New red-fleshed apple cultivars: a comprehensive review of processing effects, (poly)phenol bioavailability and biological effects.

Red-fleshed apple cultivars with an enhanced content of anthocyanins have recently attracted the interest of apple producers and consumers due to their attractive color and promising added health benefits. In this paper, we provide the first comprehensive overview of new hybrid red-fleshed apples, mainly focusing on their (poly)phenolic composition, the effect of processing, the (poly)phenolic bioavailability and the biological effects. Evidence so far from in vitro and in vivo studies supports their added beneficial effects compared to common apples on health outcomes such as cancer, cardiovascular disease, inflammation and immune function, which are mainly related to their specific (poly)phenol composition.

Metabolic Fate and Cardiometabolic Effects of Phenolic Compounds from Red-Fleshed Apple in Hypercholesterolemic Rats: A Comparative Study with Common White-Fleshed Apple. The AppleCOR Study.

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 and HFD+A, while flavan-3-ols are significantly increased after HFD+W and dihydrochalcones derivatives increased after both apples supplementation. A cardioprotective effect is observed 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 and HFD+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.

Thermal and non-thermal processing of red-fleshed apple: how are (poly)phenol composition and bioavailability affected?

The present study evaluated the impact of different thermal (infrared-drying, hot air-drying and purée pasteurization) and non-thermal (freeze-drying) processing technologies on red-fleshed apple (poly)phenolic compounds. We further investigated the processing effect on the (poly)phenol bioavailability in a crossover postprandial study where three subjects consumed three apple products (freeze-dried snack, hot air-dried snack and pasteurized purée). (Poly)phenolic compounds present in the apple products and their biological metabolites in urine were analyzed using liquid chromatography coupled to mass spectrometry (UPLC-MS/MS). When comparing different processes, infrared-drying caused important losses in most of the apple (poly)phenolics, while hot air-drying and purée pasteurization maintained approximately 83% and 65% of total (poly)phenols compared with the freeze-dried snack, respectively. Anthocyanins in particular were degraded to a higher extent, and hot air-dried apple and pasteurized purée maintained respectively 26% and 9% compared with freeze-dried apple snack. The acute intake showed that pasteurized purée exhibited the highest (poly)phenol bioavailability, followed by hot air-drying and freeze-dried snack, highlighting the impact of processing on (poly)phenols absorption. In conclusion, for obtaining affordable new red-fleshed apple products with enhanced (poly)phenol bioavailability, purée pasteurization and hot air-drying represent viable techniques. However, to obtain a red-fleshed apple snack with high anthocyanin content, freeze-drying is the technique that best preserves them.

Phytochemical Profiles of New Red-Fleshed Apple Varieties Compared with Traditional and New White-Fleshed Varieties.

This study is an exhaustive chemical characterization of the phenolic compounds, triterpenes, and organic and ascorbic acids in red-fleshed apple varieties obtained by different breeding programs and using five traditional and new white-fleshed apple cultivars as reference. To carry out these analyses, solid-liquid extraction (SLE) and ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) were used. The results showed that the red-fleshed apples contained, in either the flesh or peel, higher amounts of phenolic acids (chlorogenic acid), anthocyanins (cyanidin-3-O-galactoside), dihydrochalcones (phloretin xylosyl glucoside), and organic acids (malic acid) but a lower amount of flavan-3-ols than the white-fleshed apples. These quantitative differences could be related to an up-regulation of anthocyanins, dihydrochalcones, and malic acid and a down-regulation of flavan-3-ols (anthocyanin precursors) in both the flesh and peel of the red-fleshed apple varieties. The reported results should be considered preliminary because the complete phytochemical characterization of the red-fleshed apple cultivars will be extended to consecutive harvest seasons.

Optimisation and validation of analytical methods for the simultaneous extraction of antioxidants: application to the analysis of tomato sauces.

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.

Effect of the co-occurring components from olive oil and thyme extracts on the antioxidant status and its bioavailability in an acute ingestion in rats.

The aim of this work was to examine whether bioactives in thyme could enhance the antioxidant capacity of phenolics in virgin olive oil and their bioavailability in Wistar rats. After acute oral administration of extracts from olive cake (OE), thyme (TE) or their combination (OTE), blood samples were collected from 0 to 360 min. Plasma antioxidant status was analyzed by DPPH and FRAP in plasma and by SOD, CAT and GPx activities in erythrocytes. Plasma pharmacokinetics of the main metabolites of bioactives in olive oil and thyme were characterized. Plasma non-enzymatic antioxidant capacity was significantly modulated by OE, TE, and OTE in a time-, assay, and extract-dependent manner. OE, TE, and OTE all significantly decreased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity and catalase (CAT) activity was increased. Pharmacokinetic results showed that plasma concentration (Cmax) of the main olive phenolic metabolites in rats fed with OTE were similar to those of OE. These results indicate that an enhanced bioavailability of olive phenolic compounds could occur in the presence of thyme, although any synergistic effect was observed in the antioxidant status when both phenolic extracts were administered. Antioxidant protection by phenolics from olive and thyme against oxidative stress occurs primarily through a direct antioxidant effect and may be related to the phenolic plasmatic metabolites.

Effect of the co-occurring olive oil and thyme extracts on the phenolic bioaccessibility and bioavailability assessed by in vitro digestion and cell models.

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.

Recent advances in biologically active compounds in herbs and spices: a review of the most effective antioxidant and anti-inflammatory active principles.

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.

Distribution of procyanidins and their metabolites in rat plasma and tissues in relation to ingestion of procyanidin-enriched or procyanidin-rich cocoa creams.

BACKGROUND: Procyanidins are extensively metabolized via phase-II and microbial enzymes. However, their distribution in the body is not well characterized. AIM: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A new hydroxytyrosol metabolite identified in human plasma: hydroxytyrosol acetate sulphate.

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.

Impact of olive oil phenolic concentration on human plasmatic phenolic metabolites.

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 C(max) 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.

Plant-derived phenolics inhibit the accrual of structurally characterised protein and lipid oxidative modifications.

Epidemiological data suggest that plant-derived phenolics beneficial effects include an inhibition of LDL oxidation. After applying a screening method based on 2,4-dinitrophenyl hydrazine-protein carbonyl reaction to 21 different plant-derived phenolic acids, we selected the most antioxidant ones. Their effect was assessed in 5 different oxidation systems, as well as in other model proteins. Mass-spectrometry was then used, evidencing a heterogeneous effect on the accumulation of the structurally characterized protein carbonyl glutamic and aminoadipic semialdehydes as well as for malondialdehyde-lysine in LDL apoprotein. After TOF based lipidomics, we identified the most abundant differential lipids in Cu(++)-incubated LDL as 1-palmitoyllysophosphatidylcholine and 1-stearoyl-sn-glycero-3-phosphocholine. Most of selected phenolic compounds prevented the accumulation of those phospholipids and the cellular impairment induced by oxidized LDL. Finally, to validate these effects in vivo, we evaluated the effect of the intake of a phenolic-enriched extract in plasma protein and lipid modifications in a well-established model of atherosclerosis (diet-induced hypercholesterolemia in hamsters). This showed that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake.

Validation of determination of plasma metabolites derived from thyme bioactive compounds by improved liquid chromatography coupled to tandem mass spectrometry.

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 6h, 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 1h after the intake of the thyme extract.

Development of a phenol-enriched olive oil with both its own phenolic compounds and complementary phenols from thyme.

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.

Distribution of olive oil phenolic compounds in rat tissues after administration of a phenolic extract from olive cake.

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.

Distribution of procyanidins and their metabolites in rat plasma and tissues after an acute intake of hazelnut extract.

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.

Bioavailability of phenols from a phenol-enriched olive oil.

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.

Development of a phenol-enriched olive oil with phenolic compounds from olive cake.

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.

Rapid analysis of procyanidins and anthocyanins in plasma by microelution SPE and ultra-HPLC.

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.

Effect of the long-term regular intake of virgin olive oil on the phenolic metabolites in human fasting plasma.

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 (P<0.05). The results suggest an indirect protective mechanism of long-term regular virgin olive oil consumption related to the protection of the endogenous antioxidant system.