Targeted Analysis of the Concentration Changes of Phenolic Compounds in Persian Lime (Citrus latifolia) during Fruit Growth.

Citrus fruits possess a high content of phenolic compounds; however, few studies have focused on the changes occurring during fruit growth. In this study, the changes in the concentration of 20 flavonoids, 4 phenolic acids, and their biosynthetic precursors phenylalanine and tyrosine have been evaluated during fruit maturation (14 weeks). Extracts from all samples, obtained by ultrasound assistance, were analyzed by liquid chromatography coupled to tandem mass spectrometry with a triple quad system (LC-QqQ MS/MS). In general, the concentration of flavanones, which represented over 70% of the studied phenols, and flavones increased during fruit growth, reaching their maximum concentration around week 12. In general, flavanols and phenolic acids exhibited their maximum concentration at week 5 and then decreasing significantly during the rest of maturation. Phenylalanine and tyrosine showed a sinuous behavior during fruit growth. Partial least-squares showed a clear differentiation among fruits belonging to different maturation stages, coumaric acid derivatives being the most influential variables on the projection.

Untargeted analysis to monitor metabolic changes of garlic along heat treatment by LC-QTOF MS/MS.

Black garlic is increasing its popularity in cuisine around the world; however, scant information exists on the composition of this processed product. In this study, polar compounds in fresh garlic and in samples taken at different times during the heat treatment process to obtain black garlic have been characterized by liquid chromatography coupled to tandem mass spectrometry in high resolution mode. Ninety-five compounds (mainly amino acids and metabolites, organosulfur compounds, and saccharides and derivatives) were tentatively identified in all the analysed samples and classified as a function of the family they belong to. Statistical analysis of the results allowed establishing that the major changes in garlic occur during the first days of treatment, and they mainly affect to the three representative families. The main pathways involved in the synthesis of the compounds affected by heat treatment, and their evolution during the process were studied.

Metabolomics: A potential way to know the role of vitamin D on multiple sclerosis.

The literature about the influence of vitamin D on multiple sclerosis (MS) is very controversial, possibly as a result of the way through which the research on the subject has been conducted. The studies developed so far have been focused exclusively on gene expression: the effect of a given vitamin D metabolite on target receptors. The influence of the vitamin D status (either natural or after supplementation) on MS has been studied by measurement of the 25 monohydroxylated metabolite (also known as circulating form), despite the 1,25 dihydroxylated metabolite is considered the active form. In the light of the multiple metabolic pathways in which both forms of vitamin D (D2 and D3) are involved, monitoring of the metabolites is crucial to know the activity of the target enzymes as a function of both the state of the MS patient and the clinical treatment applied. The study of metabolomics aspects is here proposed to clarify the present controversy. In "omics" terms, our proposal is to take profit from up-stream information-thus is, from metabolomics to genomics-with a potential subsequent step to systems biology, if required.

Comparative Study of the Effect of Sample Pretreatment and Extraction on the Determination of Flavonoids from Lemon (Citrus limon).

BACKGROUND: Flavonoids have shown to exert multiple beneficial effects on human health, being also appreciated by both food and pharmaceutical industries. Citrus fruits are a key source of flavonoids, thus promoting studies to obtain them. Characteristics of these studies are the discrepancies among sample pretreatments and among extraction methods, and also the scant number of comparative studies developed so far. OBJECTIVE: Evaluate the effect of both the sample pretreatment and the extraction method on the profile of flavonoids isolated from lemon. RESULTS: Extracts from fresh, lyophilized and air-dried samples obtained by shaking extraction (SE), ultrasound-assisted extraction (USAE), microwave-assisted extraction (MAE) and superheated liquid extraction (SHLE) were analyzed by LC-QTOF MS/MS, and 32 flavonoids were tentatively identified using MS/MS information. ANOVA applied to the data from fresh and dehydrated samples and from extraction by the different methods revealed that 26 and 32 flavonoids, respectively, were significant (p≤0.01). The pairwise comparison (Tukey HSD; p≤0.01) showed that lyophilized samples are more different from fresh samples than from air-dried samples; also, principal component analysis (PCA) showed a clear discrimination among sample pretreatment strategies and suggested that such differences are mainly created by the abundance of major flavonoids. On the other hand, pairwise comparison of extraction methods revealed that USAE and MAE provided quite similar extracts, being SHLE extracts different from the other two. In this case, PCA showed a clear discrimination among extraction methods, and their position in the scores plot suggests a lower abundance of flavonoids in the extracts from SHLE. In the two PCA the loadings plots revealed a trend to forming groups according to flavonoid aglycones. CONCLUSIONS: The present study shows clear discrimination caused by both sample pretreatments and extraction methods. Under the studied conditions, liophilization provides extracts with higher amounts of flavonoids, and USAE is the best method for isolation of these compounds, followed by MAE and SE. On the contrary, the SHLE method was the less favorable to extract flavonoids from citrus owing to degradation.

Ultrasound-assisted emulsification-extraction of orange peel metabolites prior to tentative identification by LC-QTOF MS/MS.

An optimized method for extraction and characterization of compounds present in orange peel has been developed. The extraction method allows the simultaneous extraction of polar and non-polar compounds by using two immiscible extractants (a polar extractant-an 80:20 (v/v) methanol-water mixture, and a non-polar extractant-n-hexane). The method is ultrasound-assisted, thus facilitating both formation of a stable emulsion between the two immiscible extractants and favoring mass-transfer from the solid sample to the liquid phases by a wide contact surface. Optimization of the ultrasound-assisted emulsification-extraction (USAEE) led to the following values as desirability conditions for both extracts: 32% amplitude, 0.75s/s duty cycle and 7.5min of extraction time. The extracts obtained under these conditions were analyzed by LC-QTOF MS/MS in positive and negative ionization modes. Tentative identification of the most significant compounds present in each extract allowed their characterization by using high resolution tandem mass spectrometry. The optimum extracts provided by USAEE were compared by using Principal Component Analysis to those obtained by conventional extraction based on maceration. Thus, the composition of the polar extracts obtained after 7.5min ultrasonication was similar to that of conventional maceration for 4h in both the ionization modes. On the contrary, the analysis of non-polar extracts led to different results depending on the ionization mode: the ultrasound-assisted extract was similar to those of conventional maceration for 10h in negative and positive ionization. This behavior could be explained by the contribution of different groups of compounds to each ionization mode.

Global metabolomic profiling of human serum from obese individuals by liquid chromatography-time-of-flight/mass spectrometry to evaluate the intake of breakfasts prepared with heated edible oils.

The metabolic profile of human serum after intake of breakfasts prepared with different heated vegetable oils has been studied. Four oils (olive and sunflower oils, pure and enriched with natural and artificial oxidation inhibitors) were subjected to a simulated heated process prior to breakfast preparation. A metabolomics global profiling approach performed on post-basal serum samples revealed statistical differences among individuals based on breakfast intake, and identified compounds responsible for such differences. Serum samples obtained in basal state (control samples) and 2 and 4h after programmed intakes were analyzed by LC-TOF/MS. The resulting fingerprints were compared and differences between basal and post-basal states evaluated, observing that the intake of different breakfasts altered the metabolic signature of serum. Analysis models based on PLS algorithms were developed to discriminate individuals in post-basal state for each intervention breakfast. Then, Volcano tests enabled to detect significant molecular entities explaining the variability associated to each breakfast. It is worth emphasizing the importance of fatty acids, their derivatives and phospholipids for tentative identification.

The antioxidants in oils heated at frying temperature, whether natural or added, could protect against postprandial oxidative stress in obese people.

We have investigated the effects of the intake of oils heated at frying temperature in order to find an oil model for deep-frying that prevents postprandial oxidative stress. Twenty obese people received four breakfasts following a randomised crossover design consisting of different oils (virgin olive oil (VOO), sunflower oil (SFO), and a mixed seed oil (SFO/canola oil) with added dimethylpolysiloxane (SOX) or natural antioxidants from olives (SOP)), which were subjected to 20 heating cycles. The intake of SFO-breakfast reduced plasma GSH levels and the GSH/GSSG ratio, increased protein carbonyl levels, and induced a higher gene expression of the different NADPH-oxidase subunits, Nrf2-Keap1 activation, gene expression of the antioxidant enzymes in peripheral blood mononuclear cells and antioxidant plasma activities than the intake of the breakfasts prepared with VOO, SOP and SOX. Oils with phenolic compounds, whether natural (VOO) or artificially added (SOP), or with artificial antioxidant (SOX), could reduce postprandial oxidative stress compared with sunflower oil.

Short-term comparative study of the influence of fried edible oils intake on the metabolism of essential fatty acids in obese individuals.

The effect of breakfast intake of fried oils containing natural antioxidants or a synthetic autooxidation inhibitor on the metabolism of essential fatty acids focused on obese individuals. Serum levels of eicosanoids were compared in individuals before and after intake of different breakfasts. Univariate descriptive analysis was used to characterise the cohort selected for this study and multivariate analysis to reveal statistical differences of normalised eicosanoids concentrations (determined by solid-phase extraction coupled to LC-MS/MS) depending on the edible oil used for breakfast preparation. The results showed that the intake of breakfast prepared with pure sunflower oil subjected to deep frying causes an effect over the eicosanoids profile that enables discrimination versus the rest of individuals. The effect was a significant increase in the concentration of hydroxyoctadecadienoic acid (HODE) metabolites, indicative markers of the intake of fried oils. The concentration of HODE metabolites was lower when the oil contained either natural antioxidants from olive-oil pomace or a synthetic autooxidation inhibitor as dimethylsiloxane. The comparison of the effect of fried sunflower oils with fried extra virgin olive oil shows the benefits associated to the consumption of the latter.

Influence of deep frying on the unsaponifiable fraction of vegetable edible oils enriched with natural antioxidants.

The influence of deep frying, mimicked by 20 heating cycles at 180 °C (each cycle from ambient temperature to 180 °C maintained for 5 min), on the unsaponifiable fraction of vegetable edible oils represented by three characteristic families of compounds (namely, phytosterols, aliphatic alcohols, and triterpenic compounds) has been studied. The target oils were extra virgin olive oil (with intrinsic content of phenolic antioxidants), refined sunflower oil enriched with antioxidant phenolic compounds isolated from olive pomace, refined sunflower oil enriched with an autoxidation inhibitor (dimethylpolysiloxane), and refined sunflower oil without enrichment. Monitoring of the target analytes as a function of both heating cycle and the presence of natural antioxidants was also evaluated by comparison of the profiles after each heating cycle. Identification and quantitation of the target compounds were performed by gas cromatography-mass spectrometry in single ion monitoring mode. Analysis of the heated oils revealed that the addition of natural antioxidants could be an excellent strategy to decrease degradation of lipidic components of the unsaponifiable fraction with the consequent improvement of stability.

Characterization of fatty alcohol and sterol fractions in olive tree.

The determination of sterols and fatty alcohols is a part of the study of the metabolomic profile of the unsaponifiable fraction in olive tree. Leaves and drupes from three varieties of olive tree (Arbequina, Picual, and Manzanilla) were used. The content of the target compounds was studied in five ripeness stages and three harvesting periods for olive drupes and leaves, respectively. A method based on ultrasound-assisted extraction and derivatization for the individual identification and quantitation of sterols and fatty alcohols, involving chromatographic separation and mass spectrometry detection by selected ion monitoring, was used. The concentrations of alcohols and sterols in the drupes ranged between 0.1 and 1086.9 mug/g and between 0.1 and 5855.3 mug/g, respectively, which are higher than in leaves. Statistical studies were developed to show the relationship between the concentration of the target analytes and variety, ripeness stage, and harvesting period.