ABSTRACT
The Mediterranean will be one of the focal points of climate change. The predicted dry and hot summers will lead to water scarcity in agriculture, which may limit crop production and growth. The olive tree serves as a model woody plant for studying drought stress and improving water resource management; thus, it is critical to identify genotypes that are more drought tolerant and perform better under low irrigation or even rainfed conditions. In this study, the metabolomic approach was used to highlight variations in metabolites in stems and leaves of three Italian olive cultivars (previously characterized physiologically) under two and four weeks of drought stress. Phenolic and lipophilic profiles were obtained by gas chromatography-mass spectrometry and ultra-high performance liquid chromatography-mass spectrometry, respectively. The findings identified the leaf as the primary organ in which phenolic variations occurred. The Maurino cultivar exhibited a strong stress response in the form of phenolic compound accumulation, most likely to counteract oxidative stress. The phenolic compound content of 'Giarraffa' and 'Leccino' plants remained relatively stable whether they were exposed to drought or not. Variations in the lipid profile occurred in leaves and stems of all the cultivars. A high accumulation of compounds related to epicuticular wax components was observed in the leaf of 'Giarraffa', while a strong reduction of lipids and long-chain alkanes occurred in 'Maurino' when exposed to drought stress conditions.
ABSTRACT
The sweet chestnut (Castanea sativa Mill.) is subject to the progressive disappearance of its traditional chestnut groves. In the northern part of Italy, where distribution of the sweet chestnut is fragmented, many local varieties continue to be identified mostly by oral tradition. We characterised by SSRs eleven historically recognised varieties of sweet chestnut in the area surrounding Lake Como, with the goal of giving a genetic basis to the traditional classification. We performed classical analysis about differentiation and used Bayesian approaches to detect population structure and to reconstruct demography. The results revealed that historical and genetic classifications are loosely linked when chestnut fruits are just "castagne", that is, normal fruits, but increasingly overlap where "marroni" (the most prized fruits) are concerned. Bayesian classification allowed us to identify a homogeneous gene cluster not recognised in the traditional assessment of the varieties and to reconstruct possible routes used for the propagation of sweet chestnut. We also reconstructed ancestral relationships between the different gene pools involved and dated ancestral lineages whose results fit with palynological data. We suggest that conservation strategies based on a genetic evaluation of the resource should also rely on traditional cultural heritage, which could reveal new sources of germplasm.
Subject(s)
Fagaceae , Fagaceae/genetics , Fagaceae/classification , Italy , Microsatellite Repeats/genetics , Bayes Theorem , PhylogenyABSTRACT
Olive leaves are an abundant by-product of olive oil production. Olive leaf extracts (OLEs) are rich in polyphenols, which can be used for health benefits. As polyphenols are the main antioxidant molecules in plants, plants typically increase their polyphenol content when exposed to drought stress. However, the phenolic profile of OLEs can vary in relation to the origin and variety of the plant material. In this work, olive leaf extracts from three different Italian olive cultivars (Giarraffa, Leccino, and Maurino) both exposed and not exposed to drought stress were studied in terms of antioxidant properties and profile, intestinal permeation, and protection against oxidative stress of human umbilical vein endothelial cells (HUVECs), since HUVECs are considered a model to study a wide range of diseases. OLEs from stressed Maurino and Giarraffa plants showed the highest increase in antioxidant capacity compared to controls. The phenolic profile of Maurino' was mainly increased by water deficit, with a large increase in the compounds oleuropein and luteolin-7-O-rutinoside. All tested extracts exposed to a water deficit protected HUVECs against oxidative stress by reducing ROS production, and this effect was more pronounced in OLEs from Giarraffa and Maurino exposed to drought stress compared to all other extracts. Finally, OLE from the stressed Giarraffa group showed a higher apparent permeability of antioxidant molecules than that of Maurino.
ABSTRACT
The development of spectral sensors (SSs) capable of retrieving spectral information have opened new opportunities to improve several environmental and agricultural practices, e.g., crop breeding, plant phenotyping, land use monitoring, and crop classification. The SSs are classified as multispectral and hyperspectral (HS) based on the number of the spectral bands resolved and sampled during data acquisition. Large-scale applications of the HS remain limited due to the cost of this type of technology and the technical difficulties in hyperspectral data processing. Low-cost portable hyperspectral cameras (PHCs) have been progressively developed; however, critical aspects associated with data acquisition and processing, such as the presence of spectral discontinuities, signal jumps, and a high level of background noise, were reported. The aim of this work was to analyze and improve the hyperspectral output of a PHC Senop HSC-2 device by developing a general use methodology. Several signal gaps were identified as falls and jumps across the spectral signatures near 513, 650, and 930 nm, while the dark current signal magnitude and variability associated with instrumental noise showed an increasing trend over time. A data correction pipeline was successfully developed and tested, leading to 99% and 74% reductions in radiance signal jumps identified at 650 and 830 nm, respectively, while the impact of noise on the acquired signal was assessed to be in the range of 10% to 15%. The developed methodology can be effectively applied to other low-cost hyperspectral cameras.
ABSTRACT
The aim of this research was to evaluate the levels of antioxidants and polyphenols in pasta enriched with either carrot or olive leaf flours after simulating gastrointestinal digestion. Pasta samples were prepared with fixed amounts of carrot and olive leaf flours (15% and 6% of the total mixture, respectively). We measured the antioxidant capacity and polyphenol content at different stages of the pasta production process, starting from the initial flour to the cooked pasta, and tested samples of the liquid component and solid waste resulting from the digestion process. The antioxidant activity was measured by the FRAP method, while the polyphenol content was measured by the Folin-Ciocalteu method. Vitamin E contents were measured by HPLC. The pasta enriched with carrot (1.26 ± 0.05 mmol/100 g) and olive leaf (2.9 ± 0.07 mmol/100 g) exhibited higher antioxidant power compared to the unenriched pasta (0.8 ± 0.1 mmol/100 g). The polyphenol content followed a similar trend, with values of 131.23 ± 3.08 for olive flour-enriched pasta, 79.15 ± 1.11 for carrot flour-enriched pasta, and 67.5 ± 1.39 for the wheat-only pasta. The pasta samples maintained their antioxidant and polyphenol levels even after undergoing the simulated digestion process. Significantly, the liquid component of the pasta with olive leaf flours had the highest levels of antioxidants and polyphenols during all stages of the digestion process. According to the results of this study, pasta enriched with carrot and olive leaf flours shows promising potential for improving nutritional and functional properties by increasing antioxidant and polyphenol content. The samples were also evaluated by a sensory panel, which showed that fortification modified the perception of some organoleptic attributes without affecting the overall taste of the pasta.
ABSTRACT
Olive drupe traits (i.e., ripening index and pericarp water content) and minor components (i.e., phenols and pigments in both fruit and oil) are important for human health and are affected by agronomic background. The aim of this study was to investigate the relationship between fruit traits, phenols, and pigments in samples derived from different soil and water management practices. Chromatographic (UHPLC-MS/MS) and spectroscopic (1HNMR and near UV-Vis spectroscopy) techniques were employed for the characterization of olive fruits and oils. The use of various techniques allowed the identification of interesting trace compounds. We observed that most of the fruit phenols (a total of 29 compounds) were correlated with the degree of ripening: most of the phenolic acids (and their derivatives), phenolic alcohols, and secoiridoids were negatively correlated, whereas the majority of the studied flavonoids were positively correlated. The relationship between the ripening index and fruit phenolic compounds appears to be dependent on the metabolic pathway that controls the synthesis of each individual compound. Conversely, the secoiridoids and pigments in olive oil showed a negative correlation with pulp moisture, probably because of the influence of the water content on the extractability and transfer in the oil phase of these minor components.
Subject(s)
Olea , Humans , Olea/chemistry , Fruit/chemistry , Iridoids/analysis , Tandem Mass Spectrometry , Alcohols/analysis , Flavonoids/analysis , Water/analysis , Olive Oil/chemistry , Phenols/chemistry , Plant Oils/chemistryABSTRACT
Recent developments in low-cost imaging hyperspectral cameras have opened up new possibilities for high-throughput phenotyping (HTP), allowing for high-resolution spectral data to be obtained in the visible and near-infrared spectral range. This study presents, for the first time, the integration of a low-cost hyperspectral camera Senop HSC-2 into an HTP platform to evaluate the drought stress resistance and physiological response of four tomato genotypes (770P, 990P, Red Setter and Torremaggiore) during two cycles of well-watered and deficit irrigation. Over 120 gigabytes of hyperspectral data were collected, and an innovative segmentation method able to reduce the hyperspectral dataset by 85.5% was developed and applied. A hyperspectral index (H-index) based on the red-edge slope was selected, and its ability to discriminate stress conditions was compared with three optical indices (OIs) obtained by the HTP platform. The analysis of variance (ANOVA) applied to the OIs and H-index revealed the better capacity of the H-index to describe the dynamic of drought stress trend compared to OIs, especially in the first stress and recovery phases. Selected OIs were instead capable of describing structural changes during plant growth. Finally, the OIs and H-index results have revealed a higher susceptibility to drought stress in 770P and 990P than Red Setter and Torremaggiore genotypes.
ABSTRACT
An innovative low-cost device based on hyperspectral spectroscopy in the near infrared (NIR) spectral region is proposed for the non-invasive detection of moldy core (MC) in apples. The system, based on light collection by an integrating sphere, was tested on 70 apples cultivar (cv) Golden Delicious infected by Alternaria alternata, one of the main pathogens responsible for MC disease. Apples were sampled in vertical and horizontal positions during five measurement rounds in 13 days' time, and 700 spectral signatures were collected. Spectral correlation together with transmittance temporal patterns and ANOVA showed that the spectral region from 863.38 to 877.69 nm was most linked to MC presence. Then, two binary classification models based on Artificial Neural Network Pattern Recognition (ANN-AP) and Bagging Classifier (BC) with decision trees were developed, revealing a better detection capability by ANN-AP, especially in the early stage of infection, where the predictive accuracy was 100% at round 1 and 97.15% at round 2. In subsequent rounds, the classification results were similar in ANN-AP and BC models. The system proposed surpassed previous MC detection methods, needing only one measurement per fruit, while further research is needed to extend it to different cultivars or fruits.
Subject(s)
Malus , Fruit/chemistry , Malus/chemistry , Neural Networks, ComputerABSTRACT
The demand for chestnut flour is growing because of its use in gluten-free products. Previous studies have correlated the quality of chestnut flours to the drying temperature and technology applied. This work is a novel study on the role of the traditional drying method with a wood fire in a "metato" building for flour compared with a food dryer at 40 °C or 70 °C. The contents of antioxidants, total polyphenols and sugars were determined as well as the presence of toxic volatiles or aflatoxins. The flour, resulting from the traditional method, presented lower polyphenol content and antioxidant power compared to the others. The content of the sugars was similar to the flours obtained after drying with hot air, both at 40 °C and 70 °C. The toxic volatile molecules, furfural, guaiacol, and o-cresol, were found. There was no correlation between the aflatoxin content and the presence of damage in chestnut fruits. The traditional method should not be abandoned since it confers a pleasant smoky taste to the product, but it is necessary to regulate the level and steadiness of temperature. Future research needs to be directed to the quantification of harmful volatile compounds and their correlation with the quantity of smoke emitted by the wood fire.
ABSTRACT
Future climate scenarios suggest that crop plants will experience environmental changes capable of affecting their productivity. Among the most harmful environmental stresses is drought, defined as a total or partial lack of water availability. It is essential to study and understand both the damage caused by drought on crop plants and the mechanisms implemented to tolerate the stress. In this study, we focused on four cultivars of tomato, an economically important crop in the Mediterranean basin. We investigated the biochemical mechanisms of plant defense against drought by focusing on proteins specifically involved in this stress, such as osmotin, dehydrin, and aquaporin, and on proteins involved in the general stress response, such as HSP70 and cyclophilins. Since sugars are also known to act as osmoprotectants in plant cells, proteins involved in sugar metabolism (such as RuBisCO and sucrose synthase) were also analyzed. The results show crucial differences in biochemical behavior among the selected cultivars and highlight that the most tolerant tomato cultivars adopt quite specific biochemical strategies such as different accumulations of aquaporins and osmotins. The data set also suggests that RuBisCO isoforms and aquaporins can be used as markers of tolerance/susceptibility to drought stress and be used to select tomato cultivars within breeding programs.
Subject(s)
Aquaporins , Solanum lycopersicum , Droughts , Solanum lycopersicum/physiology , Plant Breeding , Plants , Ribulose-Bisphosphate CarboxylaseABSTRACT
The impact of dried apples (Malus × domestica Borkh.) addition on improving the antioxidant characteristics of dark chocolate was evaluated. The antioxidant activity was measured through DPPH scavenging activity and showed an increase in the cocoa bar with 'Nesta' dry apple (17.3% vs. 46.8%) in comparison to cocoa mass. The 15 polyphenols analyzed by UHPLC-ESI-MS/MS indicated great variability among the apple varieties. Quercetin was detected in the highest concentrations (ranged from 753.3 to 1915.5 µg g-1), while the lowest were for kaempferol 7-O-glucoside, measured only in 'Mora' and 'Nesta' cocoa bars (from 0.034 to 0.069 µg g-1, respectively). P-coumaric acid, trans-ferulic acid, and chlorogenic acid contribute largely to the antioxidant activity in cocoa bars. Principal component analysis shows that a cocoa bar with the addition of 'Nesta' dry apple differ from others due to its higher content of polyphenols (1614 ± 61.8 mg gallic acid equivalents per 100 g). In conclusion, data confirm that cocoa bars with dry apples might be considered as a polyphenol-enriched food.
ABSTRACT
The depletion of the stratospheric ozone layer due to natural and/or anthropogenic causes decreases the amount of UV-B radiation filtered, and consequently increases the risk of potential damage to organisms. In the Mediterranean region, high UV-B indices are frequent. Even for species typical of this region, such as the olive tree, the progressive increase in UV-B radiation represents a threat. This work aimed to understand how high UV-B radiation modulates the phenolic and lipophilic profile of olive varieties, and identify metabolites that enhance olive stress tolerance. Two Italian olive varieties were subjected to chronic UV-B stress, and leaves were analyzed by gas and liquid chromatography. The results indicated that the most representative phenolic and lipophilic compounds of Giarraffa and Olivastra Seggianese were readjusted in response to UV-B stress. The Giarraffa variety seemed better suited to prolonged UV-B stress, possibly due to the higher availability of flavonoids that could help control oxidative damage, and the accumulation of hydroxycinnamic acid derivatives that could provide strong UV-B shield protection. In addition, this variety contained higher levels of fatty acids (e.g., palmitic, α-linolenic, and stearic acids), which can help to maintain membrane integrity and accumulate more sorbitol (which may serve as an osmoprotectant or act a free-radical scavenger), terpenes, and long-chain alkanes, providing higher protection against UV-B stress.
ABSTRACT
BACKGROUND: This study aims to evaluate the performance, in terms of accumulation of antioxidant compounds in fruits, of nine local and three commercial Italian tomato cultivars subjected to drought stress. The same local cultivars had been previously studied at morpho-physiological level. METHODS: The present manuscript analyzes drought stress as a tool to increase the amount of secondary metabolites that can enhance fruit quality. Nutraceutical characterization of the fruits was performed by analyzing the content of antioxidants, phenols, flavonoids, lycopene, ascorbic acid (vitamin C), rutin, caffeic acid, and naringenin. At the same time, plant sensitivity to stress during the reproductive phase was monitored in terms of flower abscission, fruit drop, and seed germination. RESULTS: Perina turns out to be the tomato cultivar with the best nutraceutical properties in the absence of stress while the Quarantino cultivar is so for flavonoid content (control plants) and lycopene and vitamin C content (stressed plants). Perina and Quarantino are the cultivars with the best response to drought and Perina has the highest concentrations of bioactives. Quarantino responds most effectively to stress in the reproductive phase. CONCLUSIONS: data confirm that drought stress increases bioactive production in some local cultivars of tomato, which produce higher quality fruits.
ABSTRACT
In recent decades, atmospheric pollution led to a progressive reduction of the ozone layer with a consequent increase in UV-B radiation. Despite the high adaptation of olive trees to the Mediterranean environment, the progressive increase of UV-B radiation is a risk factor for olive tree cultivation. It is therefore necessary to understand how high levels of UV-B radiation affect olive plants and to identify olive varieties which are better adapted. In this study we analyzed two Italian olive varieties subjected to chronic UV-B stress. We focused on the effects of UV-B radiation on RubisCO, in terms of quantity, enzymatic activity and isoform composition. In addition, we also analyzed changes in the activity of antioxidant enzymes (SOD, CAT, GPox) to get a comprehensive picture of the antioxidant system. We also evaluated the effects of UV-B on the enzyme sucrose synthase. The overall damage at biochemical level was also assessed by analyzing changes in Hsp70, a protein triggered under stress conditions. The results of this work indicate that the varieties (Giarraffa and Olivastra Seggianese) differ significantly in the use of specific antioxidant defense systems, as well as in the activity and isoform composition of RubisCO. Combined with a different use of sucrose synthase, the overall picture shows that Giarraffa optimized the use of GPox and opted for a targeted choice of RubisCO isoforms, in addition to managing the content of sucrose synthase, thereby saving energy during critical stress points.
Subject(s)
Antioxidants/metabolism , Olea/metabolism , Olea/radiation effects , Plant Proteins/metabolism , Ribulose-Bisphosphate Carboxylase/metabolism , Enzymes/metabolism , Glucosyltransferases/metabolism , HSP70 Heat-Shock Proteins/metabolism , Italy , Lipid Peroxidation/radiation effects , Malondialdehyde/metabolism , Microscopy, Electron, Transmission , Olea/cytology , Plant Leaves/cytology , Plant Leaves/radiation effects , Ultraviolet RaysABSTRACT
Irrigation is fundamental for agriculture but, as climate change becomes more persistent, there is a need to conserve water and use it more efficiently. It is therefore crucial to identify cultivars that can tolerate drought. For economically relevant crops, such as tomatoes, this purpose takes on an even more incisive role and local agrobiodiversity is a large genetic reservoir of promising cultivars. In this study, nine local Italian cultivars of tomatoes plus four widely used commercial cultivars were considered. These experienced about 20 d of drought, either at vegetative or reproductive phase. Various physio-morphological parameters were monitored, such as stomatal conductance (gs), photosynthesis (A), water use efficiency (WUE), growth (GI) and soil water content (SWC). The different responses and behaviors allowed to divide the cultivars into three groups: tolerant, susceptible, and intermediate. The classification was also confirmed by a principal component analysis (PCA). The study, in addition to deepening the knowledge of local Italian tomato cultivars, reveals how some cultivars perform better under stress condition than commercial ones. Moreover, the different behavior depends on the genotype and on the growth phase of plants. In fact, the Perina cultivar is the most tolerant during vegetative growth while the Quarantino cultivar is mostly tolerant at reproductive stage. The results suggest that selection of cultivars could lead to a more sustainable agriculture and less wasteful irrigation plans.
ABSTRACT
BACKGROUND: Tomato by-products contain a great variety of biologically active substances and represent a significant source of natural antioxidant supplements of the human diet. The aim of the work was to compare the antioxidant properties of a by-product from an ancient Tuscan tomato variety, Rosso di Pitigliano (RED), obtained by growing plants in normal conditions (-Ctr) or in drought stress conditions (-Ds) for their beneficial effects on vascular related dysfunction. METHODS: The antioxidant activity and total polyphenol content (TPC) were measured. The identification of bioactive compounds of tomato peel was performed by HPLC. HUVEC were pre-treated with different TPC of RED-Ctr or RED-Ds, then stressed with H2O2. Cell viability, ROS production and CAT, SOD and GPx activities were evaluated. Permeation of antioxidant molecules contained in RED across excised rat intestine was also studied. RESULTS: RED-Ds tomato peel extract possessed higher TPC than compared to RED-Ctr (361.32 ± 7.204 mg vs. 152.46 ± 1.568 mg GAE/100 g fresh weight). All extracts were non-cytotoxic. Two hour pre-treatment with 5 µg GAE/mL from RED-Ctr or RED-Ds showed protection from H2O2-induced oxidative stress and significantly reduced ROS production raising SOD and CAT activity (* p < 0.05 and ** p < 0.005 vs. H2O2, respectively). The permeation of antioxidant molecules contained in RED-Ctr or RED-Ds across excised rat intestine was high with non-significant difference between the two RED types (41.9 ± 9.6% vs. 26.6 ± 7.8%). CONCLUSIONS: RED-Ds tomato peel extract represents a good source of bioactive molecules, which protects HUVECs from oxidative stress at low concentration.
Subject(s)
Antioxidants/pharmacology , Endothelial Cells/drug effects , Plant Extracts/pharmacology , Polyphenols/pharmacology , Solanum lycopersicum/chemistry , Antioxidants/chemistry , Droughts , Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Solanum lycopersicum/physiology , Oxidative Stress/drug effects , Plant Extracts/chemistry , Polyphenols/chemistry , Stress, PhysiologicalABSTRACT
Sweet cherry (Prunus avium L.) is a stone fruit widely consumed and appreciated for its organoleptic properties, as well as its nutraceutical potential. We here investigated the characteristics of six non-commercial Tuscan varieties of sweet cherry maintained at the Regional Germplasm Bank of the CNR-IBE in Follonica (Italy) and sampled ca. 60 days post-anthesis over three consecutive years (2016-2017-2018). We adopted an approach merging genotyping and targeted gene expression profiling with metabolomics. To complement the data, a study of the soluble proteomes was also performed on two varieties showing the highest content of flavonoids. Metabolomics identified the presence of flavanols and proanthocyanidins in highest abundance in the varieties Morellona and Crognola, while gene expression revealed that some differences were present in genes involved in the phenylpropanoid pathway during the 3 years and among the varieties. Finally, proteomics on Morellona and Crognola showed variations in proteins involved in stress response, primary metabolism and cell wall expansion. To the best of our knowledge, this is the first multi-pronged study focused on Tuscan sweet cherry varieties providing insights into the differential abundance of genes, proteins and metabolites.
ABSTRACT
Given the economical importance of the olive tree it is essential to study its responses to stress agents such as excessive UV-B radiation, to understand the defense mechanisms and to identify the varieties that are able to cope with it. In the light of the analysis carried out in this study, we argue that UV-B radiation represents a dangerous source of stress for the olive tree, especially in the current increasingly changing environmental conditions. Both the varieties considered (Giarraffa and Olivastra Seggianese), although resistant to the strong treatment to which they were exposed, showed, albeit in different ways and at different times, evident effects. The two varieties have different response times and the Giarraffa variety seems better suited to prolonged UV-B stress, possible due to a more efficient and quick activation of the antioxidant response (e.g., flavonoids use to counteract reactive oxygen species) and because of its capacity to maintain the photosynthetic efficiency as well as a relatively higher content of mannitol. Moreover, pigments reduction after a long period of UV-B exposure can also be an adaptation mechanism triggered by Giarraffa to reduce energy absorption under UV-B stress. Olivastra Seggianese seems less suited to overcome UV-B stress for a long period (e.g., higher reduction of Fv/Fm) and has a higher requirement for sugars (e.g., glucose) possible to counteract stress and to restore energy.
ABSTRACT
Drought stress is one of the most severe conditions for plants, especially in the face of the emerging problem of global warming. This issue is important when considering economically relevant crops, including the tomato. For these plants, a promising solution is the valorization of local agrobiodiversity as a source of genetic variability. In this paper we investigated how six Italian tomato varieties react to a prolonged period of water depletion. We used a multidisciplinary approach, from genetics to plant physiology and cytology, to provide a detailed overview of the response of plants to stress. The varieties analyzed, each characterized by a specific genetic profile, showed a genotype-specific response with the variety 'Fragola' being the most resistant and the variety 'Pisanello' the most susceptible. For all the parameters evaluated, 'Fragola' performed in a manner comparable to that of control plants. On the contrary, 'Pisanello' appeared to be more affected and showed an increase in the number of stomata and a drastic increase in antioxidants, a symptom of acute oxidative stress. Our work suggests the existence of a valuable reservoir of genetic biodiversity with more drought-tolerant tomato genotypes opening the way to further exploitation and use of local germplasm in breeding programs.
ABSTRACT
BACKGROUND: Endothelial dysfunction has been associated to cardiovascular outcomes in patients with cardiovascular risk factors. Circulating endothelial progenitor cells (EPCs) play an important physiological role for their reparative potential of vascular integrity, but are numerically reduced and functionally impaired in patients with cardiovascular risks. This study assesses the effects of Extra Virgin Olive Oil (EVOO) and apple-enriched dark chocolate intake on the blood levels of EPCs. Methods: Thirty volunteers with cardiovascular risk factors, enrolled in a randomised, crossover, four-weeks trial, received a solid dark chocolate bar (40 g/day) containing 10% EVOO or 2.5% dry apples. Urine samples were analyzed for endogenous metabolites. Circulating EPCs levels, clinical data and anthropometric examinations were collected. RESULTS: 26 volunteers (M/F:14/12, 51 ± 9 years of age) completed the study. Comparison of pre-post intervention revealed a significant increase in EPCs levels associated with EVOO-dark chocolate consumption. Most biochemical parameters were not significantly modified by both chocolates. CONCLUSIONS: This study shows that a daily consumption of a non fattening dose of dark chocolate enriched with EVOO improves blood levels of EPCs, a well known surrogate biologic marker for endothelial function.