/química , Masculino , Espectrometria de Massas/métodos , Medicina Tradicional , Compostos Fitoquímicos/farmacocinética , Extratos Vegetais/sangue , Extratos Vegetais/urina , Ratos Sprague-Dawley , Escopoletina/sangue , Escopoletina/farmacocinética , Umbeliferonas/sangue , Umbeliferonas/farmacocinéticaRESUMO
A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of esculin and its metabolite esculetin in rat plasma. After addition of internal standards scopoletin, the plasma sample was pretreated by solid-phase extraction (SPE), and separated on a reversed phase C(18) column with a mobile phase of 0.01% formic acid in water (solvent A) and methanol (solvent B) using isocratic elution (A:B=20:80, v/v). The detection of target compounds was done in multiple reaction monitoring (MRM) mode. The MRM detection was operated in the negative ESI mode using the transitions of m/z 339.1 ([M-H](-))â176.7 for esculetin, m/z 176.9 ([M-H](-))â133.0 and m/z 191.0 ([M-H](-))â175.9 for scopoletin. The standard curves, which ranged from 25 to 3200 ng/mL for esculin with the lowest limit of quantification (LLOQ) of 0.25 ng/mL and from 1.25 to 160 ng/mL for esculetin with the LLOQ of 1.25 ng/mL, were fitted to a 1/x weighted quadratic regression model. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and inter-day, RSD<8.73%), accuracy, recovery as well as the stability of the analyte under various conditions. The method was successfully applied to study the pharmacokinetics of esculin and its metabolite esculetin in rat plasma after oral administration of esculin at a dose of 100mg/kg.
Assuntos
Cromatografia Líquida/métodos , Esculina/sangue , Espectrometria de Massas em Tandem/métodos , Umbeliferonas/sangue , Animais , Estabilidade de Medicamentos , Esculina/química , Esculina/farmacocinética , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Umbeliferonas/química , Umbeliferonas/farmacocinéticaRESUMO
6,7-Dimethylesculetin (D), geniposide (G) and rhein (R) are the three major active ingredients of Yin-Chen-Hao-Tang (YCHT), a famous Chinese herbal formula, which has been shown to be clinically effective for treating hepatic injury (HI) syndrome. The present study was conducted to investigate the therapeutic and synergistic effects of COC (combination of D, G and R) on HI rats by combining pharmacokinetic with biochemical analysis strategy. Plasma was analyzed by using reversed-phase high performance liquid chromatography (RP-HPLC). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models were built to evaluate the therapeutic and synergistic effects of COC at the biochemical level. Here, we report that the COC combination could increase the plasma level, slow elimination rate, exert a more robust therapeutic effect than any one or two of the three individual compounds by hitting multiple targets in a rat model of HI. Overall, this beneficially accounts for the popular view that traditional Chinese medicine (TCM) formula usually takes multi-component to exert their therapeutic effects. We suggest that dissecting the mode of action of clinically effective formula to be capable of producing a sufficient effect at low doses.
Assuntos
Antraquinonas/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Iridoides/uso terapêutico , Magnoliopsida/química , Fitoterapia , Umbeliferonas/uso terapêutico , Animais , Antraquinonas/sangue , Artemisia/química , Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas/sangue , Cromatografia Líquida de Alta Pressão , Combinação de Medicamentos , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacocinética , Gardenia/química , Iridoides/sangue , Análise dos Mínimos Quadrados , Masculino , Análise de Componente Principal , Ratos , Ratos Wistar , Rheum/química , Umbeliferonas/sangueRESUMO
SCOPE: Cassia cinnamon contains high levels (up to 1 %) of coumarin. Heavy consumption of this spice may result in a dose exceeding the tolerable daily intake (TDI). In this context, the question was raised whether coumarin in the plant matrix of cinnamon has the same bioavailability as isolated coumarin. METHODS AND RESULTS: A four-way crossover study was performed, in which the same dose of 12 mg coumarin was administered in different formulations to 24 healthy volunteers. The relative extent of absorption measured as urinary excretion of the main metabolite 7-hydroxycoumarin (7OHC) was found to be 62.8% for isolated coumarin in a capsule (reference), 56.0% for cinnamon in capsules, 66.1% for cinnamon tea, and 54.7% for cinnamon in rice pudding (means, n=23, observation period 8 hours). Additionally, 7OHC plasma levels were measured for 105 minutes after administration and revealed a fast absorption of coumarin from cinnamon tea leading to the highest peak concentrations. CONCLUSION: The relative extent of absorption of coumarin from powder of cassia cinnamon is only slightly lower than that of isolated coumarin. Therefore, the TDI of coumarin can be used for risk assessment of coumarin exposure from cinnamon-containing meals.
Assuntos
Bebidas , Cinnamomum aromaticum/química , Cumarínicos/metabolismo , Alimentos , Casca de Planta/química , Adulto , Bebidas/efeitos adversos , Cápsulas , Cumarínicos/efeitos adversos , Estudos Cross-Over , Feminino , Alimentos/efeitos adversos , Glucuronídeos/sangue , Glucuronídeos/urina , Humanos , Absorção Intestinal , Cinética , Masculino , Pessoa de Meia-Idade , Pós , Medição de Risco/métodos , Especiarias/efeitos adversos , Umbeliferonas/sangue , Umbeliferonas/química , Umbeliferonas/urinaRESUMO
OBJECTIVE: To investigate whether the antimalarial drug artemisinin affects CYP2A6 activity in healthy subjects and to compare the utility of coumarin and nicotine as in vivo probe compounds for CYP2A6. METHODS: Twelve healthy male Vietnamese subjects were given coumarin or nicotine in randomized sequence before and after 5 days of a repeated oral administration of artemisinin during two different treatment periods 1 month apart. Sequential blood samples were drawn at baseline 7 days prior to artemisinin treatment and on the first and fifth day of artemisinin treatment during both treatment periods. Plasma concentrations of 7-hydroxycoumarin glucuronide (7-OHCG), nicotine, cotinine and artemisinin were analysed by high-performance liquid chromatography and those of coumarin and 7-hydroxycoumarin (7-OHC) were determined by liquid chromatography-tandem mass spectrometry. Urine, collected in two time intervals on the days of coumarin intake, was treated with beta-glucuronidase and analysed for 7-OHC levels. RESULTS: Artemisinin AUC(0-infinity) values decreased significantly to 23% [95% confidence interval (CI) 18%-28%] on the fifth day of artemisinin administration as compared with the first. The sum of renally excreted 7-OHC and 7-OHCG increased by 1.55-fold (adjusted 95% CI 1.08-2.23) in the 3- to 8-h interval compared to baseline 7 days before. The 7-OHCG/7-OHC plasma AUC(0-infinity) ratio increased by 1.72-fold (adjusted 95% CI 1.16-2.54) following 5 days of artemisinin intake. There was no significant change in the cotinine/nicotine AUC(0-11 hr) ratio between study days. CONCLUSION: Artemisinin significantly increased the sum of renally excreted 7-OHC and 7-OHCG in one of the two collection intervals, suggesting an induction of CYP2A6. A significant increase in the 7-OHCG to 7-OHC AUC(0-infinity) ratio indicates artemisinin to be an inducer of glucuronidation.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Hidrocarboneto de Aril Hidroxilases/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Oxigenases de Função Mista/efeitos dos fármacos , Adulto , Antimaláricos/farmacocinética , Área Sob a Curva , Artemisininas/farmacocinética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Cumarínicos/administração & dosagem , Cumarínicos/farmacocinética , Estudos Cross-Over , Citocromo P-450 CYP2A6 , Humanos , Masculino , Oxigenases de Função Mista/metabolismo , Nicotina/administração & dosagem , Nicotina/farmacocinética , Espectrometria de Massas em Tandem , Umbeliferonas/sangue , VietnãRESUMO
A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for the simultaneous quantitative determination of icariin and its two major metabolites, icariside I and icariside II in rat plasma. The analytes were extracted by liquid-liquid extraction with ethyl acetate after internal standard (daidzein) spiked. The separation was performed by a ZORBAX SB-C(18) column (3.5 microm, 2.1 mm x 100 mm) and a C(18) guard column (5 microm, 4.0 mm x 2.0mm) with an isocratic mobile phase consisting of acetonitrile-water-formic acid (50:50:0.05, v/v/v) at a flow rate of 0.25 mL/min. The Agilent G6410A triple quadrupole LC-MS system was operated under the multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The nominal retention times for icariin, icariside I, icariside II and daidzein were 1.21, 1.88, 2.34 and 1.35 min, respectively. The lower limits of quantification (LLOQ) of icariin, icariside I and icariside II of the method were 1.0, 0.5 and 0.5 ng/mL, respectively. The method was linear for icariin and its metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 12.5%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of icariin to rats.
Assuntos
Flavonas/sangue , Flavonoides/sangue , Flavonoides/metabolismo , Umbeliferonas/sangue , Administração Oral , Animais , Cromatografia Líquida/métodos , Epimedium/química , Flavonas/isolamento & purificação , Flavonoides/administração & dosagem , Flavonoides/isolamento & purificação , Isoflavonas/sangue , Masculino , Medicina Tradicional Chinesa , Ratos , Ratos Wistar , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos , Umbeliferonas/isolamento & purificaçãoRESUMO
Coumarin and its derivatives are naturally occurring substances with multiple biological activities. Here we demonstrate that prophylactic peroral administration of coumarin or 7-hydroxycoumarin (7-OHC) enhances resistance to subsequent lethal Salmonella enterica Serovar Typhimurium infection in mice. 7-OHC decreased bacterial load in liver and spleen, and enhanced phagocytosis and bacterial killing by macrophages when applied in vitro and in vivo. 7-OHC treatment induced significant NO release in peritoneal macrophage cultures. The observed protective effect correlated with the induction of Th1-associated cytokines, such as IL-12, IFN-gamma, and TNF-alpha. These data demonstrate a clear immunomodulatory potential of coumarins which might have important therapeutic implications to enhance resistance to infection.
Assuntos
Cumarínicos/farmacologia , Salmonella typhi/isolamento & purificação , Febre Tifoide/imunologia , Umbeliferonas/farmacologia , Animais , Cumarínicos/sangue , Cumarínicos/imunologia , Interferon gama/imunologia , Interleucina-12/imunologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Espécies Reativas de Nitrogênio/metabolismo , Salmonella typhi/imunologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/microbiologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Fator de Necrose Tumoral alfa/imunologia , Febre Tifoide/microbiologia , Febre Tifoide/prevenção & controle , Umbeliferonas/sangue , Umbeliferonas/imunologiaRESUMO
The aim of this study was to prepare and characterize a transgenic mouse model in which CYP2A6, a human P450 enzyme, is expressed specifically in the liver. CYP2A6, which is mainly expressed in human liver, is active toward many xenobiotics. Our transgene construct contained the mouse transthyretin promoter/enhancer, a full-length CYP2A6 cDNA, and a downstream neomycin-resistance gene for positive selection in embryonic stem cells. Hepatic expression of the CYP2A6 transgene was demonstrated by immunoblotting, whereas tissue specificity of CYP2A6 expression was confirmed by RNA-PCR. The transgenic mouse was further characterized after being backcrossed to the B6 strain for six generations. Hepatic microsomes from homozygous transgenic mice had activities significantly higher than those of B6 mice toward coumarin. The in vivo activity of transgenic CYP2A6 was also determined. Systemic clearance of coumarin was significantly higher in the transgenic mice than in B6 controls, consistent with the predicted role of CYP2A6 as the major coumarin hydroxylase in human liver. The CYP2A6-transgenic mouse model should be valuable for studying the in vivo function of this polymorphic human enzyme in drug metabolism and chemical toxicity.
Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Fígado/enzimologia , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Animais , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Cumarínicos/sangue , Citocromo P-450 CYP2A6 , Feminino , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxigenases de Função Mista/metabolismo , Umbeliferonas/sangueRESUMO
We developed genotyping assays for CYP2A6*7 (Ile471Thr) and CYP2A6*8 (Arg485Leu). We found higher allelic frequencies in Japanese and Chinese versus Caucasians and identified an allele in which both substitutions occur together (CYP2A6*10). We created a homology model for predicting the impact of allelic variants on enzymatic activity and subsequently tested this in vivo in a pilot kinetic study. Consistent with our homology model predictions, we found (i) that CYP2A6*7 produces an enzyme that has decreased (not inactive) activity for metabolizing nicotine and coumarin; (ii) that CYP2A6*8 is unlikely to affect catalytic activity in vivo; and (iii) that having both substitutions together on an allele (CYP2A6*10) dramatically reduces function and may be fully inactive for some substrates. In conclusion, this study identifies, at relatively high frequency in Asians, an allele with decreased activity (may be substrate selective), a fully functional allele, and an allele containing both substitutions in which function is dramatically reduced.
Assuntos
Alelos , Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Oxigenases de Função Mista/genética , Área Sob a Curva , Povo Asiático , China , Cotinina/sangue , Citocromo P-450 CYP2A6 , Variação Genética , Genótipo , Humanos , Japão , Cinética , Modelos Moleculares , Nicotina/sangue , Reação em Cadeia da Polimerase , Polimorfismo Genético , Análise de Sequência de DNA , Fatores de Tempo , Umbeliferonas/sangue , População BrancaRESUMO
Methoxsalen (8-methoxypsoralen) is an effective and selective mechanism-based inhibitor of human hepatic cytochrome P-450 (CYP)2A6 in vitro, and may have utility as a clinical probe for CYP2A6-catalyzed xenobiotic metabolism in humans in vivo. This investigation explored single-dose oral methoxsalen effects on human CYP2A6 activity in vivo, assessed by coumarin 7-hydroxylation. Eleven volunteers received 50 mg of oral coumarin on two occasions in a randomized crossover, 90 min after oral methoxsalen or nothing (controls). Plasma and urine 7-hydroxycoumarin and plasma methoxsalen concentrations were determined by HPLC. Methoxsalen pretreatment diminished area under the curve of plasma 7-hydroxycoumarin versus time by 24% (2.40 +/- 0.48 versus 3.20 +/- 0.55 microg. h. ml(-1); P <.001), and also decreased plasma 7-hydroxycoumarin C(max) (0.80 +/- 0.26 versus 1.4 +/- 0.5 microg/ml; P <.05); however, 7-hydroxycoumarin concentrations were only diminished 0.75 to 2 h after coumarin administration, but not thereafter. Methoxsalen diminished urine 7-hydroxycoumarin excretion in 0- to 1- and 1- to 2-h samples, but not thereafter, and total excretion was unchanged. Considerable individual variability in methoxsalen plasma concentrations was observed. There were significant correlations between the decrease in plasma 7-hydroxycoumarin C(max) and plasma methoxsalen C(max), but not between the decrease in plasma 7-hydroxycoumarin area under the curve and methoxsalen disposition. These results show that single-dose oral methoxsalen, in conventional doses, was a moderately effective inhibitor of human CYP2A6 activity in vivo, however, the duration of inhibition was limited. Interindividual variability in the extent of CYP2A6 inhibition appeared attributable to variability in the absorption and first-pass clearance of methoxsalen. Alternative doses, timing, and/or routes of methoxsalen administration are required for greater, longer, and more reproducible CYP2A6 inhibition than that provided by single-dose methoxsalen.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Inibidores das Enzimas do Citocromo P-450 , Metoxaleno/farmacologia , Oxigenases de Função Mista/antagonistas & inibidores , Administração Oral , Adulto , Análise de Variância , Anticoagulantes/administração & dosagem , Anticoagulantes/farmacocinética , Área Sob a Curva , Cromatografia Líquida de Alta Pressão , Cumarínicos/administração & dosagem , Cumarínicos/farmacocinética , Estudos Cross-Over , Citocromo P-450 CYP2A6 , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Humanos , Masculino , Metoxaleno/administração & dosagem , Metoxaleno/sangue , Metoxaleno/farmacocinética , Oxigenases de Função Mista/metabolismo , Sondas Moleculares , Umbeliferonas/sangue , Umbeliferonas/urinaRESUMO
Biliary excretion and intestinal reabsorption in enterohepatic circulation play major dispositional roles for some drugs. To circumvent multiple blood sampling and interruption of enterohepatic circulation in conventional biliary cannulation, the present study utilized the minimally invasive sampling technique of microdialysis in pharmacokinetics and biliary excretion studies. Microdialysis probes were inserted into the jugular vein and bile duct in the anesthetized rat for simultaneous and continuous sampling following intravenous administration of esculetin, a bioactive coumarin derivative. Placements of the microdialysis probes were designed to minimize obstruction to normal flows of the body fluids. Separation and quantitation of esculetin in the dialysates were achieved using high performance liquid chromatography (HPLC) coupled to UV detection. The results indicated higher drug concentrations in the bile than in the blood, suggesting active biliary excretion. The study also provided an example of successful application of in vivo microdialysis as an interesting and feasible alternative for pharmacokinetics and biliary drug excretion studies.
Assuntos
Antioxidantes/farmacocinética , Bile/metabolismo , Microdiálise , Umbeliferonas/sangue , Umbeliferonas/farmacocinética , Animais , Antioxidantes/isolamento & purificação , Bile/química , Sistema Biliar/química , Sistema Biliar/metabolismo , Cromatografia Líquida , Meia-Vida , Masculino , Microdiálise/métodos , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Distribuição Tecidual , Umbeliferonas/isolamento & purificaçãoRESUMO
Trematode infections have long been associated with specific types of cancer. We investigated the ability of the liver fluke Fasciola hepatica to alter host enzymes in a manner that might provide insight into the phenomenon of biologically associated cancers. Our data demonstrate an increased activity of the CYP2A5 isozyme in male mouse liver infected with F.hepatica. Induction of this enzyme was further assessed immunohistochemically. The infection affected CYP2A5 distribution in hepatic tissue. Inflammation and proliferation in liver tissue were observed at the same time that CYP2A5 activity increased. This enzyme is known to participate in the metabolism of several carcinogens which are common contaminants in environments of developing countries where parasitic infections may be prevalent.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Fasciolíase/enzimologia , Oxigenases de Função Mista/metabolismo , Animais , Citocromo P-450 CYP2A6 , Família 2 do Citocromo P450 , Suscetibilidade a Doenças , Indução Enzimática , Imuno-Histoquímica , Fígado/anatomia & histologia , Fígado/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fenobarbital/farmacologia , Fatores de Tempo , Umbeliferonas/sangue , Umbeliferonas/farmacologiaRESUMO
BACKGROUND: Disulfiram and its primary metabolite diethyldithiocarbamate are effective mechanism-based inhibitors of human liver cytochrome P450 2E1 (CYP2E1) in vitro. A single dose of disulfiram, which significantly diminishes human P450 2E1 activity in vivo, has been used to investigate the role of CYP2E1 in human drug metabolism and to prevent CYP2E1-mediated biotransformation. Nevertheless, the specificity of single-dose disulfiram toward human CYP2E1 in vivo is unknown. Because diethyldithiocarbamate also inhibits human liver CYP2A6 in vitro, this investigation explored the effect of single-dose disulfiram on human CYP2A6 activity in vivo. METHODS: CYP2A6 activity was assessed by the 7-hydroxylation of coumarin, which is catalyzed selectively by CYP2A6. Ten healthy volunteers received 50 mg oral coumarin on two occasions in a randomized crossover design, approximately 10 hours after 500 mg oral disulfiram was administered or after no pretreatment (control group). Plasma and urine 7-hydroxycoumarin and plasma coumarin concentrations were determined by HPLC. RESULTS: The area under the plasma 7-hydroxycoumarin versus time curve (2.69 +/- 0.90 micrograms.hr/ml) was not decreased after disulfiram pretreatment (3.33 +/- 0.93 micrograms.hr/ml). Furthermore, maximum plasma concentration (Cmax) of 7-hydroxycoumarin (1.4 +/- 0.5 versus 1.8 +/- 0.6 micrograms/ml) and time to reach Cmax (1.0 +/- 0.2 and 1.0 +/- 0.4 hour) were unchanged by disulfiram pretreatment. Urinary 7-hydroxycoumarin excretion over a 24-hour period (38.9 +/- 10.8 mg) was also undiminished by disulfiram pretreatment (45.2 +/- 6.6 mg). CONCLUSIONS: Single-dose disulfiram does not inhibit human CYP2A6 activity in vivo. When single-dose disulfiram is used as an in vivo probe for P450, inhibition of drug metabolism suggests involvement of CYP2E1 but not CYP2A6.