RESUMO
A high throughput method was developed to detect bioactive molecules with inhibitory activity over cyclooxygenase (COX-2) enzyme applying effect-directed analysis and planar chromatography hyphenated with bioassay and mass spectrometry. The assay was based on the indirect measurement of arachidonic acid transformation into prostaglandin with the colorimetric co-substrate N,N,N',N'-tetramethyl-p-phenylenediamine. Inhibitory zones were observed as colorless bands over a blue background. Using a central composite design the critical factors like substrate concentration, enzyme: substrate ratio, reaction time, and co-substrate concentration were optimized. Optimal conditions were achieved with 0.03 mg/mL of arachidonic acid, 0.15 U/mL of COX-2, and 8.21 mg/mL of chromogenic reagent. Method usefulness was challenged analyzing fresh Chiloe's giant garlic (Allium ampeloprasum L) ethanol: water (8:2 v/v) extract, finding COX-2 inhibitors that were preliminarily identified as the isomers γ-glutamyl-S-allyl-l-cysteine and γ-glutamyl-S-(trans-1-propenyl)-L- cysteine.
Assuntos
Bioensaio , Inibidores de Ciclo-Oxigenase 2 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2/farmacologia , Cromatografia em Camada Fina/métodos , Ácido Araquidônico , Espectrometria de Massas , Bioensaio/métodos , Extratos Vegetais/farmacologia , Extratos Vegetais/químicaRESUMO
PURPOSE: To test the performance of different algorithms that can be used in inter-laboratory comparisons based on dicentric chromosome analysis, and to evaluate the impact of considering a priori values different to calculate individual laboratory performance based on the ionizing radiation dose estimation. METHODS: Mean and standard deviation estimations in inter-laboratory comparisons are tested on simulated data and data from previously published inter-laboratory comparisons using three robust algorithms, Algorithm A, Algorithm B and Q/Hampel, all programmed in R-project language and implemented in a Shiny application. The simulated data were generated assuming three different probabilities to contaminate inter-laboratory comparisons samples with atypical dose values. Comparison between different algorithms was also done using published exercises where blood samples were irradiated at 0 and 0.7 Gy that represent a challenge for the assessment of an inter-laboratory comparison. RESULTS: The best performance was obtained with the Q/Hampel algorithm for the estimation of the dose mean and with the Algorithm B for the estimation of the dose standard deviation under the conditions tested in the simulations. The Q/Hampel algorithm showed the best performance when non-irradiated samples were evaluated and there was a high proportion of identical values. The presence identical values cause the Algorithm B to fail. Real examples illustrating the need to consider standard deviation priors, and the need to use algorithms resistant to a high proportion of identical values are presented. CONCLUSIONS: Q/Hampel algorithm is a serious candidate to estimate the dose mean in the inter-laboratory comparisons, and to estimate both parameters when the proportion of identical values equals or higher than the half of the results. When the proportion of identical values is less than the half of the results, the Algorithm B should be considered as a candidate to estimate the standard deviation in the inter-laboratory comparisons with small number of laboratories. We remark that special attention is needed to establish prior definitions of standard deviation in the assessment of inter-laboratory dicentric assay comparisons.
Assuntos
Bioensaio , Aberrações Cromossômicas , Humanos , Bioensaio/métodos , Radiação IonizanteRESUMO
Behavioral lab bioassays involving termites must be promptly performed to allow intended observations prior to death from dissecation, typical of these soft-bodied insects. To this end, topic markers have been proposed as an alternative to histological stains which, while not always toxic are inevitably lengthy to apply. Among recommended topic markers, gouache is easy to apply, dries out quickly, but it is known affect termites in the long run, being suitable only to short-term bioassays. Its alternative, colored glue, is also easy to apply, but it takes long to dry and it is too dense and heavy, being thus prone to affect termite walking patterns. Here we tested a mix of gouache and colored glue aiming to combine the qualities of both into a suitable topical marker for Cornitermes cumulans termites. Similar patterns of survival presented by marked and unmarked termites ruled out concerns about toxicity of this mixture. Such results were consistent across distinct group densities evidencing that the mixture does not interfere with, nor it is affected by, crowding effects. Because crowding regulates interindividual interactions and these underlie most behaviors, the mixture can be thought to be suitable to behavioral studies. We argue that this 1:2 glue:gouache mixture is an excellent alternative to mark termites for lab bioassays. Being atoxic, cheap, easy to apply, and non-invasive, this mixture may happen to be useful not only for termites but also in bioassaying other similarly soft-bodied insects.
Bioensaios comportamentais em laboratório com cupins devem ser realizados rapidamente a fim de garantir observações antes da morte por dissecação, típico desses insetos de corpo mole. Para este fim, marcadores tópicos têm sido propostos como uma alternativa para marcadores histológicos que, embora nem sempre tóxico, possuem uma aplicação demorada. Entre os marcadores tópicos recomendados, tinta guache é de fácil aplicação, rápida secagem, porém afeta os cupins em bioensaios longos, sendo adequado apenas para bioensaios curtos. Sua alternativa, cola colorida, também é de fácil aplicação mas leva muito tempo para secar e é muito denso e pesado, afetando os padrões de caminhamento dos cupins. No presente estudo, nós testamos uma mistura de tinta guache e cola colorida objetivando combinar as qualidades de ambos os marcadores tópicos em um marcador tópico adequado para Cornitermes cumulans. Padrões similares de sobrevivência entre cupins marcados e controle indicam a ausência de toxicidade na mistura de tinta guache e cola colorida. Tais resultados são consistentes em grupos de densidades distintas, o que comprova que a mistura não interfere, nem sofre efeitos de aglomeração. Uma vez que a aglomeração regula as interações inter-individuais e afetam a maioria dos comportamentos, a mistura pode ser adequada para estudos comportamentais. Nós argumentamos que a mistura de tinta guache e cola (1:2) é uma excelente alternativa como marcador tópico em cupins para bioensaios em laboratório. Sendo atóxico, barato, fácil de aplicar e não invasivo, esta mistura pode ser útil não só para os cupins, mas também em bioensaios com outros insetos de corpo mole.
Assuntos
Animais , Bioensaio/métodos , Bioensaio/veterinária , IsópterosRESUMO
Abstract The anecdotal use of Alternanthera sessilis L. as a relief for diabetes has been known in the Philippines for generations, and antidiabetic activity of similar varieties in other countries is likewise documented. However, the compounds responsible for this activity remain unclear. This study aims to isolate the anti-hyperglycemic fraction of local A. sessilis leaves and identify the compounds in this fraction. Methanol extract of A. sessilis leaves and its hexane, ethyl acetate (ASE), and water fractions were administered to alloxan-induced diabetic mice. ASE (250mg/kg) had the highest anti-hyperglycemic activity at 6-h post-treatment (25.81%±12.72%), with almost similar blood glucose reduction rate as metformin (30.13±3.75%, p=0.767). Repeated fractionation employing chromatographic separation techniques followed by in vivo anti-hyperglycemic assay yielded partially purified subfractions. A. sessilis ethyl acetate subfraction 4-2 (100mg/kg) displayed remarkable suppression of blood glucose rise in diabetic mice at 6-h post-treatment (26.45±3.75%, p<0.0001), with comparable activity with metformin (100mg/kg, 27.87±5.65%, p=0.652). Liquid chromatography/mass spectrometry showed eight distinct peaks, with four peaks annotated via the Traditional Chinese Medicine library and custom library for A. sessilis. Among these, luteolin, apigenin, ononin, and sophorabioside were identified as putative compounds responsible for the anti-hyperglycemic activity. This result provided basis for the reported anecdotal claims and potential utility of the local variety of A. sessilis leaves as sources of anti-hyperglycemic agents
Assuntos
Animais , Masculino , Feminino , Camundongos , Espectrometria de Massas/métodos , Bioensaio/métodos , Folhas de Planta/classificação , Amaranthaceae/efeitos adversos , Cromatografia Líquida/métodos , Apigenina/agonistasRESUMO
Among the management and control tactics of the coffee berry borer Hypothenemus hampei (Ferrari), there is the use of entomopathogenic fungi. Due to the importance of prospecting isolates of entomopathogenic fungi for the control of the coffee berry borer, the objective of this study was to evaluate the efficiency of 26 isolates of entomopathogenic fungi in the control of this insect pest in the laboratory. The coffee berry borers were immersed in a solution adjusted to the concentration of 1 to 3 × 108 conidia/mL of each isolate and the control treatment (sterilized water). After seven days total mortality and confirmed mortality were evaluated. The isolates that caused the highest mortality and two commercial isolates were selected for evaluation of lethal concentration (LC50 and LC90) and lethal time (LT50 and LT90). Coffee berry borers were treated at different conidia concentrations for lethal concentration to assess total and confirmed mortality. For a lethal time, the coffee berry borers were treated at the concentration of 108 conidia/mL of the selected isolates, and, after two days and every 24 hours until the eighth day, the number of dead individuals was verified. Among the 26 isolates evaluated, 24 presented mortality higher than the control treatment, and three presented mortality higher than 85%. In the LC50 and LC90 assays, the IBCB 353 and IBCB 364 isolates were more lethal to H. hampei. In the LT50 and LT90 assays, the IBCB 66 and IBCB 353 isolates caused lethality in a shorter time.
Assuntos
Bioensaio/métodos , Controle Biológico de Vetores , Gorgulhos , Fungos , Fabaceae/parasitologiaRESUMO
Behavioral lab bioassays involving termites must be promptly performed to allow intended observations prior to death from dissecation, typical of these soft-bodied insects. To this end, topic markers have been proposed as an alternative to histological stains which, while not always toxic are inevitably lengthy to apply. Among recommended topic markers, gouache is easy to apply, dries out quickly, but it is known affect termites in the long run, being suitable only to short-term bioassays. Its alternative, colored glue, is also easy to apply, but it takes long to dry and it is too dense and heavy, being thus prone to affect termite walking patterns. Here we tested a mix of gouache and colored glue aiming to combine the qualities of both into a suitable topical marker for Cornitermes cumulans termites. Similar patterns of survival presented by marked and unmarked termites ruled out concerns about toxicity of this mixture. Such results were consistent across distinct group densities evidencing that the mixture does not interfere with, nor it is affected by, crowding effects. Because crowding regulates interindividual interactions and these underlie most behaviors, the mixture can be thought to be suitable to behavioral studies. We argue that this 1:2 glue:gouache mixture is an excellent alternative to mark termites for lab bioassays. Being atoxic, cheap, easy to apply, and non-invasive, this mixture may happen to be useful not only for termites but also in bioassaying other similarly soft-bodied insects.(AU)
Bioensaios comportamentais em laboratório com cupins devem ser realizados rapidamente a fim de garantir observações antes da morte por dissecação, típico desses insetos de corpo mole. Para este fim, marcadores tópicos têm sido propostos como uma alternativa para marcadores histológicos que, embora nem sempre tóxico, possuem uma aplicação demorada. Entre os marcadores tópicos recomendados, tinta guache é de fácil aplicação, rápida secagem, porém afeta os cupins em bioensaios longos, sendo adequado apenas para bioensaios curtos. Sua alternativa, cola colorida, também é de fácil aplicação mas leva muito tempo para secar e é muito denso e pesado, afetando os padrões de caminhamento dos cupins. No presente estudo, nós testamos uma mistura de tinta guache e cola colorida objetivando combinar as qualidades de ambos os marcadores tópicos em um marcador tópico adequado para Cornitermes cumulans. Padrões similares de sobrevivência entre cupins marcados e controle indicam a ausência de toxicidade na mistura de tinta guache e cola colorida. Tais resultados são consistentes em grupos de densidades distintas, o que comprova que a mistura não interfere, nem sofre efeitos de aglomeração. Uma vez que a aglomeração regula as interações inter-individuais e afetam a maioria dos comportamentos, a mistura pode ser adequada para estudos comportamentais. Nós argumentamos que a mistura de tinta guache e cola (1:2) é uma excelente alternativa como marcador tópico em cupins para bioensaios em laboratório. Sendo atóxico, barato, fácil de aplicar e não invasivo, esta mistura pode ser útil não só para os cupins, mas também em bioensaios com outros insetos de corpo mole.(AU)
Assuntos
Animais , Bioensaio/métodos , Bioensaio/veterinária , IsópterosRESUMO
Thin-layer chromatography (TLC) is widely used for food analysis and quality control. As an open chromatographic system, TLC is compatible with microbial-, biochemical-, and chemical-based derivatization methods. This compatibility makes it possible to run in situ bioassays directly on the plate to obtain activity-profile chromatograms, i.e., the effect-directed analysis of the sample. Many of the properties that can be currently measured using this assay format are related to either desired or undesired features for food related products. The TLC assays can detect compounds related to the stability of foods (antioxidant, antimicrobial, antibrowning, etc.), contaminants (antibiotics, pesticides, estrogenic compounds, etc.), and compounds that affect the absorption, metabolism or excretion of nutrients and metabolites or could improve the consumers health (enzyme inhibitors). In this article, different food related TLC-assays are reviewed. The different detection systems used, the way in which they are applied as well as selected examples are discussed.
Assuntos
Anti-Infecciosos , Antioxidantes , Anti-Infecciosos/farmacologia , Antioxidantes/análise , Bioensaio/métodos , Cromatografia em Camada Fina/métodos , Inibidores Enzimáticos/farmacologiaRESUMO
A variety of microalgal species produce lipophilic toxins (LT) that are accumulated by filter-feeding bivalves. Their negative impacts on human health and shellfish exploitation are determined by toxic potential of the local strains and toxin biotransformations by exploited bivalve species. Chile has become, in a decade, the world's major exporter of mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) and has implemented toxin testing according to importing countries' demands. Species of the Dinophysis acuminata complex and Protoceratium reticulatum are the most widespread and abundant LT producers in Chile. Dominant D. acuminata strains, notwithstanding, unlike most strains in Europe rich in okadaic acid (OA), produce only pectenotoxins, with no impact on human health. Dinophysis acuta, suspected to be the main cause of diarrhetic shellfish poisoning outbreaks, is found in the two southernmost regions of Chile, and has apparently shifted poleward. Mouse bioassay (MBA) is the official method to control shellfish safety for the national market. Positive results from mouse tests to mixtures of toxins and other compounds only toxic by intraperitoneal injection, including already deregulated toxins (PTXs), force unnecessary harvesting bans, and hinder progress in the identification of emerging toxins. Here, 50 years of LST events in Chile, and current knowledge of their sources, accumulation and effects, are reviewed. Improvements of monitoring practices are suggested, and strategies to face new challenges and answer the main questions are proposed.
Assuntos
Toxinas Marinhas/toxicidade , Microalgas/metabolismo , Intoxicação por Frutos do Mar/prevenção & controle , Animais , Bioensaio/métodos , Bivalves/química , Bivalves/metabolismo , Chile , Humanos , Toxinas Marinhas/isolamento & purificação , CamundongosRESUMO
Abstract Instrumental techniques are preferred over bioassay methods for antibiotic quantification mainly due to speed and ability to quantify metabolites in biological samples; however, the potency and biological activity of these drugs cannot be assessed. Two methods - agar well diffusion (bio-assay) and spectrophotometric methods were used to evaluate amikacin sulfate injection. Agar plates were inoculated with S. aureus inoculum; zones of inhibition from its susceptibility to amikacin were obtained, while spectrophotometric absorption at 650 nm of ninhydrin- derivatized amikacin in phosphate buffer (pH 8) was measured. Methods performance showed linearity from 1 - 16 µgmL-1 (bioassay, r = 0.9994) and 10-50 µgmL-1 (spectrophotometric, r = 0.9998). Molar absorptivity was 2.595 x 104 Lmol-1cm-1. Limits of detection and quantification were 1.07 and 3.24 µgmL-1 respectively for bioassay method, while corresponding values for spectrophotometric method were 0.98 and 2.97 µg mL-1. Relative standard deviations were ≤ 2.0% for both methods, with recoveries from 95.93 - 100.25%. Amikacin in brands ranged from 97.53 ± 2.68 to 100.84 ± 1.82%, student's t-test was ≤ 2.78 (n = 4) with respect to label claim for both methods. Experimental paired t-test (t = 2.07; n = 4) and F-test (F = 3.94; n = 4) values indicated no significant difference between both methods, hence comparable and can jointly be used in quality control assessment of antibiotics
Assuntos
Injeções/classificação , Bioensaio/métodos , Preparações Farmacêuticas/classificação , Ágar/farmacologia , Aminoglicosídeos/agonistas , Antibacterianos/farmacologia , Ninidrina/administração & dosagemRESUMO
Bacterial cell surface hydrophobicity is a relevant property in determining the ability of bacteria to adhere to inert surfaces. This property has been measured using the microbial adhesion to hydrocarbon (MATH) test. Several reports in the literature establish the percentage of adhesion to hydrocarbons (PoAtH) value produced by the MATH test for a broad variety of bacteria. Discrepancies in PoAtH values reported for the same strain of a specific microorganism suggest that some method-induced variation may exist, as different research teams employ different versions of the assay. The objective of the present study was to compare the performance of different versions of the MATH test as reported in the literature, to quantify the magnitude of the method-induced variation on PoAtH values. The study demonstrated that PoAtH values are influenced twice as much by variations in the employed assay than by actual differences in cell surface composition or architecture. The two L. reuteri strains studied responded differently to changes in assay conditions showing 40 and 70% method-dependent variation for strain ATCC 53609 and 55730, respectively. These results highlight the need to properly standardize the MATH test to enable comparison of PoAtH values produced by independent research teams.