RESUMEN
Under appropriate experimental conditions, some glycoside hydrolases can catalyze transglycosylation reactions; a hypothesis associated with this is that the glycosidic linkages formed will be preferentially hydrolyzed under optimal conditions. Therefore, the hydrolytic and transglycosylation activities of isolated membranes from differentiated Caco-2 cells on sucrose, maltose and isomaltulose were evaluated. After the enzymatic reactions, the di- and trisaccharides obtained were identified by gas chromatography coupled to a mass spectrometer. Differentiated Caco-2 cell membranes exerted hydrolytic and transglycosylation activities towards the studied disaccharides. The obtained di- and trisaccharides were detected for the first time using human cell models. Due to the absence of maltase-glucoamylase complex (MGAM) in Caco-2 cells, and the known hydrolytic activity of sucrase-isomaltase (SI) towards sucrose, maltose and isomaltulose, it is plausible that the glycosidic linkages obtained after the transglycosylation reaction, mainly α-glucosyl-fructoses and α-glucosyl-glucoses, were carried out by SI complex. This approach can be used as a model to explain carbohydrate digestibility in the small intestine and as a tool to design new oligosaccharides with low intestinal digestibility.
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Disacaridasas , Maltosa , Humanos , Células CACO-2 , Hexosas , Glicósidos , SacarosaRESUMEN
This study was conducted to investigate the sweetness intensity and the potential fecal microbiome modulation of galactooligosaccharides in combination with enzymatically modified mogrosides (mMV-GOS), both generated through a patented single-pot synthesis. Sweetness intensity was performed in vivo by trained sensory panelists. The impact on the human fecal microbiome was evaluated by in vitro pH-controlled batch fermentation, and bacterial populations and organic acid concentrations were measured by qPCR and GC-FID, respectively. Significant growth (p ≤ 0.05) during the fermentation at 10 h of bacterial populations includes Bifidobacterium (8.49 ± 0.44 CFU/mL), Bacteroides (9.73 ± 0.32 CFU/mL), Enterococcus (8.17 ± 0.42 CFU/mL), and Clostridium coccoides (6.15 ± 0.11 CFU/mL) as compared to the negative control counts for each bacterial group (7.94 ± 0.27, 7.84 ± 1.11, 7.52 ± 0.37, and 5.81 ± 0.08 CFU/mL, respectively) at the same time of fermentation. Likewise, the corresponding significant increase in production of SCFA in mMV-GOS at 10 h of fermentation, mainly seen in acetate (20.32 ± 2.56 mM) and propionate (9.49 ± 1.44 mM) production compared to a negative control at the same time (8.15 ± 1.97 and 1.86 ± 0.24 mM), is in line with a positive control (short-chain fructooligosaccharides; 46.74 ± 12.13 and 6.51 ± 1.91 mM, respectively) revealing a selective fermentation. In conclusion, these substrates could be considered as novel candidate prebiotic sweeteners, foreseeing a feasible and innovative approach targeting the sucrose content reduction in food. This new ingredient could provide health benefits when evaluated in human studies by combining sweetness and prebiotic fiber functionality.
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Ácidos Grasos Volátiles , Prebióticos , Bacterias/genética , Bifidobacterium , Heces/microbiología , Fermentación , Humanos , Oligosacáridos , EdulcorantesRESUMEN
Nowadays, there is great interest in the discovery of food compounds that might inhibit gut microbial TMA production from its methylamine precursors. In this work, an innovative novel screening strategy capable of rapidly determining the differences in the metabolic response of Klebsiella pneumoniae, a bacteria producing TMA under aerobic conditions, to a library of extracts obtained from food and natural sources was developed. The proposed high-throughput screening (HTS) method combines resazurin reduction assay in 384-well plates and Gaussian Processes as a machine learning tool for data processing, allowing for a fast, cheap and highly standardized evaluation of any interfering effect of a given compound or extract on the microbial metabolism sustained by L-carnitine utilization. As a proof-of-concept of this strategy, a pilot screening of 39 extracts and 6 pure compounds was performed to search for potential candidates that could inhibit in vitro TMA formation from L-carnitine. Among all the extracts tested, three of them were selected as candidates to interfere with TMA formation. Subsequent in vitro assays confirmed the potential of oregano and red thyme hexane extracts (at 1 mg mL-1) to inhibit TMA formation in bacterial lysates. In such in vitro assay, the red thyme extract exerted comparable effects on TMA reduction (â¼40%) as 7.5 mM meldonium (â¼50% TMA decrease), a reported L-carnitine analogue. Our results show that metabolic activity could be used as a proxy of the capacity to produce TMA under controlled culture conditions using L-carnitine to sustain metabolism.
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Enfermedades Cardiovasculares , Microbioma Gastrointestinal , Carnitina/metabolismo , Microbioma Gastrointestinal/fisiología , Ensayos Analíticos de Alto Rendimiento , Humanos , Metilaminas/metabolismo , Oxazinas , Fitoquímicos , XantenosRESUMEN
Recent evidences indicate that mitochondrial genes and function are decreased in active ulcerative colitis (UC) patients, in particular, the activity of Complex I of the electron transport chain is heavily compromised. MCJ is a mitochondrial inner membrane protein identified as a natural inhibitor of respiratory chain Complex I. The induction of experimental colitis in MCJ-deficient mice leads to the upregulation of Timp3 expression resulting in the inhibition of TACE activity that likely inhibits Tnf and Tnfr1 shedding from the cell membrane in the colon. MCJ-deficient mice also show higher expression of Myd88 and Tlr9, proinflammatory genes and disease severity. Interestingly, the absence of MCJ resulted in distinct microbiota metabolism and composition, including a member of the gut community in UC patients, Ruminococcus gnavus. These changes provoked an effect on IgA levels. Gene expression analyses in UC patients showed decreased levels of MCJ and higher expression of TIMP3, suggesting a relevant role of mitochondrial genes and function among active UC. The MCJ deficiency disturbs the regulatory relationship between the host mitochondria and microbiota affecting disease severity. Our results indicate that mitochondria function may be an important factor in the pathogenesis. All together support the importance of MCJ regulation during UC.
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Bacterias/clasificación , Colitis Ulcerosa/genética , Disbiosis/genética , Proteínas del Choque Térmico HSP40/genética , Proteínas Mitocondriales/genética , Chaperonas Moleculares/genética , Proteína ADAM17/genética , Animales , Bacterias/genética , Bacterias/aislamiento & purificación , Colitis Ulcerosa/microbiología , Modelos Animales de Enfermedad , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Ratones , Microbiota , Filogenia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Índice de Severidad de la Enfermedad , Inhibidor Tisular de Metaloproteinasa-3/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The present work is aimed to develop a simple, rapid, and cost-effective CE method for the determination of trimethylamine (TMA) from bacterial origin. Optimum separation of TMA from the other components of the bacterial culture was achieved using a fused silica capillary (27 cm × 75 µm ID) and a background electrolyte solution that consisted of 0.75 M formic acid at pH 2.05. Analytical characteristics of the proposed method were evaluated through the study of its specificity, linearity, precision, accuracy, robustness, and detection/quantitation limit values. The method was linear over the range 25-2000 µM (R2 = 0.9998). The LOD and LOQ were 9 µM and 27 µM, respectively. Intra-day and inter-day RSD were ≤ 0.24% and ≤ 1.3% for migration time, respectively. Intra-day and inter-day RSD for peak area were ≤ 2.44% and ≤ 3.51%, respectively. The method showed a good accuracy with recovery percentages ranging from 95.45 to 102.21%. The method was successfully applied for the determination of microbial conversion of L-carnitine to TMA. The method shows great potential in high-throughput screening applications to assess the functionality of the gut microbiota to produce TMA. Graphical abstract.
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Análisis Costo-Beneficio , Electroforesis Capilar/métodos , Microbioma Gastrointestinal , Metilaminas/metabolismo , Bacterias/metabolismo , Electroforesis en Gel de Agar , Electroforesis Capilar/economía , Humanos , Límite de Detección , Reproducibilidad de los Resultados , Espectrofotometría UltravioletaRESUMEN
Polymethoxylated flavones (PMFs) from citrus fruits are reported to present anticancer potential. However, there is a lack of information regarding their effect on cancer stem cell (CSC) populations, which has been recognized as responsible for tumor initiation, relapse, and chemoresistance. In this study, we evaluated the effect of an orange peel extract (OPE) and its main PMFs, namely, nobiletin, sinensetin, tangeretin, and scutellarein tetramethylether in targeting cell proliferation and stemness using a 3D cell model of colorectal cancer composed of HT29 cell spheroids cultured for 7 days in stirred conditions. Soft agar assay, ALDH1 activity, and relative quantitative gene expression analysis of specific biomarkers were carried out to characterize the stemness, self-renewal, and mesenchymal features of HT29 cell spheroids. Then, the impact of OPE and PMFs in reducing cell proliferation and modulating cancer stemness and self-renewal was assessed. Results showed that, when compared with monolayer cultures, HT29 cell spheroids presented higher ALDH1 activity (81.97% ± 5.27% compared to 63.55% ± 17.49% for 2D), upregulation of CD44, PROM1, SOX9, and SNAI1 genes (1.83 ± 0.34, 2.54 ± 0.51, 2.03 ± 0.15, and 6.12 ± 1.59 times) and high self-renewal capability (352 ± 55 colonies compared to 253 ± 42 for 2D). Incubation with OPE (1 mg/mL) significantly inhibited cell proliferation and modulated cancer stemness and self-renewal ability: colony formation, ALDH1 activity, and the expression of cancer stemness biomarkers PROM1 and LGR5 were significantly reduced (0.66 ± 0.15 and 0.51 ± 0.14 times, respectively). Among all PMFs, tangeretin was the most efficient in targeting the CSC population by decreasing colony formation and the expression of PROM1 and LGR5. Scutellarein tetramethylether was shown to modulate markers of mesenchymal/metastatic transition (increasing CDH1 and reducing ZEB1 and SNAI1) and nobiletin was capable of downregulating PROM1 and SNAI1 expression. Importantly, all PMFs and OPE were shown to synergistically interact with 5-fluorouracil, improving the antiproliferative response of this drug.
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Antineoplásicos Fitogénicos/uso terapéutico , Citrus/química , Neoplasias Colorrectales/tratamiento farmacológico , Flavonas/uso terapéutico , Fluorouracilo/uso terapéutico , Células Madre Neoplásicas/efectos de los fármacos , Fitoterapia , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos Fitogénicos/farmacología , Proliferación Celular , Células Cultivadas , Neoplasias Colorrectales/metabolismo , Flavonas/farmacología , Frutas , Células HT29 , Humanos , Proteínas de Neoplasias/metabolismo , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéuticoRESUMEN
Rosemary diterpenes have demonstrated diverse biological activities, such as anti-cancer, antiinflammatory, as well as other beneficial effects against neurological and metabolic disorders. In particular, carnosic acid (CA), carnosol (CS) and rosmanol (RS) diterpenes have shown interesting results on anti-cancer activity. However, little is known about the toxic effects of rosemary diterpenes at the concentrations needed to exert their antiproliferative effect on cancer cells. In our study, CA, CS and RS exhibited a concentration-dependent effect on cell viability of two human colon cancer cell lines (HT-29 and HCT116) after 24â¯h exposure. HT-29â¯cell line was more resistant to the inhibitory effect of the three diterpenes than HCT116â¯cell line. Among the three diterpenes, RS exerted the strongest effect in both cell lines. To investigate the hepatotoxicity of CA, CS and RS, undifferentiated and differentiated HepaRG cells were exposed to increasing concentrations of the diterpenes (from 10 to 100⯵M). Differentiated cells were found to be more resistant to the toxic activity of the three diterpenes than undifferentiated HepaRG, probably related to a higher detoxifying function of differentiated HepaRG cells compared with the undifferentiated cells. The metabolic profiles of differentiated HepaRG cells in response to CA, CS and RS were examined to determine biochemical alterations and deepen the study of the effects of rosemary phenolic diterpenes at molecular level. A multiplatform metabolomics study based on liquid- and gas-chromatography hyphenated to high resolution mass spectrometry revealed that rosemary diterpenes exerted different effects when HepaRG cells were treated with the same concentration of each diterpene. RS revealed a greater metabolome alteration followed by CS and CA, in agreement with their observed cytotoxicity. Metabolomics provided valuable information about early events in the metabolic profiles after the treatment with the investigated diterpenes from rosemary.
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Diterpenos/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Metabolómica , Rosmarinus/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromatografía Liquida , Diterpenos/química , Cromatografía de Gases y Espectrometría de Masas , Células HCT116 , Células HT29 , HumanosRESUMEN
Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73.
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Tolerancia Inmunológica/efectos de los fármacos , Inmunomodulación/efectos de los fármacos , Inmunosupresores/farmacología , Proteínas y Péptidos Salivales/farmacología , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/etiología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Inmunoglobulina G/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transcripción GenéticaRESUMEN
The antiproliferative activity of Rosemary (Rosmarinus officinalis) has been widely studied in different in vitro and in vivo models, which demonstrate that rosemary extracts inhibit the cellular proliferation due to its ability to interact with a wide spectrum of molecular targets. However, a comprehensive proteomics study in vivo has not been carried out yet. In the present work, the effects of rosemary extract on xenograft tumor growth has been studied and, for the first time, a shotgun proteomic analysis based on nano-LC-MS/MS together with stable isotope dimethyl labeling (DML) has been applied to investigate the global protein changes in vivo. Our results show that the daily administration of a polyphenol-enriched rosemary extract reduces the progression of colorectal cancer in vivo with the subsequent deregulation of 74 proteins. The bioinformatic analysis of these proteins indicates that the rosemary extract mainly alters the RNA Post-Transcriptional Modification, the Protein Synthesis and the Amino Acid Metabolism functions and suggests the inactivation of the oncogene MYC. These results demonstrate the high utility of the proposed analytical methodology to determine, simultaneously, the expression levels of a large number of protein biomarkers and to generate new hypothesis about the molecular mechanisms of this extract in vivo.
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Carcinogénesis/efectos de los fármacos , Extractos Vegetales/farmacología , Proteómica/métodos , Rosmarinus/química , Animales , Proliferación Celular/efectos de los fármacos , Células HT29 , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Extractos Vegetales/química , Polifenoles/farmacología , Espectrometría de Masas en TándemRESUMEN
Carnosic acid (CA) and carnosol (CS) are two structurally related diterpenes present in rosemary herb (Rosmarinus officinalis). Although several studies have demonstrated that both diterpenes can scavenge free radicals and interfere in cellular processes such as cell proliferation, they may not necessarily exert the same effects at the molecular level. In this work, a shotgun proteomics study based on stable isotope dimethyl labeling (DML) and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) has been performed to identify the relative changes in proteins and to gain some light on the specific molecular targets and mechanisms of action of CA and CS in HT-29 colon cancer cells. Protein profiles revealed that CA and CS induce different Nrf2-mediated response. Furthermore, examination of our data revealed that each diterpene affects protein homeostasis by different mechanisms. CA treatment induces the expression of proteins involved in the unfolded protein response in a concentration dependent manner reflecting ER stress, whereas CS directly inhibits chymotrypsin-like activity of the 20S proteasome. In conclusion, the unbiased proteomics-wide method applied in the present study has demonstrated to be a powerful tool to reveal differences on the mechanisms of action of two related bioactive compounds in the same biological model.
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Abietanos/farmacología , Cromatografía Liquida/métodos , Neoplasias del Colon/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HT29 , Homeostasis/efectos de los fármacos , Humanos , Marcaje Isotópico , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
In the present work, four green processes have been compared to evaluate their potential to obtain rosemary extracts with in vitro anti-proliferative activity against two colon cancer cell lines (HT-29 and HCT116). The processes, carried out under optimal conditions, were: (1) pressurized liquid extraction (PLE, using an hydroalcoholic mixture as solvent) at lab-scale; (2) Single-step supercritical fluid extraction (SFE) at pilot scale; (3) Intensified two-step sequential SFE at pilot scale; (4) Integrated PLE plus supercritical antisolvent fractionation (SAF) at pilot scale. Although higher extraction yields were achieved by using PLE (38.46% dry weight), this extract provided the lowest anti-proliferative activity with no observed cytotoxic effects at the assayed concentrations. On the other hand, extracts obtained using the PLE + SAF process provided the most active rosemary extracts against both colon cancer cell lines, with LC50 ranging from 11.2 to 12.4 µg/mL and from 21.8 to 31.9 µg/mL for HCT116 and HT-29, respectively. In general, active rosemary extracts were characterized by containing carnosic acid (CA) and carnosol (CS) at concentrations above 263.7 and 33.9 mg/g extract, respectively. Some distinct compounds have been identified in the SAF extracts (rosmaridiphenol and safficinolide), suggesting their possible role as additional contributors to the observed strong anti-proliferative activity of CA and CS in SAF extracts.
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Abietanos/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Rosmarinus/química , Compuestos de Bifenilo/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía Liquida , Neoplasias del Colon/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Células HCT116 , Células HT29 , Humanos , Espectrometría de Masas , Picratos/metabolismoRESUMEN
Quality and safety assessment as well as the evaluation of other nutritional and functional properties of foods imply the use of robust, efficient, sensitive, and cost-effective analytical methodologies. Among analytical technologies used in the fields of food analysis and foodomics, capillary electrophoresis (CE) has generated great interest for the analyses of a large number of compounds due to its high separation efficiency, extremely small sample and reagent requirements, and rapid analysis. The introductory section of this chapter provides an overview of the recent applications of capillary electrophoresis (CE) in food analysis and foodomics. Relevant reviews and research articles on these topics are tabulated including papers published in the period 2011-2014. In addition, to illustrate the great capabilities of CE in foodomics the chapter describes the main experimental points to be taken into consideration for a metabolomic study of the antiproliferative effect of carnosic acid (a natural diterpene found in rosemary) against HT-29 human colon cancer cells.
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Electroforesis Capilar/métodos , Análisis de los Alimentos/métodos , Metabolómica/métodos , Células HT29 , Humanos , Espectrometría de Masas/métodos , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificaciónRESUMEN
In this work, a proteomics strategy based on nanoliquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) using an Orbitrap high-resolution mass spectrometer together with stable isotope dimethyl labeling (DML) is applied to quantitatively examine relative changes in the protein fraction of HT-29 human colon cancer cells treated with different concentrations of a polyphenol-enriched rosemary extract over the time. The major objective of this study was to gain insights into the antiproliferative mechanisms induced by rosemary polyphenols. Using this methodology, 1909 and 698 proteins were identified and quantified in cell extracts. The polyphenol-enriched rosemary extract treatment changed the expression of several proteins in a time- and concentration-dependent manner. Most of the altered proteins are implicated in the activation of Nrf2 transcription factor and the unfolded protein response. In conclusion, rosemary polyphenols induced proteomic changes that were related to the attenuation of aggresome formation and activation of autophagy to alleviate cellular stress.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/patología , Extractos Vegetales/farmacología , Proteoma/efectos de los fármacos , Proteómica/métodos , Rosmarinus/química , Autofagia/efectos de los fármacos , Cromatografía Liquida , Neoplasias del Colon/tratamiento farmacológico , Células HT29 , Humanos , Marcaje Isotópico , Factor 2 Relacionado con NF-E2/metabolismo , Polifenoles/farmacología , Espectrometría de Masas en Tándem , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
A number of studies have demonstrated a strong association between the antioxidant properties of rosemary polyphenols and their chemoprotective activity. However, the prooxidant effects of rosemary polyphenols have been rarely reported. In this work, a foodomics study is performed to investigate the in vitro autooxidation of carnosic acid (CA), carnosol (CS) and a polyphenol-enriched rosemary extract (SC-RE) in cell culture conditions. The results revealed that rosemary polyphenols autooxidation in culture medium generated H2 O2 at different rates. Generated H2 O2 levels by SC-RE and CA, but not CS, were correlated with intracellular reactive oxygen species (ROS) generation in HT-29 cells, and were partially involved in their anti-proliferative effect in this cell line. These compounds also induced different effects on glutathione metabolism. Results also indicated that high extracellular H2 O2 concentrations, resulting of using high (45 µg/mL) SC-RE concentration in culture media, exerted some artifactual effects related with cell cycle, but they did not influence the expression of relevant molecular biomarkers of stress.
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Proliferación Celular/efectos de los fármacos , Análisis de los Alimentos , Peróxido de Hidrógeno/metabolismo , Polifenoles/farmacología , Rosmarinus/química , Ciclo Celular/efectos de los fármacos , Medios de Cultivo , Células HT29 , Humanos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
This review work presents and discusses the main applications of capillary electromigration methods in food analysis and Foodomics. Papers that were published during the period February 2013-February 2015 are included following the previous review by Garcia-Cañas et al. (Electrophoresis, 2014, 35, 147-169). Analysis by CE of a large variety of food-related molecules with different chemical properties, including amino acids, hazardous amines, peptides, proteins, phenols, polyphenols, lipids, carbohydrates, DNAs, vitamins, toxins, contaminants, pesticides, residues, food additives, as well as small organic and inorganic compounds. This work includes recent results on food quality and safety, nutritional value, storage, bioactivity, as well as applications of CE for monitoring food processing. The use, among other CE developments, of microchips, CE-MS, and chiral CE in food analysis and Foodomics is also discussed.
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Electroforesis Capilar , Análisis de los Alimentos/métodos , Electroforesis Capilar/métodos , Electroforesis Capilar/tendenciasRESUMEN
In the present work, the phlorotannin composition of different Sargassum muticum samples collected at different locations along the North Atlantic coasts as well as the bioactivities related to these components were investigated. After pressurized liquid extraction, the samples collected at the extreme locations of a latitudinal gradient from Portugal and Norway, were found to be the richest on total phenols and, particularly, on phlorotannins, containing up to 148.97 and 5.12mg phloroglucinol equivalents g(-1), respectively. The extracts obtained from these locations were further purified and chemically characterized using a modified HILIC×RP-DAD-MS/MS method. The application of this methodology allowed the tentative identification of a great variability of phlorotannins with different degrees of polymerization (from 3 to 11) and structures, determined for the first time in S. muticum. The most-abundant phlorotannins on these samples were fuhalols, hydroxyfuhalols and phlorethols, showing also particularities and important differences depending on the geographical location. Afterwards, the antiproliferative activity of these extracts against HT-29 adenocarcinoma colon cancer cells was studied. Results revealed that the richest S. muticum samples in terms of total phlorotannins, i.e., those from Norway, presented the highest activity, showing a good cytotoxic potential at concentrations in the medium micromolar range.
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Cromatografía Liquida , Sargassum/química , Espectrometría de Masas en Tándem , Taninos/química , Taninos/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Océano Atlántico , Proliferación Celular/efectos de los fármacos , Células HT29 , Humanos , Fenoles/química , Fenoles/farmacología , Floroglucinol/química , Floroglucinol/farmacologíaRESUMEN
Foodomics has been defined as a global discipline in which advanced analytical techniques and bioinformatics are combined to address different questions in food science and nutrition. There is a growing number of works on the development and application of non-targeted omics methods in foodomics, which reflects that this emerging discipline is already considered by the scientific community to be a valuable approach to assess food safety, quality, and traceability as well as for the study of the links between food and health. As a result, there is a clear need for more rapid, high-throughput MS approaches for developing and applying non-targeted studies. Nowadays, direct MS analysis is one of the main choices to achieve high throughput, generating a set of information from the largest possible number of samples in a fast and straightforward way. The use of high- and ultrahigh-resolution MS greatly improves the analytical performance and offers a good combination of selectivity and sensitivity. By using a range of methods for direct sample introduction/desorption/ionization, high-throughput and non-target analysis of a variety of samples can be obtained in a few seconds by HRMS analysis. In this review, a general overview is presented of the main characteristics of direct HRMS-based approaches and their principal applications in foodomics.
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Análisis de los Alimentos , Espectrometría de Masas/métodosRESUMEN
In this work, the optimization of an effective protocol for cell metabolomics is described with special emphasis in the sample preparation and subsequent analysis of intracellular metabolites from adherent mammalian cells by capillary electrophoresis-mass spectrometry. As case study, colon cancer HT-29 cells, a human cell model to investigate colon cancer, are employed. The feasibility of the whole method for cell metabolomics is demonstrated via a fast and sensitive profiling of the intracellular metabolites HT-29 cells by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF MS). The suitability of this methodology is further corroborated through the examination of the metabolic changes in the polyamines pathway produced in colon cancer HT-29 cells by difluoromethylornithine (DFMO), a known potent ornithine decarboxylase inhibitor. The selection of the optimum extraction conditions allowed a higher sample volume injection that led to an increase in CE-TOF MS sensitivity. Following a non-targeted metabolomics approach, 10 metabolites (namely, putrescine, ornithine, gamma-aminobutyric acid (GABA), oxidized and reduced glutathione, 5'-deoxy-5'-(methylthio)adenosine, N-acetylputrescine, cysteinyl-glycine, spermidine and an unknown compound) were found to be significantly altered by DFMO (p<0.05) in HT-29 cells. In addition to the effect of DFMO on polyamine metabolism, minor modifications of other metabolic pathways (e.g., related to intracellular thiol redox state) were observed.
Asunto(s)
Adenocarcinoma/metabolismo , Adhesión Celular , Neoplasias del Colon/metabolismo , Electroforesis Capilar , Metabolómica/métodos , Poliaminas/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Eflornitina/farmacología , Estudios de Factibilidad , Células HT29 , Humanos , Inhibidores de la Ornitina Descarboxilasa/farmacologíaRESUMEN
Metabolomic-based approaches are increasingly applied to analyse genetically modified organisms (GMOs) making it possible to obtain broader and deeper information on the composition of GMOs compared to that obtained from traditional analytical approaches. The combination in metabolomics of advanced analytical methods and bioinformatics tools provides wide chemical compositional data that contributes to corroborate (or not) the substantial equivalence and occurrence of unintended changes resulting from genetic transformation. This review provides insight into recent progress in metabolomics studies on transgenic crops focusing mainly in papers published in the last decade.
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Productos Agrícolas/metabolismo , Metabolómica/métodos , Plantas Modificadas Genéticamente/metabolismo , Productos Agrícolas/química , Productos Agrícolas/genética , Alimentos Modificados Genéticamente , Metabolómica/instrumentación , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genéticaRESUMEN
In this work, the contribution of carnosic acid (CA) and carnosol (CS), two major compounds present in rosemary, against colon cancer HT-29 cells proliferation is investigated using a comprehensive Foodomics approach. The Foodomics study reveals that CA induces transcriptional activation of genes that encode detoxifying enzymes and altered the expression of genes linked to transport and biosynthesis of terpenoids in the colon cancer cell line. Functional analysis highlighted the activation of the ROS metabolism and alteration of several genes involved in pathways describing oxidative degradation of relevant endogenous metabolites, providing new evidence about the transcriptional change induced by CA in HT-29 cells. Metabolomics analysis showed that the treatment with CA affected the intracellular levels of glutathione. Elevated levels of GSH provided additional evidence to transcriptomic results regarding chemopreventive response of cells to CA treatment. Moreover, the Foodomics approach was useful to establish the links between decreased levels of N-acetylputrescine and its degradation pathway at the gene level. The findings from this work and the predictions based on microarray data will help explore novel metabolic processes and potential signaling pathways to further elucidate the effect of CA in colon cancer cells.