RESUMO
PURPOSE: The rhizome of Kaempferia parviflora (KP) is used in traditional Thai medicine. In this study, we investigated the effects of an ethanol KP extract and two of its components [5,7-dimethoxyflavone (DMF) and 5-hydroxy-3,7,3',4'-tetramethoxyflavone (TMF)] on monocyte adhesion and cellular reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVECs), which provide an in vitro model of events relevant to the development and progression of atherosclerosis. METHODS: RAW264.7 mouse macrophage-like cells were incubated with various concentrations of KP extract or polymethoxyflavonoids and stimulated with lipopolysaccharide prior to measuring nitrite levels in the culture media. Monocyte adhesion was evaluated by measuring the fluorescently labeled human monocytic leukemia THP-1 cells that is attached to tumor necrosis factor-α (TNF-α)-stimulated HUVECs. Cellular ROS production was assessed by measuring cellular antioxidant activity using pyocyanin-stimulated HUVECs. RESULTS: KP extract and DMF reduced nitrite levels (as indicator of nitric oxide production) in LPS-stimulated RAW264.7 cells and also inhibited THP-1 cell adhesion to HUVECs. These treatments induced mRNA expression of endothelial nitric oxide synthase in TNF-α-stimulated HUVECs and downregulated that of various cell adhesion molecules, inflammatory mediators, and endothelial function-related genes. Angiotensin-converting enzyme activity was inhibited by KP extract in vitro. Furthermore, KP extract, DMF, and TMF inhibited the production of cellular ROS in pyocyanin-stimulated HUVECs. CONCLUSION: KP extract, DMF, and TMF showed potential anti-inflammatory and antioxidant effects in these in vitro models, properties that would inhibit the development and progression of atherosclerosis.
Assuntos
Adesão Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Zingiberaceae/química , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Regulação para Baixo , Flavonoides/farmacologia , Humanos , Lipopolissacarídeos/metabolismo , Camundongos , Monócitos/citologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Bone homeostasis is maintained by balancing bone formation and bone resorption, but an imbalance between them is associated with various bone-related diseases such as osteoporosis and rheumatoid arthritis. We found that 5,6-dehydrokawain (DK) and dihydro-5,6-dehydrokawain (DDK), which were isolated as promising compounds from Alpinia zerumbet rhizomes, promote differentiation of osteoblastic MC3T3-E1 cells. DK and DDK increased the alkaline phosphatase activity and matrix mineralization of MC3T3-E1 cells. DK exerts larger effects than DDK. The gene expression of runt-related transcription factor 2 and osterix, which are essential transcription factors in the early period of osteoblast differentiation, was significantly increased by DK treatment. The mRNA level of distal-less homeobox 5 was also enhanced by DK treatment, and DK activated the p38 mitogen-activated protein kinase pathway. Therefore, DK may have clinical potential for preventing osteoporosis, and could be considered as a potential anabolic therapeutic agent.
Assuntos
Alpinia/química , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Pironas/farmacologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/agonistas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/agonistas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Extratos Vegetais/química , Pironas/isolamento & purificação , RNA Mensageiro/agonistas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rizoma/química , Fator de Transcrição Sp7 , Fatores de Transcrição/agonistas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Toddalia asiatica (L.) Lam. (T. asiatica) has been utilized traditionally for medicinal purposes such as the treatment of diabetes. Currently, the extract is considered to be a good source of anti-diabetic agents, but the active compounds have yet to be identified. In this study, we investigated the effects of fractionated T. asiatica extracts on the differentiation of 3T3-L1 preadipocytes and identified aculeatin as a potential active agent. When 3T3-L1 preadipocytes were treated with aculeatin isolated from T. asiatica in the presence of insulin, aculeatin increased cellular triglyceride levels and glycerol-3-phosphate dehydrogenase activity. This indicated that aculeatin could enhance the differentiation of preadipocytes into adipocytes. Further analyses using a DNA microarray and real-time quantitative reverse-transcription PCR showed an increase in the expression of peroxisome proliferator-activated receptor-γ target genes (Pparg, Ap2, Cd36, Glut4 and Adipoq) by aculeatin, suggesting that aculeatin enhances the differentiation of 3T3-L1 cells by modulating the expression of genes critical for adipogenesis. Interestingly, after treatment of differentiated adipocytes with aculeatin, glucose uptake and lipolysis were enhanced. Overall, our results suggested that aculeatin is an active compound in T. asiatica for enhancing both differentiation and lipolysis of adipocytes, which are useful for the treatment of lipid abnormalities as well as diabetes.
Assuntos
Células 3T3-L1/citologia , Células 3T3-L1/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Cumarínicos/farmacologia , Lipólise/fisiologia , Rutaceae/química , Células 3T3-L1/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Cicloexanonas/farmacologia , Lipólise/efeitos dos fármacos , Camundongos , Extratos Vegetais/farmacologiaRESUMO
The rhizome of Kaempferia parviflora has been used in traditional Thai medicine. In this study, we identified and compared specific compounds from the hexane extract of K. parviflora with those from other Zingiberaceous plants by using gas chromatography-mass spectrometry. We identified 5,7-dimethoxyflavone (DMF), 5-hydroxy-3,7,3',4'-tetramethoxyflavone (TMF), estimated 3,5,7-trimethoxyflavone, 5-hydroxy-7,4'-dimethoxyflavone, 3,5,7,4'-tetramethoxyflavone, and investigated their anti-inflammatory effects in rat basophilic leukemia (RBL-2H3) cells stimulated with an IgE antigen or a calcium ionophore. We found that DMF and TMF more potently inhibited antigen-induced degranulation than did nobiletin, a well-known anti-inflammatory agent. In addition, compared to RBL-2H3 cells stimulated with a calcium ionophore, those treated with DMF and TMF showed more marked inhibition of the degranulation and the production and mRNA expression of inflammatory mediators. These results suggest that DMF and TMF inhibit an early step in the high-affinity IgE receptor signaling cascade rather than intracellular calcium release and protein kinase C activation.
Assuntos
Anti-Inflamatórios/análise , Anti-Inflamatórios/farmacologia , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Zingiberaceae/química , Animais , Anti-Inflamatórios/isolamento & purificação , Degranulação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia Líquida , Flavonoides/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Hexanos/química , Mediadores da Inflamação/metabolismo , Extratos Vegetais/isolamento & purificação , RatosRESUMO
A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.
Assuntos
Antibacterianos/química , Cromatografia de Afinidade/métodos , Fluoroquinolonas/química , Mel/análise , Estrutura Molecular , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.
Assuntos
Glycine max/genética , Reação em Cadeia da Polimerase/métodos , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Glicina/análogos & derivados , Herbicidas , Japão , Laboratórios , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/estatística & dados numéricos , GlifosatoRESUMO
Real-time polymerase chain reaction (PCR)-based quantitative methods were previously developed and validated for genetically modified (GM) maize or soy. In this study, the quantification step of the validated methods was modified, and an interlaboratory study was conducted. The modification included the introduction of the PCR system SSIIb 3 instead of SSIIb 1 for the detection of the taxon-specific sequence of maize, as well as the adoption of colE1 as a carrier included in a reference plasmid solution as a replacement for salmon testis. The interlaboratory study was conducted with the ABI PRISM 7700 and consisted of 2 separate stages: (1) the measurement of conversion factor (Cf) value, which is the ratio of recombinant DNA (r-DNA) sequence to taxon-specific sequence in each genuine GM seed, and (2) the quantification of blind samples. Additionally, Cf values of other instruments, such as the ABI PRISM 7900 and the ABI PRISM 7000, were measured in a multilaboratory trial. After outlier laboratories were eliminated, the repeatability and reproducibility for 5.0% samples were <15.8 and 20.6%, respectively. The quantitation limits of these methods were 0.5% for Bt11, T25, and MON810, and 0.1% for GA21, Event176, and RR soy. The quantitation limits, trueness, and precision of the current modified methods were equivalent to those of the previous methods. Therefore, it was concluded that the modified methods would be a suitable replacement for the validated methods.
Assuntos
Glycine max/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/métodos , Padrões de Referência , Zea mays/genética , Sequência de Bases , Elementos de DNA Transponíveis , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , DNA de Plantas/normas , Dados de Sequência Molecular , Plasmídeos/genética , Reprodutibilidade dos TestesRESUMO
A novel liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed for the trace residue determination of chloramphenicol (CAP) in bee pollen. CAP was extracted from bee pollen with a mixture of methanol and 1% metaphosphoric acid solution, followed by a 2-stage solid-phase extraction enrichment and cleanup. The first stage involved a polymeric cartridge, and the second stage involved an alumina neutral cartridge. The LC separation was performed on a C18 column with 10 mM ammonium formate-acetonitrile (7 + 3) as the mobile phase and MS detection with negative-ion electrospray ionization. CAP-d5 was used as the internal standard. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear between 0.1 and 5.0 ng/mL, and overall recoveries ranged from 98 to 113%. Decision limits (CCalpha) ranged from 0.05 to 0.07 microg/kg, and detection capabilities (CCbeta) ranged from 0.08 to 0.12 microg/kg. The developed method was applied to 11 samples.
Assuntos
Antibacterianos/análise , Cloranfenicol/análise , Resíduos de Drogas/análise , Pólen/química , Animais , Abelhas , Calibragem , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes , Reprodutibilidade dos Testes , Soluções , Espectrometria de Massas em TandemRESUMO
A simple and rapid LC/MS method for simultaneous determination of sedecamyin (SCM) and terdecamycin (TDM) in livestock products has been developed. SCM and TDM were extracted with acetonitrile. The extract was washed with n-hexane and then evaporated to dryness. The residue was dissolved in methanol, and injected into the LC/MS. The mass spectrometer was operated in the positive electrospray ionization (ESI) mode. LC separation was performed on a high-pH-resistant C18 column with 10 mmol/L carbonic acid-ammonia buffer (pH 10.0)-acetonitrile as a mobile pahse. The recoveries from swine muscle and liver fortified at the levels of 0.01 and 0.05 microg/g were 77-88%, and those from poultry muscle and liver fortified at the levels of 0.01 and 0.3 microg/g were 51-93%. The quantitation limits of SCM and TDM were 0.008 microg/g and 0.005 microg/g, respectively.
Assuntos
Cromatografia Líquida/métodos , Macrolídeos/análise , Espectrometria de Massas/métodos , Carne/análise , Drogas Veterinárias/análise , Animais , Galinhas , SuínosRESUMO
Okadaic acid (OA), a lipophilic shellfish toxin, was accurately quantified using quantitative nuclear magnetic resonance with internal standards for the development of an authentic reference standard. Pyridine and the residual proton in methanol-d4 were used as removable internal standards to limit any contamination. They were calibrated based on a maleic acid certified reference material. Thus, the concentration of OA was traceable to the SI units through accurate quantitative NMR with an internal reference substance. Signals from the protons on the oxygenated and unsaturated carbons of OA were used for quantification. A reasonable accuracy was obtained by integrating between the lower and upper (13)C satellite signal range when more than 4 mg of OA was used. The best-determined purity was 97.4% (0.16% RSD) when 20 mg of OA was used. Dinophysistoxin-1, a methylated analog of OA having an almost identical spectrum, was also quantified by using the same methodology.
Assuntos
Dinoflagellida/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Toxinas Marinhas/análise , Ácido Okadáico/análise , Piranos/análise , Frutos do Mar/análise , Calibragem , Padrões de Referência , Sensibilidade e EspecificidadeRESUMO
Osteoporosis with bone loss is widely recognized as a major health problem. Bone homeostasis is maintained by balancing bone formation and bone resorption. The imbalance caused by increased bone resorption over bone formation can lead to various bone-related diseases such as osteoporosis and rheumatoid arthritis. Osteoclasts are the principal cells responsible for bone resorption and the main targets of anti-resorptive therapies. However, excessive inhibition of osteoclast differentiation may lead to inhibition of osteoblast differentiation. Therefore, it is important to screen for new compounds capable of inhibiting bone resorption and enhancing bone formation. Toddalia asiatica (L.) Lam. has been utilized traditionally for medicinal purposes such as the treatment of rheumatism. Currently, the extract is considered to be a good source of pharmacological agents for the treatment of bone-related diseases, but the active compounds have yet to be identified. We investigated whether toddaculin, derived from Toddalia asiatica (L.) Lam., affects both processes by inhibiting bone resorption and enhancing bone formation. Towards this end, we used pre-osteoclastic RAW 264 cells and pre-osteoblastic MC3T3-E1 cells. We found that toddaculin not only inhibited the differentiation of osteoclasts via activation of the NF-κB, ERK 1/2, and p38 MAPK signaling pathways, but it also induced differentiation and mineralization of osteoblasts by regulating differentiation factors. Thus, toddaculin might be beneficial for the prevention and treatment of osteoporosis.
Assuntos
Cumarínicos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Osteogênese/efeitos dos fármacos , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Extracts of sausage, sauce, cookie, cereal and pasta sauce spiked with milk standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of the three ELISA methods using a Milk Protein Casein ELISA Kit (Casein kit), a Milk Protein Beta-Lactoglobulin ELISA Kit (Beta-Lactoglobulin kit) and a FASTKIT Milk ELISA Kit (Milk ELISA kit) were mostly below 10%. Mean recoveries of the milk standard protein from the food extracts were over 40% in the three ELISA methods with a few excertions. The recoveries of milk standard protein from the sauce extract in Casein kit were improved by adjusting the extract to neutrality before the Casein kit assay. The recoveries of milk standard protein from cookie, cereal and pasta sauce were improved by the increasing the amount of antibody coated in the Milk ELISA kit. The detection limits of all the ELISA methods were 1 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of milk protein levels in extracts of sausage, sauce, cookie, cereal and pasta sauce.
Assuntos
Alérgenos/análise , Ensaio de Imunoadsorção Enzimática/métodos , Análise de Alimentos/métodos , Proteínas do Leite/análise , Kit de Reagentes para Diagnóstico/normas , Estudos Multicêntricos como Assunto , Reprodutibilidade dos TestesRESUMO
Extracts of sausage, sauce, pasta sauce, fish paste and cereal spiked with wheat standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of both ELISA methods using a Wheat Protein ELISA Kit (Gliadin kit) and a FASTKIT Wheat ELISA Kit (Wheat ELISA kit) were mostly below 10%. Mean recoveries of the wheat standard protein from the food extracts were over 40% in the two ELISA methods except those from cereal extract determined using the Wheat ELISA kit. Repeatability relative standard deviations of wheat standard protein in the five food extracts were in the ranges of 16-26.9% and 3.7-36.2% for the Gliadin kit and the Wheat ELISA kit, respectively. Reproducibility relative standard deviations of wheat standard protein in the five food extracts were 21.6-38.5%, 29.7-53.8% for the Gliadin kit and the Wheat ELISA kit, respectively. The recoveries of wheat standard protein from the cereal extract were improved by the increasing the amount of antibody coated on the plate in the Wheat ELISA kit. The detection limits of both ELISA methods were 1 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of wheat protein levels in extracts of sausage, sauce, pasta sauce, fish paste and cereal.
Assuntos
Alérgenos/análise , Ensaio de Imunoadsorção Enzimática/métodos , Análise de Alimentos/métodos , Proteínas de Plantas/análise , Kit de Reagentes para Diagnóstico/normas , Triticum , Estudos Multicêntricos como Assunto , Reprodutibilidade dos TestesRESUMO
Inter-laboratory evaluation studies were conducted for the notified ELISA methods for allergic substances (Egg). Standard extracts of egg spiked in extracts of sausage, sauce, cookie, bread and cereal at a level of 5-20 ng/mL as the sample solution were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of all three ELISA methods using an Egg Protein ovalbumin ELISA Kit (ovalbumin kit), an Egg Protein ovomucoid ELISA Kit (ovomucoid kit) and a FASTKIT Egg ELISA kit (Egg ELISA kit) were mostly less than 10%. Mean recoveries of the standard extract of egg were over 40% in the three ELISA methods. Repeatability relative standard deviations of egg standard solution in five food extracts were in the ranges of 18.7-25.5%, 18.6-41.8%, 21.3-43.3% for the ovalbumin kit, the ovomucoid kit and the Egg ELISA kit, respectively. Reproducibility relative standard deviations of egg standard solution in five food extracts were 16.8-35.1%, 19.6-35.8%, 24.7-51.1% for the ovalbumin kit, the ovomucoid kit and the Egg ELISA kit, respectively. The detection limits of all the ELISA methods were 4-5 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of egg protein levels in extracts of sausage, sauce, cookie, bread and cereal.
Assuntos
Alérgenos/análise , Proteínas do Ovo/análise , Ensaio de Imunoadsorção Enzimática/normas , Análise de Alimentos/normas , Reprodutibilidade dos TestesRESUMO
Inter-laboratory evaluation studies were conducted for the ELISA methods for allergic substances (buckwheat). Extracts of snack, bun and udon spiked with buckwheat standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate at 10 laboratories. Coefficients of variation (CVs) of the ELISA methods using a Buckwheat Protein ELISA Kit (Buckwheat kit) and a FASTKIT Buckwheat ELISA kit (Buckwheat ELISA kit) were mostly below 10%. Mean recoveries of the buckwheat standard protein from the food extracts were over 40% in the two ELISA methods. Repeatability relative standard deviations of buckwheat standard protein in three food extracts were in the ranges of 6.8-78.5% and 5.0-33.9% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. Reproducibility relative standard deviations of buckwheat standard protein in three food extracts were 11.9-69.5% and 16.5-34.1% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. The detection limits of both ELISA methods were 1 ng/mL in sample solutions. These results suggest that the notified ELISA methods are reliable and reproducible for the inspection of buckwheat protein levels in extracts of snack, bun and udon.
Assuntos
Alérgenos/análise , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Fagopyrum/química , Análise de Alimentos/métodos , Análise de Alimentos/normas , Japão , Laboratórios , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Inter-laboratory evaluation studies were conducted for ELISA methods for allergic substances (peanuts). Extracts of biscuit, sauce, chocolate and butter spiked with peanut standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of the ELISA methods using a Peanut Protein ELISA Kit (Peanut kit) and a FASTKIT Peanut ELISA kit (Peanut ELISA kit) were mostly below 10%. Mean recoveries of the peanut standard protein from the food extracts were over 40% in the two ELISA methods. Repeatability relative standard deviations of peanut standard protein in four food extracts were in the ranges of 15.2-49.7% and 3.0-28.3% for the Peanut kit and the Peanut ELISA kit, respectively. Reproducibility relative standard deviations of peanut standard protein in four food extracts were 23.5-44.4%, 9.6-28.4% for the Peanut kit and the Peanut ELISA kit, respectively. The detection limits of both ELISA methods were 2-2.5 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of peanut protein levels in extracts of biscuit, sauce, chocolate and butter.
Assuntos
Alérgenos/análise , Arachis/química , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Análise de Alimentos/métodos , Japão , Laboratórios , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A soybean certified reference material for pesticide residue analysis was developed by the National Metrology Institute of Japan. Three organophosphorus (diazinon, fenitrothion, chlorphyrifos) and one pyrethroid (permethrin) pesticides were sprayed on soybeans three times before harvest. These soybeans were freeze pulverized, homogenized, bottled, and sterilized by γ-irradiation to prepare the candidate material. Three isotope-dilution mass spectrometric methods that varied in terms of the solvents used for extraction of the target pesticides, the clean-up procedure, and the injection techniques and columns used for quantification via gas chromatography/mass spectrometry were applied to the characterization. Each target pesticide was quantified by two of these analytical methods, and the results were in good agreement. Homogeneity and stability assessment of the material demonstrated that the relative standard uncertainties due to the inhomogeneity and the instability for an expiry date of 55 months were 1.89-4.00% and 6.65-11.5%, respectively. The certified pesticide concentrations with expanded uncertainties (coverage factor k=2, approximate 95% confidence interval) calculated using the results of the characterization and the homogeneity and stability assessment were 21.7 ± 3.2 µg/kg for diazinon, 88 ± 21 µg/kg for fenitrothion, 11.1 ± 3.2 µg/kg for chlorpyrifos, and 20.1 ± 4.3 µg/kg for permethrin (as the sum of the constituent isomers).
Assuntos
Glycine max/química , Resíduos de Praguicidas/análise , Padrões de Referência , Espectrometria de Massas/métodos , IncertezaRESUMO
Corn is a major ingredient of mixed feed, and it has been reported that corn may be contaminated with deoxynivalenol (DON). There is also a possibility of contamination with other trichothecenes. Recently, corn-derived products such as Distiller's Dried Grains with Solubles (DDGS), corn gluten feed and corn gluten meal have been introduced and used for mixed feed. However the actual occurrence of trichothecenes has not been sufficiently investigated. So, in this study, we analyzed DON and 6 other trichothecenes, i.e., 3-acetyl-deoxynivalenol (3AcDON), 15-acetyl-deoxynivalenol (15AcDON), T-2 toxin, HT-2 toxin, nivalenol, and fusarenon-X, in DDGS, corn gluten feed, corn gluten meal, and mixed feed containing corn-derived ingredients. The major trichothecenes identified in the samples tested were DON, 3AcDON and 15AcDON. In particular, DON, 3AcDON and 15AcDON were detected in most DDGS and corn gluten feed samples. Most samples of mixed feed contained DON and 15AcDON, but only one mixed feed sample contained 3AcDON. In contrast, corn gluten meal was contaminated with lower levels of these compounds than the other samples tested. Among 36 corn gluten meal samples, DON was detected in 24 and 15AcDON was detected in 20 samples. 3AcDON was not detected in any of the corn gluten meal samples.
Assuntos
Ração Animal/análise , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Tricotecenos/análise , Zea mays/química , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
An apple certified reference material for the analysis of pesticide residues was issued by the National Metrology Institute of Japan. Organophosphorus and pyrethroid pesticides were sprayed on apples, and these were used as raw materials of certified reference material. The harvested apples were cut into small pieces, freeze-dried, pulverized, sieved, placed into 200 brown glass bottles (3g each), and sterilized by γ-irradiation. Stability and homogeneity assessment was performed, and the relative uncertainties due to instability (for an expiry date of 32 months) and inhomogeneity were 10.3-25.0% and 4.0-6.8%, respectively. The characterization was carried out using multiple analytical methods to ensure the reliability of analytical results; the values of target pesticides were obtained by isotope dilution mass spectrometry. Certified values were 2.28 ± 0.82 mg/kg for diazinon, 3.14 ± 0.79 mg/kg for fenitrothion, 1.55 ± 0.81 mg/kg for cypermethrin, and 2.81 ± 0.70 mg/kg for permethrin.
Assuntos
Malus/química , Espectrometria de Massas/normas , Compostos Organofosforados/análise , Resíduos de Praguicidas/análise , Piretrinas/análise , Contaminação de Alimentos/análise , Espectrometria de Massas/métodos , Padrões de ReferênciaRESUMO
We examined whether immunochemical-based test kits designed for quantitative analysis of deoxynivalenol (DON) screening in grain crops are applicable to corn processing by-products. Commercially available test kits (two types of immunochromatographic kits and three types of ELISA kits) were used to assay three types of corn processing by-products and mixed feed. The results obtained with some kits were significantly different from those of LC-MS analysis. Since the differences might be caused by insufficient extraction of DON from samples, the extraction time of all kits was set to be 20 minutes, based on a study of the dependence of the amount of DON extracted on the shaking time. Moreover, the extract of corn processing by-products was acidic, resulting in inhibition of the antigen-antibody reaction, so neutralization and centrifugation processes were introduced to prevent denaturation of antibody. After these modifications, the recovery for all kits in assays of corn gluten meal was within the range of 80-120%, and all kits showed acceptable accuracy. The relative standard deviation (RSD) of repeatability tests for all kits was less than 11.3% for analyses of both corn processing by-products and mixed feeds, indicating good precision. The above results showed that the kits studied were applicable to the quantitative assay of DON in corn processing by-products and mixed feed after modifications as described in this paper.