RESUMO
Although genomes from many edible mushrooms are sequenced, studies on fungal micro RNAs (miRNAs) are scarce. Most of the bioinformatic tools are designed for plants or animals, but the processing and expression of fungal miRNAs share similarities and differences with both kingdoms. Moreover, since mushroom species such as Agaricus bisporus (A. bisporus, white button mushroom) are frequently consumed as food, controversial discussions are still evaluating whether their miRNAs might or might not be assimilated, perhaps within extracellular vesicles (i.e., exosomes). Therefore, the A. bisporus RNA-seq was studied in order to identify potential de novo miRNA-like small RNAs (milRNAs) that might allow their later detection in diet. Results pointed to 1 already known and 37 de novo milRNAs. Three milRNAs were selected for RT-qPCR experiments. Precursors and mature milRNAs were found in the edible parts (caps and stipes), validating the predictions carried out in silico. When their potential gene targets were investigated, results pointed that most were involved in primary and secondary metabolic regulation. However, when the human transcriptome is used as the target, the results suggest that they might interfere with important biological processes related with cancer, infection and neurodegenerative diseases.
Assuntos
Agaricus , MicroRNAs , Agaricus/genética , Biologia Computacional/métodos , MicroRNAs/genética , RNA Fúngico , RNA-SeqRESUMO
In many occasions it is necessary to use fast and simple methods, different to the chromatographic techniques, for the quantification of biomolecules such as flavonoids. Also, the flavonoid levels in some foodstuffs can be influenced by industrial extraction processes such as pressing and squeezing, resulting in modification of their functional value. For this purpose, we have developed a rapid method to analyze flavonoids, based on a coupling reaction between ABTS and flavonoid mediated by peroxidase. The present method can be used to detect and measure flavonoids with hydroxyl moieties on A- or B-rings, not adjacent to methoxy or oxo substitutions. The visible spectrum of the ABTS-flavonoid complex, the calibration curve (within the range 5-50 µM) and the molar absorption coefficients for isosakuranetin, isonaringin, rhoifolin, hyperoside, rutin, hesperetin, quercetin, kaempherol and naringenin are given. The method has been applied to complex culture media and is sensitive, accurate, quick and easy to apply. This method can be used in laboratories that do not have sophisticated and expensive techniques such as liquid chromatography and also as a quick, simple and inexpensive technique for student practice laboratories.
Assuntos
Benzotiazóis/química , Colorimetria/métodos , Flavonoides/análise , Peroxidase/química , Ácidos Sulfônicos/química , Cromatografia Líquida de Alta PressãoRESUMO
PURPOSE: To investigate the effect of two extracts obtained from Agaricus bisporus on the mRNA expression of cholesterol-related genes. One of the extracts contained ergosterol and other fungal sterols (SFE) and the other contained ß-glucans and fungal sterols (EßG). METHODS: Firstly, the dietary mixed micelles (DMMs) generated after in vitro digestion of standards and SFE were applied to Caco2 cells. Then, the lower compartment after a Caco2-transport experiment was applied to HepG2 cells. The mRNA expression was assessed in both cell lines by low-density arrays (LDA). Mice received the extracts, ergosterol or control drugs after 4 weeks of a high-cholesterol diet. The lipid profile of plasma, liver and feces was determined. LDA assays were performed in liver and intestines. RESULTS: The DMM fraction of SFE up-regulated the LDLR mRNA expression in Caco2 cells. The lower compartment after Caco2-transport experiments up-regulated LDLR and modulated several other lipid-related genes in HepG2 cells. In mice, SFE decreased TC/HDL ratio and reduced hepatic triglycerides paralleled with down-regulation of Dgat1 expression, while EßG did it without transcriptional changes. Addition of SFE or ergosterol induced in jejunum a similar transcriptional response to simvastatin and ezetimibe; they all down-regulated Srebf2 and Nr1h4 (FXR) genes. CONCLUSION: Ergosterol-containing extracts from A. bisporus lowered hepatic triglyceride and modify the mRNA expression of cholesterol-related genes although the transcriptional regulation was unrelated to changes in plasma lipid profile. These extracts may be useful limiting hepatic steatosis and as bioactive ingredients to design novel functional foods preventing lifestyle-related diseases such as non-alcoholic fatty liver disease.
Assuntos
Agaricus/química , Ergosterol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Células CACO-2 , Colesterol na Dieta/administração & dosagem , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Dieta Hiperlipídica , Regulação para Baixo , Ezetimiba/farmacologia , Fezes/química , Células Hep G2 , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Sinvastatina/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Esteróis/farmacologia , Triglicerídeos/sangue , Regulação para Cima , beta-Glucanas/farmacologiaRESUMO
BACKGROUND: Agaricus bisporus mushrooms were able to lower cholesterol levels in hypercholesterolaemic rats and it was suggested that dietary fibre might inhibit cholesterol absorption. However, A. bisporus extracts were also able to inhibit the 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR, the key enzyme in the cholesterol biosynthetic pathway) and this might also contribute to the observed lowering of cholesterol levels in serum. RESULTS: The methanol-water extracts obtained from A. bisporus were able to inhibit up to 60% the HMGCR activity using an in vitro assay. The HMGCR inhibitory capacities depended on cultivation conditions, strains, etc. The potential inhibitors were not statins, they might be ß-glucans able to scavenge the substrate and impair the enzymatic reaction. They were present during all mushroom developmental stages and similarly distributed through all the tissues including the parts discarded as a by-product. Accelerated solvent extractions using 1:1 ethanol-water as pressurised solvent (10.7 MPa, 25°C, five cycles of 5 min) were more effective in the extraction of the HMGCiR inhibitor(s) than supercritical fluid extractions (9 MPa, 40°C) using CO2 with 10% ethanol. CONCLUSION: A mushroom cultivation and two extraction procedures were optimised to obtain fractions from A. bisporus with high HMGCR inhibitory activities to design novel ingredients for hypocholesterolaemic functional foodstuffs.
Assuntos
Agaricus/química , Fracionamento Químico/métodos , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Selênio/químicaRESUMO
In the present work, an HPLC method is proposed to simultaneously detect and quantify water- and fat-soluble vitamins, phenolic compounds, carotenoids and chlorophylls in a single run, by using an ultradeactivated C18 column and gradient separation using trifluoroacetic acid, water and methanol. It is shown that the HPLC method provides baseline separation of all these compounds with good resolution values in 40 min. Moreover, other figures of merit of the method show a good linear response and low detection limits for all the compounds considered in the present study. Furthermore, the usefulness of this method is demonstrated via its successful application to the analysis of different beverages from different natural origin (orange, strawberry, apple, peach pineapple, plum and blackcurrant juices, soybean milk, beers) without the need of any previous sample preparation. A good correlation is also found by comparing the total phenol content (measured by Folin-Ciocalteu method) with the sum of total phenolic compounds obtained using the proposed HPLC method. By using statistical tools, the main compounds associated with antioxidant activity of the extracts (measured by 1,1-diphenyl-2-picrylhydrazyl radical scavenging) were assessed.
Assuntos
Bebidas/análise , Compostos Orgânicos/análise , Antioxidantes/análise , Compostos de Bifenilo/química , Calibragem , Cromatografia Líquida de Alta Pressão , Flavonoides/análise , Análise de Alimentos , Sequestradores de Radicais Livres/química , Hidrazinas/química , Análise dos Mínimos Quadrados , Fenóis/análise , Picratos , Pigmentos Biológicos/análise , Polifenóis , Padrões de Referência , Vitaminas/análiseRESUMO
The physiological role of the tripeptide glutathione (GSH) and its oxidized form (GSSG) was investigated during the initial steps of dimorphism (formation of germ-tubes), which is induced by human serum in exponential yeast-like cells (blastoconidia) of the Candida albicans strain CAI-4 (wild type) and its congenic tps1/tps1 mutant, deficient in trehalose synthesis. The content of glutathione, measured both as GSH and the ratio GSH/GSSG, underwent a moderate drop in parallel with the induction of a significant degree of germ-tube emergence. Whereas the supply of exogenous glutathione did not affect the degree of dimorphic transition, depletion of intracellular glutathione by addition of 1-chloro-2,4 dinitrobenzene (CDNB) caused a clear reduction in the percentage of hyphae formation; although this effect must be due to the severe cell mortality produced by CDNB. Simultaneous measurements of GSH-metabolizing activities revealed a moderate decrease of glutathione reductase concomitant with the activation of glutathione peroxidase. In turn, catalase activity did not show noticeable changes. The putative correlation between the redox status of glutathione and the dimorphic conversion in C. albicans is discussed.
Assuntos
Sangue , Candida albicans/metabolismo , Glutationa/metabolismo , HumanosRESUMO
Interest in food matrices supplemented with mushrooms as hypocholesterolemic functional foods is increasing. This study was to (i) investigate the hypocholesterolemic activity of lard functionalized with mushroom extracts (LF) including fungal ß-glucans, water-soluble polysaccharides, or ergosterol and (ii) examine the LF influence on transcriptional mechanisms involved in cholesterol metabolism. mRNA levels of 17 cholesterol-related genes were evaluated in jejunum, cecum, and liver of high cholesterol-fed mice. The four tested LFs decreased plasma cholesterol by 22-42%, HDLc by 18-40%, and LDLc by 27-51%, and two of them increased mRNA levels of jejunal Npc1l1 and Abcg5 and hepatic Npc1l1. mRNA levels of other cholesterol-related genes were unchanged. These findings suggest that LF may have potential as a dietary supplement for counteracting diet-induced hypercholesterolemia and could be a source for the development of novel cholesterol-lowering functional foods. However, the cholesterol-lowering effect was unrelated to transcriptional changes, suggesting that post-transcriptional mechanisms could be involved.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Agaricales/química , Anticolesterolemiantes/administração & dosagem , Gorduras na Dieta/administração & dosagem , Hipercolesterolemia/tratamento farmacológico , Lipoproteínas/genética , Proteínas de Membrana Transportadoras/genética , beta-Glucanas/administração & dosagem , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Colesterol/sangue , Expressão Gênica/efeitos dos fármacos , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Lipoproteínas/metabolismo , Masculino , Proteínas de Membrana Transportadoras/metabolismo , CamundongosRESUMO
A water extract from Lentinula edodes (LWE) showed HMG-CoA reductase inhibitory activity but contained no statins. NMR indicated the presence of water-soluble α- and ß-glucans and fucomannogalactans. Fractions containing derivatives of these polysaccharides with molecular weight down to approximately 1 kDa still retained their inhibitory activity. Once digested LWE was applied to Caco2 in transport experiments, no significant effect was noticed on the modulation of cholesterol-related gene expression. But, when the lower compartment of the Caco2 monolayer was applied to HepG2, some genes were modulated (after 24 h). LWE was also administrated to normo- and hypercholesterolemic mice, and no significant lowering of serum cholesterol levels was observed; but reduction of triglycerides in liver was observed. However, LWE supplementation modulated the transcriptional profile of some genes involved in the cholesterol metabolism similarly to simvastatin, suggesting that it could hold potential as a hypolipidemic/hypocholesterolemic extract, although further dose-dependent studies should be carried out.
Assuntos
Colesterol/genética , Colesterol/metabolismo , Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Cogumelos Shiitake/química , Animais , Células CACO-2 , Colesterol/sangue , Fucose/análise , Galactanos/análise , Glucanos/análise , Glucanos/química , Células Hep G2 , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/análise , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Fígado/química , Masculino , Manose/análise , Camundongos , Camundongos Endogâmicos C57BL , Solubilidade , Triglicerídeos/análise , ÁguaRESUMO
Mushrooms are a source of dietary fiber (DF) with a cholesterol-lowering effect. However, their underlying mechanisms are poorly understood. The effect of DF-enriched fractions from three mushrooms species on cholesterol-related expression was studied in vitro. The Pleurotus ostreatus DF fraction (PDF) was used in mice models to assess its potential palliative or preventive effect against hypercholesterolemia. PDF induced a transcriptional response in Caco-2 cells, suggesting a possible cholesterol-lowering effect. In the palliative setting, PDF reduced hepatic triglyceride likely because Dgat1 was downregulated. However, cholesterol-related biochemical data showed no changes and no relation with the observed transcriptional modulation. In the preventive setting, PDF modulated cholesterol-related genes expression in a manner similar to that of simvastatin and ezetimibe in the liver, although no changes in plasma and liver biochemical data were induced. Therefore, PDF may be useful reducing hepatic triglyceride accumulation. Because it induced a molecular response similar to hypocholesterolemic drugs in liver, further dose-dependent studies should be carried out.
Assuntos
Colesterol/genética , Fibras na Dieta/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipercolesterolemia/dietoterapia , Pleurotus/química , Agaricus/química , Animais , Peso Corporal/efeitos dos fármacos , Células CACO-2/efeitos dos fármacos , Colesterol/sangue , Colesterol/metabolismo , Suplementos Nutricionais , Fezes , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Cogumelos Shiitake/químicaRESUMO
A pressurized water extraction (PWE) method was developed in order to extract ß-glucans with bile acids-binding capacities from cultivated mushrooms (Agaricus bisporus, Lentinula edodes, and Pleurotus ostreatus) to be used as supplements to design novel foods with hypocholesterolemic properties. Extraction yields were higher in individual than sequential extractions being the optimal extraction parameters: 200°C, 5 cycles of 5 min each at 10.3 MPa. The crude polysaccharide (PSC) fractions, isolated from the PWE extracts contained mainly ß-glucans (including chitooligosaccharides deriving from chitin hydrolysis), α-glucans, and other PSCs (hetero-/proteo-glucans) depending on the extraction temperature and mushroom strain considered. The observed bile acids-binding capacities of some extracts were similar to a ß-glucan enriched fraction obtained from cereals.
Assuntos
Agaricales/química , Ácidos e Sais Biliares/metabolismo , Fracionamento Químico/métodos , Polissacarídeos Fúngicos/isolamento & purificação , Glucanos/isolamento & purificação , Análise de Variância , Polissacarídeos Fúngicos/análise , Polissacarídeos Fúngicos/química , Polissacarídeos Fúngicos/metabolismo , Glucanos/análise , Glucanos/química , Glucanos/metabolismo , Pressão , Solventes , ÁguaRESUMO
An antioxidant-enriched extract (RE) was obtained from rosemary (Rosmarinus officinalis) by supercritical fluid extraction to be used as an ingredient to design functional foods. The optimized mixture (42 mg RE g(-1) sunflower oil) was submitted to in vitro digestion and absorption tests (using Caco2 cells) to investigate the effect of these processes on its DPPH scavenging activity and also whether its major abietanes (tricyclic diterpenes) might be bioaccessible and bioavailable. Results indicated that supplementation of the rosemary extract with sunflower oil and lecithin (37 mg g(-1)) enhanced abietanes micellation (almost 2-fold). In vitro digestion of the mixture including RE, sunflower oil, and lecithin reduced 50% the bioaccesibility in terms of antioxidant activity. Bioavailability was 31%. It was evidenced that this activity was not due to the original levels of carnosol, carnosic acid, and methyl carnosate (which only 47% remained after digestion) but due to their derivatives and digestion products.
Assuntos
Abietanos/metabolismo , Antioxidantes/metabolismo , Digestão , Extratos Vegetais/metabolismo , Rosmarinus/química , Abietanos/análise , Antioxidantes/análise , Disponibilidade Biológica , Células CACO-2 , Humanos , Modelos Biológicos , Extratos Vegetais/análiseRESUMO
Nutritionists encourage improving the diet by combining meat products with fish or other sea-related foods, in order to equilibrate the omega-6/omega-3 ratio. Strong scientific evidence supports the beneficial health effects of a balanced omega-6/omega-3 PUFA (poly unsaturated fatty acids) diets. In the present work, the scientific bases of new functional meat products with both a balanced omega-6/omega-3 ratio and a synergic combination of antioxidants are discussed. The aim is to contribute to the dietary equilibrium omega-6/omega-3 and to increase the antioxidant intake. Conventional meat products supplemented with a specific fatty acids and antioxidants combination led to functional foods with healthier nutritional parameters.
Assuntos
Ácidos Graxos Ômega-3/química , Ácidos Graxos Ômega-6/química , Produtos da Carne , Alimentos Marinhos , Animais , Antioxidantes/química , Dieta , Tecnologia de Alimentos , Alimentos Orgânicos , Extratos Vegetais/química , Salmão , Substâncias Reativas com Ácido Tiobarbitúrico/químicaRESUMO
Spirulina platensis microalga has been extracted on a pilot scale plant using supercritical fluid extraction (SFE) under various extraction conditions. The extraction yield and the antioxidant activity of the extracts were evaluated in order to select those extracts with both the highest antioxidant capacity and a good extraction yield. These extracts were characterized using LC coupled to diode array detection (DAD) and LC coupled to mass spectrometry (MS) with two different interfaces, atmospheric pressure chemical ionization (APCI) and electrospray (ESI) which allowed us to perform tandem MS by using an ion trap analyzer. The best extraction conditions were as follows: CO2 with 10% of modifier (ethanol) as extraction solvent, 55 degrees C (extraction temperature) and 220 bar (extraction pressure). Fractionation was achieved by cascade depressurization providing two extracts with different activity and chemical composition. Several compounds have been identified in the extracts, corresponding to different carotenoids previously identified in Spirulina platensis microalga along with chlorophyll a and some degradation products. Also, the structure of some phenolic compounds could be tentatively identified. The antioxidant activity of the extracts could be attributed to some of the above mentioned compounds.
Assuntos
Antioxidantes/análise , Cromatografia Líquida/métodos , Cromatografia com Fluido Supercrítico/métodos , Cianobactérias/química , Espectrometria de Massas/métodos , Espectrofotometria Ultravioleta/métodosRESUMO
Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone-7-O-neohesperidosides responsible, in part, for their characteristic taste. Non-bitter species, such as mandarin and orange, accumulate only tasteless flavanone-7-O-rutinosides. The key flavor-determining step of citrus flavanone-glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7-O-neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non-bitter species, was not cross-reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone-neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaT on a separate gene cluster together with the only other functionally characterized flavonoid-glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides.