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An improved chromatographic method was developed using ultra-performance liquid chromatography-tandem mass spectrometry to identify and quantify phenolic compounds and alkaloids, theobromine and caffeine, in carob flour samples. The developed method has been validated in terms of speed, sensitivity, selectivity, peak efficiency, linearity, reproducibility, limits of detection, and limits of quantification. The chromatographic method allows the identification and quantification of 20 phenolic compounds, that is, phenolic acids, flavonoids, and their aglycone and glucoside forms, together with the determination of the alkaloids, caffeine and theobromine, at low concentration levels all in a short analysis time of less than 20 min.

This work describes the applicability of an in vitro digestion model for the evaluation of the digestibility and bioaccessibility of cocoa polyphenols (procyanidins, phenolic acids, and flavones) and for the study of the food matrix effect in relation with the fat content. For this purpose, two cocoa samples, cocoa liquor ( approximately 50% fat content) and cocoa powder ( approximately 15% fat content), were used. The results showed an important increase of the concentration of procyanidin (monomers and dimers), probably due to the hydrolysis of procyanidins with a high degree of polymerization (pentamers to nonamers) submitted to the digestion procedure. In relation to flavones, the concentration of aglycone forms remained almost constant after the digestion steps; in contrast, the concentration of the glycoside forms an increase in the digestion mixtures mainly after the duodenal step, probably as a result of the partial digestion of the dietary fiber present in the cocoa. The higher fat content in the cocoa liquor seemed to have a protective effect, probably related with a better micellarization that favors the stability of polyphenols during digestion.

Olive cake, the most important byproduct of olive oil extraction by the two-phase centrifugation system, was used to obtain phenolic extracts. The extracts were obtained using the two constituents of this waste, vegetative water and solid residue, to maximize the extraction of all phenolic compounds. Different extraction procedures were studied, a simple and rapid extraction procedure being developed from the solid residue using an accelerated solvent extractor (ASE). Afterward, the phenolic extracts were fractionated using semipreparative HPLC to study the antioxidant activity of the different components. The identification of the phenolic compounds was carried out with an ultraperformance liquid chromatograph coupled to tandem mass spectrometry equipment (UPLC-ESI-MS/MS). With this method, a complete list of the polyphenols from the extract was obtained. Finally, the antioxidant activity of the phenolic extracts and the isolated fractions was evaluated, showing great antioxidant capacities, between 3450 and 17900 micromol of Trolox equivalents/g of extract. With regard to the isolated fractions the most antioxidant were those that contained hydroxytyrosol and 3,4-DHPEA-EDA. The suitability of the solid residue extract obtained by the ASE procedure was demonstrated given the great range of phenolic compounds and the feasibility of production on an industrial scale.

The aim of this study was to characterise the pigment profile (chlorophylls and carotenoids) of monovarietal virgin olive oils from six Italian olive cultivars (Ghiacciolo, Giarraffa, Sant’Agatese, Biancolilla, Nocellara del Belice and Gentile di Larino), from three Italian olive-growing regions (Emilia-Romagna, Sicily and Molise). The results show that the qualitative study of the pigment chlorophyll and carotenoid fractions of virgin olive oils demonstrated a common pattern that did not depend on the olive cultivar or growing region. Besides these, mutatoxanthin was only detected in oils from the Nocellara del Belice and Gentile di Larino cultivars, and pheophorbide a was only detected in oils from the Ghiacciolo and Sant’Agatese cultivars. However, significant quantitative differences were observed in the composition of the pigment fractions. The principal component discriminate analysis showed that the first two discriminate functions of the statistical analysis explained 78.9% of the total variance. The plot of discriminate functions showed an effect of the olive cultivar and growing area on the chlorophyll and carotenoid contents of the virgin olive oils. The results of this study are a contribution to the pigment characterization of the Italian monovarietal olive oils, which should be completed with more samples obtained from different crop seasons and different growing areas.

The aim of this study was to characterize antioxidant activities of phenolic compounds that appear in olive pulp and olive oils using both radical scavenging and antioxidant activity tests. Antiradical and antioxidant activities of olive pulp and olive oil phenolic compounds were due mainly to the presence of a 3,4-dihydroxy moiety linked to an aromatic ring, and the effect depended on the polarity of the phenolic compound. Glucosides and more complex phenolics exhibited higher antioxidant activities toward oxidation of liposomes, whereas in bulk lipids aglycons were more potent antioxidants with the exception of oleuropein. Lignans acted as antioxidants only in liposomes, which could partly be due to their chelating activity, because liposome oxidation was initiated by cupric acetate. The antioxidant activity of virgin olive oil is principally due to the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), a secoiridoid derivative (peak RT 36, structure unidentified), and luteolin.

Procyanidins are present in a wide range of dietary foods and their metabolism is well known. Nevertheless, the biological target and their distribution are topics lacking information. The purpose of the present work was to study the metabolism and distribution of procyanidins and their metabolites in rat plasma and different tissues, such as liver, brain, lung, kidney, intestine, testicle, spleen, heart and thymus, after 2 h of an acute intake of hazelnut extract rich in procyanidins (5 g kg(-1) of rat body weight). The interest of an acute intake of procyanidins instead of repeated low doses from daily ingestion of is to achieve a concentration of metabolites in the tissues that allows their detection and quantification. The results showed that catechin and epicatechin-glucuronide, methyl catechin and epicatechin-glucuronide and methyl catechin and epicatechin-sulphate were detected in plasma samples at the μmol level. On the other hand, catechin-glucuronide, methyl catechin-glucuronide and methyl catechin-sulphate were identified in some tissues, such as thymus, intestine, lung, kidney, spleen and testicle at the nmol level. Procyanidins with a low grade of polymerization (dimers and trimers) were detected in plasma samples and the intestine. Additionally, a wide range of simple aromatic acids from fermentation by the colonic microflora was detected in all tissues studied.

An understanding of causal relations between phenol intake and its beneficial effects on health is hindered by the lack of robust biological markers of its exposure. This is particularly relevant in mid/long-term nutritional intervention studies. An analytical methodology based on UPLC MS/MS has been developed to determine the metabolites of the phenolic compounds from olive oil and thyme in biological fluids after a sustained intake of two phenol-enriched olive oils for their further use as compliance biomarkers. In a randomized, double-blind, controlled, cross-over trial, 33 hypercholesterolemic volunteers received during 3 weeks 25 mL/day of (1) raw Virgin Olive Oil with a low phenolic content as a control (80 mg total phenols / kg oil; VOO), (2) Functional Virgin Olive Oil enriched with its own phenolics (500 mg total phenols / kg oil; FVOO), and (3) Functional Virgin Olive Oil enriched with its own phenolics plus complementary phenolics from Thyme (500 mg total phenols / kg oil, 50% from olive oil and 50% from thyme respectively; FVOOT). Plasma and 24 h-urine samples were collected. The results showed that somehydroxytyrosol (HT)metabolites presented lowspecificity as biomarkers of intake. However, hydroxytyrosol sulfate and hydroxytyrosol acetate sulfate appeared to be suitable biomarkers for monitoring compliancewith olive oil intake as their values in plasmaor/and 24-h urinewere significantly higher after FVOO compared to baseline pre-intervention concentrations. They were also significantly correlated with the monitored level of compliance. On the other hand, metabolites derived from thyme were more specific, thymol sulfate and hydroxyphenylpropionic acid sulfate being the metabolites with the largest increase in both plasma and 24-h urine, whereas urinary p-cymene-diol glucuronide presented the greatest increase post-treatment. Their urinary excretion values also displayed significant correlations with the level of compliance and they were defined as FVOOT compliance biomarkers. This study enabled robust quantitative and qualitative compliance biomarkers after the ingestion of two phenol-enriched olive oils to be determined and provided a thorough analysis of the true phenolic exposure after a sustained consumption that could be further related to expected biological effects.

Three food barley genotypes differing in the presence or absence of husks were sequentially pearled and their fractions analyzed for ash, proteins, bioactive compounds and antioxidant capacity in order to identify potential functional food ingredients. Husks were high in ash, arabinoxylans, procyanidin B3, prodelphinidin B4 and p-coumaric, ferulic and diferulic bound acids, resulting in a high antioxidant capacity. The outermost layers provided a similar content of those bioactive compounds and antioxidant capacity that were high in husks, and also an elevated content of tocols, representing the most valuable source of bioactive compounds. Intermediate layers provided high protein content, β-glucans, tocopherols and such phenolic compounds as catechins and bound hydroxybenzoic acid. The endosperm had very high β-glucan content and relative high levels of catechins and hydroxybenzoic acid. Based on the spatial distribution of the bioactive compounds, the outermost 30% pearling fractions seem the best option to exploit the antioxidant capacity of barley to the full, whereas pearled grains supply β-glucans enriched flours. Current regulations require elimination of inedible husks from human foods. However, due to their high content in bioactive compounds and antioxidant capacity, they should be considered as a valuable material, at least for animal feeds. 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).

The present study aims to investigate the metabolic fate and the cardiometabolic effects of phenolic compounds provided by a red-fleshed apple variety biofortified in anthocyanins (ACN). Wistar rats are fed with high-fat diet (HFD) to induce hypercholesterolemia and supplemented with red-fleshed apple (HFD+R), white-fleshed apple (HFD+W), or an ACN-rich infusion from aronia fruit (HFD+A) providing matched content and profile of ACN. Plasma biochemical parameters, histological analysis, and phenol biological metabolites are determined. Plasma, urine, and feces show a significant increase of ACN metabolites after HFD+R andHFD+A, while flavan-3-ols are significantly increased after HFD+W and dihydrochalcones derivatives increased after both apples supplementation. A cardioprotective effect sobserved after both apples and aronia infusion supplementation in the reduction of aortic thickness. The kidney function is improved after all supplementations and a decrease in insulin plasma concentration after both apples supplementation (HFD+R andHFD+W) is also observed. The findings support that ACN without apple matrix can induce cardioprotective effects. ACN or flavan-3-ols, together with dihydrochalcones, compose a phenolic phytocomplex in red- and white-fleshed apples, respectively, which can act synergistically in the attenuation of cardiovascular outcomes in hypercholesterolemic rats., 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). I.A.L. enjoyed 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 grant from the University of Lleida. Ú.C. has a Pla estratègic de recerca i innovació en salut (PERIS) post-doctoral grant (SLT002/16/00239; Catalunya, Spain) from Generalitat de Catalunya. A.P. enjoys a post-doctoral grant (PTQ-15-08068; Spain). L.R. is a Serra Húnter Fellow. In addition, the authors were grateful to NUFRI SAT (Mollerussa, Lleida, Catalonia, Spain) for providing the red-fleshed apples; to Ana Martínez (Department of Medicine, University of Lleida) for helpful with the histological stains; and finally, to SCT-Estabulari of the University of Lleida where the animal experiment was carried out. A few spelling and layout mistakes were corrected on May 14, 2021.