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The present study aimed to evaluate the metabolism and bioavailability of anthocyanins (ACN) and other phenolics from red-fleshed apple (RFA) and to define the intake biomarkers compared to common white-fleshed apple (WFA). Acute and sustained (6-week) interventions were combined in a randomized, controlled and parallel study with 121 hypercholesterolemic subjects. Another arm consuming ACN-rich infusion from aronia fruit (ARO) provided matched content and profile of ACN. Plasma, urine and faeces samples were analyzed using UPLC-MS/MS. Results showed higher bioavailability of ACN after ARO compared to RFA, showing a clear apple matrix effect. The dihydrochalcone phloretin-2¿ -O-glucuronide was the most discriminant intake biomarker of both apples. The urinary peonidin-3-O-galactoside was a good biomarker after both ARO and RFA intakes, whereas peonidin-O-arabinoside was reported to be specific from ARO. The elucidation of the phenolic metabolism and the selection of intake biomarkers is a promising approach to relate phenolic compounds and human health., This study was supported by the Spanish Ministry of Industry, Economy and Competitiveness through the AGL2016-76943-C2-1-R and AGL2016-76943-C2-2-R projects (co-funded by the European Social Fund, European Union); M.J. Motilva thanks to Consejo Superior de Investigaciones Científicas-CSIC for partial funding throught the “Ayudas incorporacion ´ a escalas científicas CSIC, 2018” (Reference 201870I129). L.R. was Sara Borrell postdoctoral contract and she is a Serra Húnter Fellow. I.A.L. enjoys a post-doctoral contract (2017PMFPOST2-19) from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement and from the Universitat Rovira i Virgili (URV). S.Y. was supported by a pre-doctoral grant from the University of Lleida. The authors are grateful to CALIDAD PASCUAL (Aranda de Duero, Burgos, Spain) for generously providing the Bezoya mineral water to prepare daily Aronia infusion.

High temperatures at the end of the season are frequent under Mediterranean conditions, affecting final grain quality. This study determined the deposition patterns throughout grain filling of dry matter, dietary fiber, phenolic compounds and antioxidant capacity for four barley genotypes under two contrasting temperatures. Deposition pattern for dietary fiber followed that of grain weight. Genotypic differences for duration were more significant than for rate. Anthocyanins followed a second-degree polynomial pattern, reaching a maximum before grain maturation. Free and bound phenols decreased as grain developed, suggesting that they are synthesized in early stages. Rate of bound phenols deposition was more sensitive to genotypic changes. Overall, antioxidant capacity decreased over time, the decay being less steep under stress for all genotypes. Heat stress negatively affected grain weight. It did not alter the profile of β-glucans and arabinoxylans deposition but positively changed the accumulation of some phenolic compounds, increasing the antioxidant capacity differentially across genotypes. These results support the growing of food barley in high-temperature stress-prone areas, as some bioactive compound and antioxidant capacity will increase, regardless of the smaller grain size. Moreover, if a market develops for food-barley ingredients, early harvesting of non-mature grain to maximize antioxidant capacity should be considered.

BACKGROUND: Barley (Hordeum vulgare L.) is a healthy grain because of its high content of dietary fibre and phenolic compounds. It faces periods of high temperature during grain filling, frequently reducing grain weight. Heat stress may also affect some of the bioactive compounds present in the grain. To produce quality grains that provide nutritional and health benefits, it is important to understand the effect of environmental stresses on the quantity and quality of bioactive compounds. RESULTS: We have studied the effect of post-anthesis thermal stress on barley bioactive compounds and antioxidant capacity under Mediterranean field conditions during two consecutive growing seasons in four barley genotypes. Thermal stress affected grain weight and size and changed the relative composition of bioactive compounds. The relationship between heat stress and grain ⊎-glucans and arabinoxylans content was indirect, as the resulting increases in concentrations were due to the lower grain weight under stress. Conversely, heat stress had a significant direct impact on somephenolic compounds, increasing their concentrations differentially across genotypes, which contributed to an improvement in antioxidant capacity of up to 30%. CONCLUSION: Post-anthesis thermal stress had a significant effect on ⊎-glucans, arabinoxylans, phenolic compound concentration and antioxidant capacity of barley grains. Final grain quality could, at least partially, be controlled in order to increase the bioactive concentrations in the barley grain, by cultivation in growing areas prone to heat stress. Late sowings or late flowering genotypes could also be considered, should a premium be implemented to compensate for lower yields. This work was funded by project AGL 2015–69435-C3-1 from the Spanish Ministry of Economy and Competitiveness. Mariona Martínez-Subirà was supported by a pre-doctoral fellowship (BES-2016-078654/AGL 2015-69435-C3-1).

Hypertension is an independent and preventable risk factor for the development of cardiovascular diseases, however, little is known about the impact of gut microbiota composition in its development. We carried out comprehensive gut microbiota analysis and targeted metabolomics in a cross-sectional study of 29 non-treated hypertensive (HT) and 32 normotensive (NT) subjects. We determined fecal microbiota composition by 16S rRNA gene sequencing and bacterial functions by metagenomic analysis. The microbial metabolites analysed were short chain fatty acids (SCFA) both in plasma and feces, and trimethylamine N-oxide (TMAO) in plasma. The overall bacterial composition and diversity of bacterial community in the two groups were not significantly different. However, Ruminococcaceae NK4A214, Ruminococcaceae_UCG-010, Christensenellaceae_R-7, Faecalibacterium prausnitzii and Roseburia hominis were found to be significantly enriched in NT group, whereas, Bacteroides coprocola, Bacteroides plebeius and genera of Lachnospiraceae were increased in HT patients. We found a positive correlation between the HT-associated species and systolic and diastolic blood pressure after adjusted for measured confounders. SCFA showed antagonistic results in plasma and feces, detecting in HT subjects significant higher levels in feces and lower levels in plasma, which could indicate a less efficient SCFA absorption. Overall, our results present a disease classifier based on microbiota and bacterial metabolites to discriminate HT individuals from NT controls in a first disease grade prior to drug treatment. We wish to acknowledge the support of the Institut d’Investigació Sanitària Pere Virgili (IISPV) and the EURECAT-Centre Tecnològic de Catalunya, Unitat de Nutrició i Salut (Reus, Spain), the University of Lleida through the SY grant, the LR Sara Borrell postdoctoral grant (CD14/00275), and AP Torres Quevedo contract (Subprograma Estatal de Incorporación, Plan Estatal de Investigación Científica y Técnica y de Innovación). LC was granted by Ayudas Para La Promoción De Empleo Joven E Implantación De La Garantía Juvenil En I + D + I (PEJ-2014-A-67028). NFOC-Salut group is a consolidated research group of Generalitat de Catalunya, Spain (2017 SGR522).

The analysis of the fat-soluble vitamins A and E and lipid micronutrients in blood, such as carotenoids, is an important parameter to monitor the micronutrient status in humans. Although the potential of dried blood spot (DBS) cards, the use of this technique for blood sampling and subsequent analysis of these fat-soluble micronutrients has been poorly or not studied. An analytical method based on DBS cards (FTA® DMPK-A) combined with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been developed and validated for the determination of carotenoids (lutein, zeaxanthin, β-cryptoxanthin and β-carotene), tocopherols (α-tocopherol, γ-tocopherol and δ-tocopherol) and all-trans-retinol in human blood. Under optimum DBS card extraction conditions, the extraction recoveries of the studied compounds were higher than 72%, the sample matrix effect lower than 17%, and the detection limits at hundred nM concentration levels. The developed method was applied to the analysis of human blood, and the concentration ranges obtained fell within the expected ranges previously reported in healthy adults. Moreover, the influence of hematocrit effect was investigated in a range of 25–55% in order to compare the obtained results to those reported in the literature for the analysis of plasma samples. This method represents an improvement over current techniques reported in the literature due to the use of a non-invasive blood collection method, and moreover, this methodology was for the first time 1) validated for the analysis of all-trans-retinol, tocopherols and carotenoids, and 2) applied for the determination of tocopherols in human blood samples., This study was supported by the Spanish Ministry of Industry, Economy and Competitiveness through the AGL2016-76943-C2-1-R and AGL2016-76943-C2-2-R projects (co-funded by the European Social Fund, European Union); M.J. Motilva thanks to Consejo Superior de Investigaciones Científicas-CSIC for partial funding throught the “Ayudas incorporación a escalas científicas CSIC, 2018” (Reference 201870I129). I.A.L. enjoys a post-doctoral contract (2017PMF-POST2-19) from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement and from the Universitat Rovira i Virgili (URV). S.Y. was supported by a pre-doctoral grant from the University of Lleida.

Forward osmosis (FO) and emulsification with dynamic membranes of tunable pore size (DMTS) were assessed to concentrate and encapsulate polyphenol extracts from carob pulp. In the FO step, a feed solution temperature of 40 °C resulted in the fastest concentration and the highest polyphenol yield. Moreover, FO at 35 °C enabled to concentrate up to about 20 times in 3 h using a pilot scale unit of 0.5 m2 of membrane area. Phenolic compounds were subsequently encapsulated in the inner water phase (W1) of water-in-oil-in-water (W1/O/W2) emulsions produced with DMTS, which enabled to obtain emulsions with a monomodal droplet size distribution (span

In the present study, 27 barley genotypes (Hordeum vulgare L.) grown in two geographic origins (Czech Republic and Spain) were analysed for their contents of β-glucan, tocols and phenolic compounds (free and bound). The samples included hulled, hull-less and coloured genotypes. The results showed that concentrations of β-glucan range from 2.40 to 7.42 g/100 g. Total tocol content of the barley samples ranged between 39.9 and 81.6 μg/g. A total of 64 compounds were identified in the barley samples. These included 19 phenolic acids and aldehydes, 9 flavan 3-ols, 9 flavone glycosides, and 27 anthocyanins. The results showed a wide range of phenolic concentrations in the barley samples, highlighting the presence of considerable amounts of anthocyanins in purple barley genotypes. In synthesis, barley should be considered a good source of bioactive components, especially because of the broad spectrum of phytochemicals with potential health benefits besides the soluble fibre (β-glucan).

This study investigated and compared the absorption, metabolism, and subsequently, the tissue distribution and excretion of hydroxytyrosol (HT) administered either in its free form or through its naturally occurring esterified precursors, namely oleuropein (OLE) and its aglycone forms known as secoiridoids (SEC). Here, rats were fed a diet supplemented with the equivalent of 5 mg phenol/kg/day for 21 days and the HT metabolites in the gastrointestinal digesta (stomach, small intestine and caecum), plasma, urine and metabolic tissues (liver and kidney) were analysed. Compared to HT and SEC, OLE showed greater stability during digestion, and, consequently, the bioavailability based on the urine excretion of HT metabolites was higher. OLE, as a glycoside molecule, reached the colon unaltered generating more diverse microbial metabolites. In terms of bioavailability, findings suggest that OLE might be the most suitable precursor of HT for incorporation into foods or nutraceutical formulations. This study was supported by the Spanish Ministry of Education and Science (AGL2012-40144-C03-03 and AGL2012-40144-C03-02 projects) and by the University of Lleida through the M.C. López de las Hazas grant.

BACKGROUND Rainfed viticulture, mainly in semi-arid environments, is limited by environmental variability, particularly precipitation and its seasonal distribution, and soil water availability, thus ultimately determining the final quality of grape and wine. Studies on the feasibility of practices such as canopy management to adapt plant growth and yield to soil water availability open up possibilities to preserve wine quality and reinforce the characteristics of the terroir. RESULTS Principal components analysis was used to identify the relationships between a large set of variables, including soil, plant, canopy management, and wine characteristics. Canopy management was found to have a predominant influence on plant response to soil water by modifying plant water status, changing the amino acid profile in berries and, concomitantly, altering the sensorial attributes of the wine obtained. CONCLUSIONS Grapevine canopy management strategies, such as reiterate shoot trimming to restrict growth during early phases, are effective in adapting plant response to soil water availability. Such strategies affect berry and wine quality, mainly the amino acid profile and sensorial attributes of the wine, without changing yield or grape harvest quality control parameters. Also, in such conditions, nitrogen does not make a significant contribution to grapevine growth or yield or to grape quality. © 2015 Society of Chemical Industry

Olive oils flavoured with edible herbs have grown in popularity because of their added value and potential health benefits. However, the combined presence of different phytochemicals from olive oil and herbs requires study of their possible interactions during intestinal transport and metabolism. The aim of this study was firstly to evaluate the effect on bioaccessibility of the co-occurring bioactive compounds from olive oil and thyme through an in vitro digestion model of three extracts: olive extract (OE), thyme extract (TE) and a combination of both (OTE). The bioaccessible fractions were exposed to Caco-2 and HepG-2 cell models, as well as to a co-culture of both of these. Results indicated that the bioaccessibility of hydroxytyrosol was enhanced when OTE was digested. After Caco-2 cells exposure, no significant differences were observed in hydroxytyrosol transport, whereas the main flavonoids from thyme seemed to undergo an enhanced basolateral permeation when both phenolic sources where exposed.

The growing interest in β-glucans and the dietary recommendations of an exact daily intake will require rapid and accurate quantification methods of β-glucans that can be used routinely by the food industry. The objective of the present study was to adapt the standard enzymatic procedure (Megazyme method) to quantify (1-3)(1-4)-β-D-glucans to micro-plate format and further application to analyze cereal based samples with a wide range of (1-3)(1-4)-β-D-glucan content (from 0.27–75%). The samples used in this study included two breads (wheat and barley/wheat), barley flours (4% and 8% β-glucans) and two samples of oat bran (28% and 75% β-glucans). Results showed that there was no significant differences in the quantification of β-(1,3)(1,4)-D-glucans in different samples by using the Megazyme method or the micro-method. The methodology developed was also compared in terms of sensitivity and reproducibility with the results obtained by the Megazyme kit method and no differences were observed. In conclusion, the developed method allows the β-glucan quantification (specifically for mixed-linkage [(1-3)(1-4)]-β-D-glucan) to be conducted rapidly and by an efficient and sensitive micro-method in a wide range of concentrations.

Flavonoids are metabolized by the gut microflora giving rise to a wide range of phenolic acids that may exert systemic effects in the body. Nevertheless, the colonic metabolism pathways and the function of the metabolites formed are poorly studied. In the present study, the individual colonic metabolism of three subclasses of flavonoids (flavonols, flavones and flavanones) and phenolic acids was evaluated. For this, seven standards of flavonoids (quercetin, quercetin–rhamnoside, quercetin–rutinoside, myricetin, luteolin, naringenin and kaempferol–rutinoside) and two phenolic acids (protocatechuic acid and gallic acid) were submitted to an in vitro fermentation model using rat colonic microflora. Simultaneously, a nuts–cocoa cream enriched with these standards of flavonoids was incubated and the colonic metabolism of these compounds was evaluated. The results showed that the greatest number of colonic metabolites came from the fermentation of quercetin and quercetin–rhamnoside, and the maximum concentration of fermentation products was observed after 48 h of fermentation. On the other hand, a smaller number of fermentation products were observed after the colonic fermentation of kaempferol–rutinoside, naringenin, luteolin and myricetin. The phenolic acids were slightly metabolized by the colonic microflora.

Phenolic compounds are one of the main reasons behind the healthy properties of virgin olive oil (VOO). However, their daily intake from VOO is low compared with that obtained from other phenolic sources. Therefore, the intake of VOO enriched with its own phenolic compounds could be of interest to increase the daily dose of these beneficial compounds. To evaluate the effectiveness of enrichment on their bioavailability, the concentration of phenolic compounds and their metabolites in human plasma (0, 60, 120, 240 and 300 min) from thirteen healthy volunteers (seven men and six women, aged 25 and 69 years) was determined after the ingestion of a single dose (30 ml) of either enriched virgin olive oil (EVOO) (961·17 mg/kg oil) or control VOO (288·89 mg/kg oil) in a cross-over study. Compared with VOO, EVOO increased plasma concentration of the phenol metabolites, particularly hydroxytyrosol sulphate and vanillin sulphate (P,0·05). After the consumption of VOO, the maximum concentration of these peaks was reached at 60 min, while EVOO shifted this maximum to 120 min. Despite these differences, the wide variability of results indicates that the absorption and metabolism of olive oil phenols are highly dependent on the individual. The present study was supported by the Spanish Ministry of Education and Science financing the projects AGL2005- 07881-C02-01/ALI and AGL2005-07881-C02-02/ALI; Health Ministry (FIS; PI021307) and CIBERDEM; and the grant received by Manuel Suarez (BES-2006-14136).

Rapid, selective and sensitive methods were developed and validated to determine procyanidins, anthocyanins and alkaloids in different biological tissues, such as liver, brain, the aorta vein and adipose tissue. For this purpose, standards of procyanidins (catechin, epicatechin, and dimer B(2)), anthocyanins (cyanidin-3-glucoside and malvidin-3-glucoside) and alkaloids (theobromine, caffeine and theophylline) were used. The methods included the extraction of homogenized tissues by off-line liquid-solid extraction, and then solid-phase extraction to analyze alkaloids, or microelution solid-phase extraction plate for the analysis of procyanidins and anthocyanins. The eluted extracts were then analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, using a triple quadrupole as the analyzer. The optimum extraction solution was water/methanol/phosphoric acid 4% (94/4.5/1.5, v/v/v). The extraction recoveries were higher than 81% for all the studied compounds in all the tissues, except the anthocyanins, which were between 50 and 65% in the liver and brain. In order to show the applicability of the developed methods, different rat tissues were analyzed to determine the procyanidins, anthocyanins and alkaloids and their generated metabolites. The rats had previously consumed 1g of a grape pomace extract (to analyze procyanidins and anthocyanins) or a cocoa extract (to analyze alkaloids) per kilogram of body weight. Different tissues were extracted 4h after administration of the respective extracts. The analysis of the metabolites revealed a hepatic metabolism of procyanidins. The liver was the tissue which produced a greater accumulation of these metabolites.

Procyanidins are metabolised by endogenous gastrointestinal microflora into several small molecules that may exert systemic effects. However, the metabolic pathways of procyanidins are still largely uncharacterised. The aim of this study was to evaluate the colonic metabolism of procyanidins (catechin, epicatechin, epicatechin gallate, epigallocatechin gallate and dimer B2) and alkaloids (theobromine and caffeine) by an in vitro colonic fermentation model using rat colonic microflora. Simultaneously, a nuts-cocoa cream was incubated and the colonic metabolism of procyanidins and alkaloids was evaluated. Results showed that most of the procyanidins tested were catabolised after 24–48 h of fermentation. Fermentation of the dimer B2 did not produce the same catabolic compounds as epicatechin fermentation and differences were observed between the fermentation of catechin and epicatechin. The alkaloids were not metabolised by the colonic microflora and this fact was verified in vivo. The results showed an intense metabolism of procyanidins and poor metabolism of alkaloids.

The use of an emulsifier to stabilize the phenolic compounds added in the preparation of an enriched olive oil was evaluated. Two emulsifiers, lecithin and monoglyceride, were studied. The results showed lecithin to be the most convenient, due to the increase in the value of the oxidative stability of the phenol-enriched oils in relation to the enrichments prepared with monoglycerides. After that, the shelf life of the prepared oils was evaluated during a period of 256 days of storage at 25°C in the dark. Oil quality parameters, total phenolic content, bitterness index and oxidative stability were studied during the storage period. Additionally, the phenolic composition and antioxidant capacity (by using the ORAC assay) were evaluated at the end of the storage. The phenolic enrichment of the oils allowed the shelf life of the oils to be extended compared with the control (virgin olive oil without phenol addition), delaying the appearance of peroxides and improving their oxidative stability. In addition, the higher content of phenolic compounds in the oils at all stages of storage is desirable in order to increase the intake of these beneficial compounds. Practical applications: The preparation of phenol-enriched olive oils with a higher phenolic content than the commercial virgin olive oils is of special interest to increase the ingestion of these healthy compounds the daily intake of which is limited due to the high caloric value of olive oil. There are two key points in the development of this product: (i) the dispersion and stabilization of the phenol extract in the oil matrix and (ii) the stability of the phenols in the prepared oils to guarantee the phenol concentration during their shelf life. It is important to study the use of emulsifiers to determine if they allow an improvement in the dispersion of the phenolic extract, and their stabilization in the final product. In addition, the emulsifiers could mask the bitter taste of the enriched oils, which is desirable to increase consumer acceptance of the enriched oil.

Carob flour (CF) and washed carob flour (WCF) (without the water soluble dietary fraction) were submitted to an in-vitro digestion method to evaluate the dietary soluble fibre (DF) and soluble sugars content on the digestibility and bioaccessibility of polyphenols. In addition, combinations of the raw materials (CF and WCF) with hazelnut oil (HO) were proposed in order to evaluate the protection of a vegetable oil rich in polyunsaturated fatty acids (PUFA) on the phenolic compounds submitted to in-vitro digestion conditions. The results showed that the soluble dietary fraction enhanced the stability of the phenolic compounds during the duodenal digestion phase. Similarly, the lipid fraction (rich in PUFA) showed a protective effect on the recovery of the phenolic compounds during duodenal digestion. Disruption of the natural matrix under digestion conditions led to the release of DF and PUFA, which could establish interactions with certain polyphenol compounds enhancing their recovery and stability during digestion.

In the analysis of biological samples, such as plasma or serum, the quantity of sample available is a critical parameter in most cases. A good approach is the use of the microelution SPE (μSPE) plates as sample pre-treatment technique in which the loaded sample volume is low. An off-line μSPE and ultra-performance LC-ESI-MS/MS (UPLC-ESI-MS/MS) method was developed and validated to determine procyanidins and anthocyanins in spiked plasma samples. The sample pre-treatment μSPE allowed the simultaneous determination of procyanidins and anthocyanins from plasma by using a small sample volume (350 μL) and without an evaporation step previous to the chromatographic analysis. Moreover, the use of UPLC technique allowed to determine the studied compounds at low concentration levels in a short analysis time (12.5 min approximately). Then, the developed method was applied to determine the studied compounds, procyanidins and anthocyanins, and their metabolites in rat plasma samples. Previously, the rats had consumed 5000 mg/kg of a grape pomace extract and the plasma was extracted 4 h after administration. The procyanidins catechin and epicatechin glucuronide, methyl catechin and epicatechin glucuronide, and methyl catechin and epicatechin sulphate were detected at μM concentration level, and the parent anthocyanins at nM.

An off-line solid-phase extraction (SPE) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining procyanidins, catechin, epicatechin, dimer, and trimer in plasma samples. In the validation procedure of the analytical method, linearity, precision, accuracy, detection limits (LODs), quantification limits (LOQs), and the matrix effect were studied. Recoveries of the procyanidins were higher than 84%, except for the trimer, which was 65%. The LODs and LOQs were lower than 0.003 and 0.01 microM, respectively, for all the procyanidins studied, except for the trimers, which were 0.8 and 0.98 microM, respectively. This methodology was then applied for the analysis of rat plasma obtained 2h after ingestion of grape seed phenolic extract. Monomers (catechin and epicatechin), dimer and trimer in their native form were detected and quantified in plasma samples, and their concentration ranged from 0.85 to 8.55 microM. Moreover, several metabolites, such as catechin and epicatechin glucuronide, catechin and epicatechin methyl glucuronide, and catechin and epicatechin methyl-sulphate were identified. These conjugated forms were quantified, in reference to the respective unconjugated form, showing concentrations between 0.06 and 23.90 microM.

An improved liquid chromatography (LC) tandem mass spectrometry (MS/MS) method has been developed for the determination of phenolic compounds (phenyl alcohols, phenyl acids, secoiridoid derivatives, lignans and flavonoids) in virgin olive oil. The used LC technique was ultra-performance LC with columns packed with 1.7 microm particles. The obtained results (retention times, linearity, reproducibility, detection limits (LODs) and quantification limits (LOQs) for the analysis of 14 phenolic compounds in standard solutions were compared with those obtained by high-performance LC (HPLC)-fluorescence and UPLC-diode array detection (DAD). When the 1.7 microm column was used, the retention times were decreased three times with respect to conventional HPLC (5 microm). The reproducibility of these methods, expressed as relative standard deviation (RSD) in terms of concentration ranged from 0.4-5.0%. In general, the LODs and LOQs were lower in UPLC-MS/MS than the other two methodologies for all the analytes, with the exception of vanillic acid and pinoresinol which values of LODs and LOQs by HPLC-fluorescence were similar to the values obtained by UPLC-MS/MS. Afterwards, the improved UPLC-MS/MS methodology was used to determine the studied compounds in spiked refined olive oil (ROO) by combining a liquid-liquid extraction (LLE) as a sample pre-treatment technique. The recoveries of the analytes were higher than 70%, with the exception of pinoresinol and 3,4-DHPEA-EDA, which were 61% and 67%, respectively. The LODs and the LOQs ranged from 0.44-127.78 microg/kg, and from 1.11-427.78 microg/kg, respectively for all the analytes. The reproducibility of the method was lower than 3.2%. The LLE-UPLC-MS/MS was successfully applied to analyze phenolic compounds in a virgin olive oil sample within 18 min.

Monovarietal virgin olive oils are labelled with the olive varieties giving them their distinctive character. There are numerous studies focussed on the characterisation and quantification of the minor fractions of virgin olive oils that have generated databases on varietal olive oils. However, few studies have focussed on the components of the pigment fraction of virgin olive oils. The aim of this work was to quantify the components of the chlorophyll and carotenoid fractions of the monovarietal virgin olive oils from the Arbequina cultivar, growing in the Spanish area of Catalonia, during two consecutive crop seasons. Additionally the pigment changes occurring during 24 months of oil storage were evaluated. The results of this study showed minor qualitative differences between monovarietal virgin olive oils from two consecutive seasons. The quantitative differences could be attributed to the harvest period in each season rather than to the year's weather conditions. Storage of the monovarietal virgin olive oils probably caused an important loss of the chlorophyll fraction, mainly chlorophyll a, during the first 6 months of storage. On the other hand, the carotenoid fraction was very stable and the retention of provitamin A was close to 80%, even after 24 months of storage.

The aim of this study was to evaluate several cocoa sources to obtain a rich phenol extract for use as an ingredient in the food industry. Two types of phenolic extracts, complete and purified, from different cocoa sources (beans, nibs, liquor, and cocoa powder) were investigated. UPLC-MS/MS was used to identify and quantify the phenolic composition of the extracts, and the Folin-Ciocalteu and vanillin assays were used to determine the total phenolic and flavan-3-ol contents, respectively. The DPPH and ORAC assays were used to measure their antioxidant activity. The results of the analysis of the composition of the extracts revealed that the major fraction was procyanidins, followed by flavones and phenolic acids. From the obtained results, the nib could be considered the most interesting source for obtaining a rich phenolic cocoa extract because of its rich phenolic profile content and high antioxidant activity in comparison with the other cocoa sources.

The main objective of this research was to determine the extent to which irrigation practices affect the partitioning of phenolic compounds between olive paste, pomace, olive oil and wastewater. The current paper also aimed to study the effect of technological natural micro-talc (NMT) addition during the oil extraction process on the partitioning of the phenolic compounds between solid and liquid phases. The results obtained in this study showed that irrigation applied to olive trees let to a considerable decrease in the phenol content of the olive paste. The water status of the trees affected the phenol synthesis in the olive fruit, and consequently the phenol content of the olive paste, more than the partitioning of the phenolic compounds during the olive oil extraction process. The most remarkable point of the phenol partitioning was related to the simple phenols. While in the samples from non-irrigated trees the greater proportion of these phenols partitioned into the pomace, in samples from irrigated trees most of them were lost in the wastewater. After comparison of the results obtained from the experiments with and without NMT addition, it was concluded that the use of that co-adjuvant did not significantly alter either the phenolic profile of the oil phase obtained or the content of the individual phenolic compounds.

During the olive drupes ( Olea europaea L.) development we have observed significant correlations ( r = 0.82, r = 0.82 and r = 0.64 for Arbequina , Farga and Morrut varieties) between l -phenylalanine ammonia-lyase enzymatic activity (PAL, EC 4.3.1.5) and the olive drupe phenolic content. l -Phenylalanine ammonia-lyase activity and the total phenol content in olive drupes suffered a decrease when the maturation processes began. Total phenol content decrease could be used as a differential since it decreased regularly in the Arbequina cv. during maturation, while their content in the Farga and Morrut cv. underwent an important decrease due probably to high variability on ripening index of Arbequina variety. Oleuropein could be used as maturation parameter at harvesting period since in Morrut and Farga cv. showed ten and six times more oleuropein than Arbequina cv. It also could be used as a maturation parameter luteolin and luteolin-7-glucoside, which had higher values in Arbequina cv. (653–62 mg kg −1 , respectively) than in Farga (222–25 mg kg −1 ) and Morrut cv. (201–not detected mg kg −1 ) at harvesting period. The data obtained during the fruit development indicate than phenol content and composition, in particular oleuropein, luteolin and luteolin-7-glucoside will be useful for biochemical characterisation of olive drupes of different O. europaea L. varieties.

The purpose of the work was to investigate the effect of the maturation process of the olive fruit on the phenolic fraction of drupes and oils from Arbequina, Farga, and Morrut cultivars. The level in the phenolic content of olive drupes declines rapidly during the black maturation phase. A general decreasing trend was observed too in the phenolic content of olive oils during the ripening process in the three varieties studied. Important differences in the high-performance liquid chromatography profile between varieties were observed. These included the presence of very low amounts of lignans in olive oils proceeding from the Morrut cultivar, and the presence of three peaks after elution of 3,4-DHPEA-EDA in the Farga and Morrut cultivars, which could be used as differentiating parameters. Sensory profile differences were observed between olive cultivars and due to the ripening process. Keywords: Olive fruit; virgin olive oil; phenolic compounds

The aim of this work was to study the effect of freeze damage during the harvest period on the composition and quality indices of olive oil from the Arbequina cultivar. No differences were found in the quality indices of oils extracted from olives affected by freeze injuries. On the other hand, slight differences were observed in the carotenoid and chlorophyll contents. These were reflected in higher luminosity values ( L ∗ ), and an important decrease in the concentrations of secoiridoid derivatives and 4-(acetoxyethyl)-1,2-dihydroxybenzene (3,4-DHPEA-AC), giving rise to a lower oil stability and sensory changes, such as the absence of bitter taste, and less pungent taste of oils extracted from olives after frost damage. Slight rises were also observed in the concentrations of simple phenols, such as vanillic acid and vanillin, that gave rise to sweeter oils.

Spices, like vegetables, fruit, and medicinal herbs, are known to possess a variety of antioxidant effects and other biological activities. Phenolic compounds in these plant materials are closely associated with their antioxidant activity, which is mainly due to their redox properties and their capacity to block the production of reactive oxygen species. More recently, their ability to interfere with signal transduction pathways involving various transcription factors, protein kinases, phosphatases, and other metabolic enzymes has also been demonstrated. Many of the spice-derived compounds which are potent antioxidants are of great interest to biologists and clinicians because they may help protect the human body against oxidative stress and inflammatory processes. It is important to study the bioactive compounds that can modulate target functions related to defence against oxidative stress, and that might be used to achieve health benefits individually. In the present review, an attempt has been made to summarize the most current scientific evidence about the in vitro and in vivo effects of the bioactive compounds derived from herbs and spices, focused on anti-inflammatory and antioxidant effects, in order to provide science-based evidence for the traditional uses and develop either functional foods or nutraceuticals.

In the present study, simultaneous extraction of natural antioxidants (phenols and carotenoids) in complex matrices, such as tomato sauces, is presented. The tomato sauce antioxidant compounds studied were the phenolics hydroxytyrosol, from virgin olive oil, quercetin and its derivatives, from onions, and quercetin-rutinoside as well as the carotenoid, lycopene (cis and trans), from tomatoes. These antioxidant compounds were extracted simultaneously with n-hexane/acetone/ethanol (50/25/25, v/v/v). The phenolics were analysed by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS), and lycopene (cis- and trans-forms) was analysed using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). After studying the parameters of these methods, they were applied to the analysis of virgin olive oil, fresh onion, tomato concentrate and tomato powder, and commercial five tomato sauces. Subsequently, the results obtained in our laboratory were compared with those from the Gallina Blanca Star Group laboratory.

In the present study, a selective and sensitive method, based on microelution solid-phase extraction (μSPE) plate and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) was validated and applied to determine the plasma metabolites of the bioactive compounds of thyme. For validation process, standards of the more representative components of the phenolic and monoterpene fractions of thyme were spiked in plasma samples and then the quality parameters of the method were studied. Extraction recoveries (%R) of the studied compounds were higher than 75%, and the matrix effect (%ME) was lower than 18%. The LODs ranged from 1 to 65 μg/L, except for the thymol sulfate metabolite, which was 240 μg/L. This method was then applied for the analysis of rat plasma obtained at different times, from 0 to 6 h, after an acute intake of thyme extract (5 g/kg body weight). Different thyme metabolites were identified and were mainly derived from rosmarinic acid (coumaric acid sulfate, caffeic acid sulfate, ferulic acid sulfate, hydroxyphenylpropionic acid sulfate, dihydroxyphenylpropionic acid sulfate and hydroxybenzoic acid) and thymol (thymol sulfate and thymol glucuronide). The most abundant thyme metabolites generated were hydroxyphenylpropionic acid sulfate and thymol sulfate, their respective concentrations in plasma being 446 and 8464 μM 1 h after the intake of the thyme extract.

Besides affecting the oil’s sensorial characteristics, the presence of herbs and spices has an impact on the nutritional value of the flavored oils. The aim of the study was to develop a new product based on the phenol-enrichment of a virgin olive oil with both its own phenolic compounds (secoiridoid derivatives) plus additional complementary phenols from thyme (flavonoids). We studied the effect of the addition of phenolic extracts (olive cake and thyme) on phenolic composition, oxidative stability, antioxidant activity, and bitter sensory attribute of olive oils. Results showed that flavonoids from thyme appeared to have higher transference ratios (average 89.7%) from the phenolic extract to oil, whereas secoiridoids from olive presented lower transference ratios (average 35.3%). The bitter sensory attribute of the phenol-enriched oils diminished with an increase of the concentration of phenols from thyme, which might denote an improvement in the consumer acceptance.

Procyanidins are extensively metabolized via phase-II and microbial enzymes. However, their distribution in the body is not well characterized. This study investigates the distribution of procyanidins (monomers and dimers) and their phase-II metabolites in plasma and tissues (thymus, heart, liver, testicle, lung, kidney, spleen and brain). Wistar rats were fed with 1 g of cocoa cream (CC), 50 mg of procyanidin hazelnut skin extract (PE) and 50 mg PE in 1 g CC (PECC). The rats were killed at 0, 1, 1.5, 2, 3, 4 and 18 h after gavage, and the plasma and tissues were analyzed by UPLC–MS/MS. Epicatechin–glucuronide was the main metabolite in the plasma after the CC intake, with C max at 423 nM and t max at 2 h, and methyl catechin–glucuronide (301 nM, 2 h) was the main metabolite in the plasma after the PE intake. As a result of the PECC enrichment, epicatechin–glucuronide (452 nM, 1.5 h) and catechin–glucuronide (297 nM, 2 h) were the main metabolites in the plasma. Methyl catechin–glucuronide was found in the liver after PE (8 nmol/g tissue, 4 h) and PECC (8 nmol/g, 1.5 h). The kidney was found to contain a high concentration of phase-II metabolites of procyanidins and is therefore thought to be the main site of metabolism of the compounds. Methyl catechin–sulfate (6.4 nmol/g, 4 h) was only quantified in the brain and after PE intake. Catechin metabolites were not found in the spleen or heart. Phenolic acids were detected in all tissues. The formulation of a product enriched or fortified with procyanidins is a way to increase their bioavailability, with clear effects on the plasmatic pharmacokinetics, and a greater accumulation of phenolic metabolites in such tissues as the liver, kidney, lung and brain.

An improved method, based on ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS/MS), has been developed to determine the bioactive dipeptides carnosine (CAR) and anserine (ANS) in chicken broth. These analytes are hydrophilic (polar) and in order to improve their retention, the chromatographic mode used was hydrophilic interaction chromatography (HILIC) (1.7 μm particle size). In order to remove the salt before the chromatographic analysis of the chicken broth (0.8%, w/w), an exhaustive sample pre-treatment strategy was necessary since the salt is not volatilized and could block the ionization source and lead to signal suppression. The chicken broth was firstly centrifuged to remove the fat and chicken proteins, and then was pretreated by off-line solid-phase extraction (SPE), using traditional cartridges, or off-line μElution plate (μSPE), using microplates, and the results were compared. Due to the high polar character of the dipeptides studied and the sample matrix, these compounds were not retained in the sorbent hydrophilic-lipophilic balanced (HLB) and were eluted in the load step, whereas the salt was retained in the sorbent. This fact was observed by the addition of silver nitrate in the chicken broth extract, where before the SPE or μSPE a white precipitate (silver chloride) was formed and after the SPE or μSPE this precipitate was not observed. By using these sample pre-treatment strategies, the extraction recoveries were higher than 80%, and the matrix effect was lower than 12%. Once the improved method was developed, the quality parameters of the method were studied. The LODs and LOQs of the CAR and ANS were lower than 6 and 1.8 μg/l, respectively. Then, the method was applied to analyse a commercial chicken broth. This improved method allowed determining CAR and ANS between 6 and 10mg dipeptide/l chicken broth in 10 min (sample pre-treatment and chromatographic analysis). Therefore, the proposed improved method is concluded to be rapid, sensitive and selective for the determination of polar compounds by MS in samples that contain salt.

10.1016/j.foodchem.2012.07.085 Three different functional phenol-enriched virgin olive oils (FVOO) were prepared with a phenolic content of 250 (L-FVOO), 500 (M-FVOO), and 750 mg (H-FVOO) total phenols/kg. In a randomised, cross-over study with 12 healthy volunteers, the pharmacokinetics of phenolic biological metabolites was assessed. An increasing linear trend was observed for hydroxytyrosol sulfate, the main phenolic metabolite quantified in plasma, with Cmax values of 1.35, 3.32, and 4.09 µmol/l, and AUC mean values of 263.7, 581.4, and 724.4 µmol/min for L-FVOO, M-FVOO, and H-FVOO, respectively. From our data an acute intake of phenol-enriched olive oils promotes a dose-dependent response of phenol conjugate metabolites in human plasma. Also, we point out for the first time hydroxytyrosol acetate sulfate as a main biological metabolite of hydroxytyrosol from olive oil ingestion.

We report progress in the study of olive oil phenolic metabolites in humans and identify a new hydroxytyrosol metabolite called hydroxytyrosol acetate sulphate, which was determined using tandem MS, after ingestion of 30 ml of olive oil with a high phenolic content (500 mg/kg oil), reaching a maximum concentration of 1.63 μM. In order to understand and explain the generation of this metabolite, two different pathways are proposed.

Scope The distribution and accumulation of olive oil phenolic compounds in the body are topics lacked of information. The aim of this study was to evaluate the bioavailability, metabolism and distribution of phenolic compounds from olive cake. Methods and results The metabolism and distribution of phenolic compounds were examined by UPLC-MS/MS after an acute intake of a phenolic extract from olive cake, analyzing plasma and tissues (heart, brain, liver, kidney, spleen, testicle and thymus) 1, 2 and 4 h after ingestion using Wistar rats as the in vivo model. The results showed a wide distribution of phenolic compounds and their metabolites in the tissues, with a main detoxification route through the kidneys. Highlighting the quantification of the free forms of some phenolic compounds, such as oleuropein derivative in plasma (Cmax 4 h: 24 nmol/L) and brain (Cmax 2 h: 2.8 nmol/g), luteolin in kidney (Cmax 1 h: 0.04 nmol/g), testicle (Cmax 2 h: 0.07 nmol/g) and heart (Cmax 1 h: 0.47 nmol/g); and hydroxytyrosol in plasma (Cmax 2 h: 5.2 nmol/L), kidney (Cmax 4 h: 3.8 nmol/g) and testicle (Cmax 2 h: 2.7 nmol/g). Conclusion After a single ingestion of olive oil phenolic compounds, these were absorbed, metabolized and distributed through the blood stream to practically all parts of the body, even across the blood-brain barrier.

The recent information regarding the healthy properties of virgin olive oil phenols and the interest in increasing the value of byproducts from the oil extraction processs support the standardized development of phenol-enriched olive oil. Accordingly, the aim of this research work was to evaluate strategies for the development of a virgin olive oil enriched with phenolic compounds obtained from olive cake to increase phenolic ingestion without the drawback of a higher calorie intake. For this proposal, different combinations of phenolic extracts were evaluated at a range of concentrations to obtain the best prototype of enriched olive oil. To study the functionality of the phenol enrichments, the total phenolic content and the oxidative stability were determined by the Folin-Ciocalteu and Rancimat tests, respectively. In addition, the phenolic composition and antioxidant capacity (ORAC assay) of the oils were studied. Finally, the stability and potential bioaccesibility of the phenolic fraction of the enriched oils were tested by an in vitro gastrointestinal digestion model. Results of the study showed different strategies to select the best prototype of enriched olive oil, taking into consideration not only their phenolic content but also other important factors such as the feasibility of implementing the preparation process in the food industry.

Publisher Summary Maturity is one of the most important factors associated with the quality evaluation of fruits and vegetables. One of the attributes for judging maturity is the skin color. In general, during fruit ripening, the chlorophylls, which are present in all unripe fruit, break down following the transformation of chloroplasts into chromoplasts, and carotenogenesis takes over. The olive fruit, Olea europaea, a well-known and widespread species of the Oleaceae family, is a green, fleshy, edible drupe. During the ripening process, photosynthetic activity decreases and the concentrations of chloroplast pigments, chlorophylls and carotenoids, decrease progressively. At the end of the maturation process, the violet or purple color of the olive fruit is due to the formation of anthocyanins. The composition of the chlorophyll fraction of the olive fruit comprises chlorophylls a and b, and the carotenoids that typically accompany the chlorophylls in the chloroplast: lutein, β-carotene, violaxanthin, neoxanthin, and antheraxanthin. During the olive ripening, the skin color changes from green (G) at the beginning of the harvest period, light-green (LG), small reddish spots (SRS), turning color (TC), purple (P), to black (B) at the end of the harvest period. During the ripening process, the gradual disintegration of the chloroplasts involves the disappearance of the chloroplast pigments. At the beginning of the ripening period, when the olive drupe color turns from green to light green, chlorophylls a and b are the major pigments, decreasing during the process. This decrease of the chlorophylls is more marked than in the carotenoid fraction, although both pigment fractions reach similar concentration at the end of the ripening period (black stage).

Among procyanidins (PC), monomers, such as catechin and epicatechin, have been widely studied, whereas dimer and trimer oligomers have received much less attention, despite their abundance in our diet. Recent studies have showed that as dimers and trimers could be important in determining the biological effects of procyanidin-rich food, understanding their bioavailability and metabolism is fundamental. The purpose of the present work is to study the stability of PC under digestion conditions, the metabolism and the bioavailability by using a combination of in vitro and in vivo models. Simultaneously, the matrix effect of a carbohydrate-rich food on the digestibility and bioavailability of PC is investigated. The results show a high level of stability of PC under gastric and duodenal digestion conditions. However, the pharmacokinetic study revealed limited absorption. Free forms of dimers and trimers have been detected in rat plasma, reaching the maximum concentration 1 h after oral intake of a grape seed extract. The present work was supported by the CENIT program from the Spanish Minister of Industry and by a consortium of companies led by La Morella Nuts S.A. (Reus, Catalonia, Spain) with the following companies: Shirota Functional Foods, S.L.; KRAFT; BTSA, Biotecnologı´as Aplicadas, S.L.; Seleccio ´n Batalle´, S.A.; Industrial Te´cnica Pecuaria, S.A.; Neuron BioPharma, S.A.; Grupo Leche Pascual, S.A.U.; Innaves, S.A. The present work was also supported by the Spanish Ministry of Education and Science financing the project AGL2008-00 387.

Publisher Summary Virgin olive oil composition is greatly influenced by climatic conditions. In a limited area, small differences in maximum and minimum temperatures could be the cause of significant differences in fatty acid profile. This chapter aims to examine, among other things, the relationship between the sensory attributes of virgin olive oil and the freeze conditions, and to connect these to chemical composition. Differences are detected between oils in aroma and mouthfeel. In the study, the sensory notes of artichoke, tomato, and almond were perceived in before-frost oils, and no unpleasant aroma or flavor was detected. After-frost olive oils were qualified as non-extra virgin olive oil. Defects were defined as frozen olives by some panelists, while others defined these oils as thicker, softer, and with the term rancid tallow. A less bitter and pungent taste should be related to the noticeable reduction of oil phenolic compounds, especially secoiridoid compounds.

The effect of repeated consumption of virgin olive oil on endogenous phenolic metabolites of fasting plasma is unknown. For this reason, we hypothesized that regular long-term virgin olive oil intake could have an indirect protection effect on the endogenous phenols. Thus, the aim of the study was to determine the phenolic profile of human plasma in a fasting state of long-term regular virgin olive oil consumers, using the fasting plasma of non-consumers as a natural control. Forty participants living in the area of Reus (Catalonia, Spain) were selected, 20 life-long regular consumers of virgin olive oil and a natural control of 20 non-consumers, the latter being Rumanians who dislike the taste of olive oil. The diet was obtained from 3-day food records. The results showed similar phenolic composition of fasting plasmas of the two volunteer groups. Of special interest is that more of the compounds quantified showed higher concentration in fasting plasma from habitual virgin olive oil consumers. The compounds were semi-quantified using caffeic acid as the calibration standard. The quantification of fasting consumer's plasma showed higher concentration of a hydroxyflavanone type compound (2.90+/-0.04 microM vs 1.5+/-0.04 microM) and a catecholamine derivative (0.70+/-0.03 microM vs 0.56+/-0.03 microM) than the plasma of non-consumers (P0.05). The results suggest an indirect protective mechanism of long-term regular virgin olive oil consumption related to the protection of the endogenous antioxidant system.

Two methods based on solid-phase extraction (SPE) using traditional cartridges and microelution SPE plates (muSPE) as the sample pre-treatment, and an improved liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) were developed and compared to determine the phenolic compounds in virgin oil olive from plasma samples. The phenolic compounds studied were hydroxytyrosol, tyrosol, homovanillic acid, p-coumaric acid, 3,4-DHPEA-EDA, p-HPEA-EDA, luteolin, apigenin, pinoresinol and acetoxypinoresinol. Good recoveries were obtained in both methods, and the LOQs and LODs were similar, in the range of low muM. The advantage of muSPE, in comparison with SPE cartridges, was the lack of the evaporation step to pre-concentrate the analytes. The muSPE-UPLC-ESI-MS/MS method developed was then applied to determine the phenolic compounds and their metabolites, in glucuronide, sulphate and methylated forms, in human plasma after the ingestion of virgin olive oil.