RESUMO
This work aimed to carry out phytochemical prospecting and evaluate the antioxidant potential of Diplopterys pubipetala, a species of Malpighiaceae family that has not yet been studied.In qualitative analyses of hydroethanolic extracts of leaves and stems were detected the presence of flavonoids, alkaloidsand terpenes. The histochemical evaluation evidenced a greater distribution of these compounds in the tissues of leaf when compared with those of stem. The analysis by mass spectrometry allowed the identification of prenylated xanthones and glycoside flavonoids that have not yet been reported in the literature. The antioxidant activity of the stem extract was considered moderate (IAA = 0.79), but the leaves presented a strong antioxidant activity (IAA = 1.6). In this work we present information about the phytochemicals of D. pubipetala, showing that the species is promising in obtaining compounds with medicinal potential mainly antioxidant potential.
Assuntos
Malpighiaceae , Antioxidantes/química , Flavonoides/química , Malpighiaceae/química , Compostos Fitoquímicos/análise , Extratos Vegetais/química , Folhas de Planta/químicaRESUMO
The aim of this study was to evaluate in vitro the antifungal, antibiofilm and antiproliferative activities of the extract from the leaves of Guapira graciliflora Mart. The phytochemical characterization of the extract was performed using electrospray ionization mass spectrometry (ESI-MS). The antimicrobial activity of the extract and its fractions was evaluated using the broth microdilution method against species of Candida. The inhibition of C. albicans biofilm was evaluated based on the number of colony-forming units (CFU) and metabolic activity (MTT). The antiproliferative activity of the extract and its fraction was evaluated in the presence of human tumor and non-tumor cells, and the cytotoxicity of the extract was determined on the RAW 264.7 macrophage line - both using the sulforhodamine B method. The phytochemical characterization indicated the presence of the flavonoids rutin and kaempferol. The extract and the methanol fraction exhibited moderate antifungal activity against C. albicans, C. krusei, and C. glabrata, and strong activity against C. dubliniensis. In the biofilms at 24 and 48 hours, the concentration of 12500 µg/mL of the extract was the most effective at reducing the number of CFU s/mL (44.4% and 42.9%, respectively) and the metabolic activity of C. albicans cells (34.6% and 52%, respectively). The extract and its fractions had no antiproliferative effect on the tumor lines tested, with mean activity (log GI50) equal to or greater than 1.71 µg/mL. Macrophage cell viability remained higher than 80% for concentrations of the extract of up to 62.5 µg/mL. G. graciliflora has flavonoids in its chemical composition and demonstrates potential antifungal and antibiofilm activity, with no evidence of a significant change in the viability of human tumor and non-tumor cell lines.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Nyctaginaceae/química , Extratos Vegetais/farmacologia , Antifúngicos/isolamento & purificação , Biofilmes/crescimento & desenvolvimento , Sobrevivência Celular/efeitos dos fármacos , Dose Letal Mediana , Testes de Sensibilidade MicrobianaRESUMO
Abstract: The aim of this study was to evaluate in vitro the antifungal, antibiofilm and antiproliferative activities of the extract from the leaves of Guapira graciliflora Mart. The phytochemical characterization of the extract was performed using electrospray ionization mass spectrometry (ESI-MS). The antimicrobial activity of the extract and its fractions was evaluated using the broth microdilution method against species of Candida. The inhibition of C. albicans biofilm was evaluated based on the number of colony-forming units (CFU) and metabolic activity (MTT). The antiproliferative activity of the extract and its fraction was evaluated in the presence of human tumor and non-tumor cells, and the cytotoxicity of the extract was determined on the RAW 264.7 macrophage line - both using the sulforhodamine B method. The phytochemical characterization indicated the presence of the flavonoids rutin and kaempferol. The extract and the methanol fraction exhibited moderate antifungal activity against C. albicans, C. krusei, and C. glabrata, and strong activity against C. dubliniensis. In the biofilms at 24 and 48 hours, the concentration of 12500 µg/mL of the extract was the most effective at reducing the number of CFU s/mL (44.4% and 42.9%, respectively) and the metabolic activity of C. albicans cells (34.6% and 52%, respectively). The extract and its fractions had no antiproliferative effect on the tumor lines tested, with mean activity (log GI50) equal to or greater than 1.71 µg/mL. Macrophage cell viability remained higher than 80% for concentrations of the extract of up to 62.5 µg/mL. G. graciliflora has flavonoids in its chemical composition and demonstrates potential antifungal and antibiofilm activity, with no evidence of a significant change in the viability of human tumor and non-tumor cell lines.
Assuntos
Candida/efeitos dos fármacos , Extratos Vegetais/farmacologia , Biofilmes/efeitos dos fármacos , Nyctaginaceae/química , Proliferação de Células/efeitos dos fármacos , Antifúngicos/farmacologia , Testes de Sensibilidade Microbiana , Sobrevivência Celular/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Dose Letal Mediana , Antifúngicos/isolamento & purificaçãoRESUMO
PURPOSE: The goal for the present study was to implement a technique for protein extraction and identification in human cumulus cells (CCs). METHODS: Forty samples of CCs were collected after ovum pick-up from patients undergoing intracytoplasmic sperm injection (ICSI). Samples were split into the blastocyst group (n = 10), including patients in which all embryos converted into blastocysts, and the non-blastocyst group (n = 10), including patients in which none of the embryos reached the blastocyst stage or the positive-pregnancy (n = 10) and negative-pregnancy group (n = 10). Proteins were extracted and injected into a liquid chromatography system coupled to a mass spectrometer. The spectra were processed and used to search a database. RESULTS: There were 87 different proteins in samples from the blastocyst and non-blastocyst groups, in which 30 were exclusively expressed in the blastocyst group and 17 in the non-blastocyst group. Among the 72 proteins detected in the pregnancy groups, 19 were exclusively expressed in the positive, and 16 were exclusively expressed in the negative-pregnancy group. CONCLUSIONS: CC proteomics may be useful for predicting pregnancy success and the identification of patients that should be included in extended embryo culture programs.
Assuntos
Blastocisto/metabolismo , Células do Cúmulo/metabolismo , Desenvolvimento Embrionário/genética , Biossíntese de Proteínas/genética , Blastocisto/fisiologia , Cromatografia Líquida , Células do Cúmulo/fisiologia , Transferência Embrionária/métodos , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Oócitos/metabolismo , Oócitos/fisiologia , Gravidez , ProteômicaRESUMO
OBJECTIVE: To identify lipid markers of blastocyst implantation and ongoing pregnancy by day three culture medium mass spectrometry (MS) fingerprinting. METHODS: For this study, 33 culture media samples were harvested on day three, from 22 patients undergoing day five embryo transfers. All embryos achieved the blastocyst stage and were split into groups based on their implantation (Negative Implantation, n= 14 and Positive Implantation, n= 19). The positive implantation cycles resulted in successful ongoing pregnancies. The lipid extraction was performed by the Bligh-Dyer protocol and mass spectra were obtained with a direct infusion into a Q-Tof mass spectrometer. The data obtained was analyzed by Principal Component Analysis (PCA) and Partial Least Square Discrimination Analysis (PLS-DA). The statistical analysis was performed using the Metabo-Analyst 2.0. RESULTS: The variable importance in the projection (VIP) plot of the PLS-DA provided a list of four ions, in the positive mode, with an area under the curve (AUC) of 73.5%; and eight ions, in the negative mode, with and AUC of 72.0%. For both positive and negative modes, possible biomarkers for the negative implantation were identified by the lipidmaps: phosphoethanolamine, dicarboxylic acids, glycerophosphoglycerol, glycerophosphocholine, glicerophosphoinositol, phosphoethanolamine and unsaturated fat acids. The other ions were not identified. These lipids are involved in the GPI anchor biosynthesis and synthesis of lycerophospholipids and phosphate inositol. CONCLUSION: MS fingerprinting is useful to identify blastocysts that fail to implant, and therefore this technique could be incorporated into the laboratory routine, adjunct to morphology evaluation to identify embryos that should not be transferred.
RESUMO
OBJECTIVE: To identify lipid markers of blastocyst formation by day three culture medium mass spectrometry (MS) fingerprinting. METHODS: For this study, 50 embryo samples from culture media were harvested on day three, from patients undergoing embryo transfers on day five. Samples were split into groups based on their degree of expansion and hatching status on day five (Complete-Blastocyst, n=25 and No-Blastocyst, n=25) and its secretomes were analysed by MS. Mass spectra fingerprinting was acquired using a Q-Tof spectrometer (LC-MS, Agilent 6550 iFunnel Q-TOF) equipped with an automated injector. The data was analysed using the principal component analysis (PCA) followed by a partial least square discrimination analysis (PLS-DA), combined with variable influence in the projection (VIP) scores. RESULTS: In total, there were 1,657 ions found, in which 165 ions were differently expressed between groups, with a fold chance ≥ 4x and P<0.001, in the t-test. PLS-DA showed a clear separation between the groups and among 15 VIPs selected by the program, 13 of them were highly expressed in the Complete-Blastocyst Group and two were expressed in the No-Blastocyts Group. Besides embryo status on day five, the PLS-DA was also able to classify samples according to patients' age. Lipids supposedly highly expressed in the Complete-Blastocyst Group included: isoprenoids, diacylglycerols, sterols, fatty esters, secosteroids, phosphosphingolipids, glycerophosphates and diacylglycerophosphates, while fatty amides were suggested to be highly expressed in the No-Blastocysts Group. CONCLUSIONS: Day three culture medium MS is a promising approach for the identification of embryos that should be cultured until day five.
RESUMO
This study has evaluated the performance of a multivariate statistical model to predict embryo implantation potential by processing data from the chemical fingerprinting of culture medium samples used for human embryo culture. The culture medium for 113 embryos from 55 patients undergoing ICSI was collected after embryo transfer. The samples were split into positive (n=29) and negative (n=84) implantation groups according their implantation outcomes (100% or 0% implantation). The samples were individually diluted and analyzed by electrospray ionization mass spectrometry (ESI-MS). The m/z ratios and relative abundances of the major ions in each spectrum were considered for partial least square discriminant analysis. Data were divided into two subsets (calibration and validation), and the models were evaluated and applied to the validation set. A total of 5987 ions were observed in the groups. The multivariate statistical model described more than 82% of the data variability. Samples of the positive group were correctly identified with 100% probability and negative samples with 70%. The culture media used for embryos that were positive or negative for successful implantation showed specific biochemical signatures that could be detected in a fast, simple, and noninvasive way by ESI-MS. To our knowledge, this is the first report that uses MS fingerprinting to predict human embryo implantation potential. This biochemical profile could help the selection of the most viable embryo, improving single-embryo transfer and thus eliminating the risk and undesirable outcomes of multiple pregnancies.
Assuntos
Ectogênese , Implantação do Embrião , Transferência Embrionária , Proteínas Fetais/análise , Modelos Biológicos , Zigoto/metabolismo , Adulto , Calibragem , Estudos de Casos e Controles , Meios de Cultivo Condicionados/química , Análise Discriminante , Feminino , Proteínas Fetais/química , Proteínas Fetais/metabolismo , Humanos , Infertilidade Feminina/etiologia , Infertilidade Feminina/terapia , Infertilidade Masculina/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mapeamento de Peptídeos , Espectrometria de Massas por Ionização por Electrospray , Injeções de Esperma IntracitoplásmicasRESUMO
A aterosclerose encontra-se em primeiro lugar dentre as várias causas de morte no mundo ocidental; distribuída em todo mundo, ela vem alcançando proporções epidêmicas alarmantes. A determinação laboratorial das concentrações de triglicerídeos,colesterol total (CT), HDL-colesterol (HDL-C) e LDL-colesterol (LDL-C) é pré-requisito importante na avaliação do risco que um indivíduo apresenta de desenvolver doenças relacionadas à aterosclerose. O objetivo deste trabalho foi avaliar a variabilidade interlaboratorial na determinação destes analitos. Para isso, foram enviados aos laboratórios de análise clínicas (LACs) participantes do estudo 10 amostras de soro de origem humana, para determinação de triglicerídeos, CT, HDL-C e LDL-C. A metodologia foi planejada a fim de eliminar variáveis pré-analíticas, permitindo assim quantificar a variação da mesma determinação entre os LACs participantes. Adeterminação de CT foi a que apresentou a menor dispersão de resultados, sendo este dado evidenciado pela inexpressiva quantidade de valores fora da linha de ± 2SD. A análise de dispersão de HDL-C mostrou que 50 dos indivíduos analisados receberia resultados com interpretações clínicas diferentes por diferentes LACs. Esta porcentagem foi de 40 no LDL-C e de 40 para triglicerídeos. Os resultados obtidos permitem concluir que a variabilidade laboratorial na determinação de lipídeos pode ser um fator importante na interpretação de resultados seqüenciais.
Assuntos
Humanos , Aterosclerose , Laboratórios , Lipídeos , Medição de Risco , TriglicerídeosRESUMO
Background : A broader view of living systems complexity is bringing important contributions to biological sciences, since the genome expression is affected by other classes of molecules, which in their turn interact themselves in cellular metabolic pathways and biochemical networks. This level of information has been made possible by the emergence of the omic strategies, such as proteomics, metabolomics and lipidomics, that are mainly based on mass spectrometry (MS) platforms. MS has presented an incredible development over the last years, evolving to a powerful and universal analytical technique. Its ability to analyze proteins and small molecules such as lipids, sugars and metabolites at the structural level, with sensitivity and speed inconceivable a few years ago, is the major driving force in the omic fields. The development of electrospray and matrix-assisted laser desorption/ionization (MALDI) ionization techniques has decisively contributed to the many applications of this technology nowadays. Herein, we present and discuss omic concepts and strategies, as well as detail basic principles of MS. Applications and future perspectives of these approaches are focused in the reproductive medicine area. Review: The omic technologies propose global characterization of specific classes of target biomolecules of cellular systems as a strategy to achieve comprehensive understanding of biological functions. The genomics, aimed at performing the entire genetic sequencing of organisms, represented the seminal step towards the understanding of the complex logic that orchestrates the function of all organisms or the defects leading to diseases. But to express the phenotype, information needs to flow from DNA via carrier biomolecules through processes that are being addressed by new omic sciences such as the transcriptomics, proteomics, metabolomics, glycomics, lipidomics, and fluxomics. Mass spectrometry (MS) is nowadays the most powerful technique for the structural characterization of biomolecules, and has therefore become the central technique for the omic sciences. Using revolutionary ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI), a wide range of biomolecules such as peptides, proteins, lipids and sugars are efficiently transferred in intact ionized forms to the gas phase for MS analysis. The development of ESI-MS and MALDI-MS has been awarded the Nobel Prize for Chemistry in 2002, rocketing the application of MS in the omic sciences. More recently, ambient ionization MS techniques, such as desorption electrospray ionization (DESI) and easy ambient sonic-spray ionization (EASI), have been developed for ionization in the open atmosphere, in a workup free and high throughput fashion directly from sample in their original environments. For the more complex samples, the coupling with separation techniques such as liquid chromatrography (LC) as well as the use of tandem MS (LC-MS/MS) has allowed comprehensive mixture characterization of major biomolecules. Conclusion: This manuscript describes recent advances of MS in the proteomics, metabolomics and lipidomics for biological sciences, and points out the relevant contributions that MS is likely to bring to fundamental and applied research in human and animal embryo biotechnologies.