Bioavailability of phenolics from an oleuropein-rich olive (Olea europaea) leaf extract and its acute effect on plasma antioxidant status: comparison between pre- and postmenopausal women.

PURPOSE: Preclinical studies suggest a potential protective effect of oleuropein in osteoporosis, and one of the proposed mechanisms is the modulation of the oxidative stress. Oleuropein bioavailability and its effect on antioxidant status in pre- and postmenopausal women are unknown. The aim of the present study was to investigate the oral bioavailability of an olive leaf extract rich in oleuropein (40 %) and its effect on antioxidant status in postmenopausal women compared to premenopausal women. METHODS: Premenopausal (n = 8) and postmenopausal women (n = 8) received 250 mg of olive leaf extract, blood samples (t = 0, 1, 2, 3, 4, 6, 8, 12, 16 and 24 h) were taken, and 24-h urine divided into five fractions was collected. Olive-leaf-extract-derived metabolites were analyzed in plasma and urine by HPLC-ESI-QTOF and UPLC-ESI-QqQ, and pharmacokinetics parameters were determined. Ferric reducing antioxidant ability and malondialdehyde levels were measured in plasma. RESULTS: Plasma levels of hydroxytyrosol glucuronide, hydroxytyrosol sulfate, oleuropein aglycon glucuronide and oleuropein aglycon derivative 1 were higher in postmenopausal women. MDA levels were significantly decreased (32%) in postmenopausal women and inversely correlated with hydroxytyrosol sulfate levels. Postmenopausal women excreted less sulfated metabolites in urine than premenopausal women. CONCLUSIONS: Our results suggest that postmenopausal women could be a target population for the intake of olive phenolics in order to prevent age-related and oxidative stress-related processes such as osteoporosis.

How Diet and Lifestyle Can Fine-Tune Gut Microbiomes for Healthy Aging.

Many physical, social, and psychological changes occur during aging that raise the risk of developing chronic diseases, frailty, and dependency. These changes adversely affect the gut microbiota, a phenomenon known as microbe-aging. Those microbiota alterations are, in turn, associated with the development of age-related diseases. The gut microbiota is highly responsive to lifestyle and dietary changes, displaying a flexibility that also provides anactionable tool by which healthy aging can be promoted. This review covers, firstly, the main lifestyle and socioeconomic factors that modify the gut microbiota composition and function during healthy or unhealthy aging and, secondly, the advances being made in defining and promoting healthy aging, including microbiome-informed artificial intelligence tools, personalized dietary patterns, and food probiotic systems.

4-Hydroxydibenzyl: a novel metabolite from the human gut microbiota after consuming resveratrol.

Resveratrol (RSV) was known to be metabolised by the gut microbiota to dihydroresveratrol, lunularin (LUNU), and (or) 3,4'-dihydroxy-trans-stilbene (DHST). We describe here for the first time that LUNU can be further dehydroxylated, but only at the 3-position, to yield 4-hydroxydibenzyl, a novel metabolite found in human urine after RSV intake in 41 out of 59 healthy participants. In contrast, DHST was not further dehydroxylated, and thus, 4-hydroxy-trans-stilbene was not detected as a gut microbial metabolite of RSV. Faecal in vitro incubations confirmed the in vivo results.

Lack of effect of oral administration of resveratrol in LPS-induced systemic inflammation.

PURPOSE: The high mortality index due to sepsis and the lack of an effective treatment requires the search for new compounds that can serve as therapy for this disease. Resveratrol, a well-known anti-inflammatory natural compound, might be a good candidate for the treatment of sepsis. The aim of this work was to study the effects of oral administration of resveratrol, before and after sepsis initiation, on inflammation markers in a murine model of endotoxin-induced sepsis. METHODS: Sprague-Dawley male rats were treated with resveratrol the 3 days prior to LPS administration and 45 min later. Hematological parameters, TNF-α, IL-1ß and CINC-1, FRAP and TBARS levels were determined. Resveratrol and resveratrol-derived metabolites profile in plasma was compared after oral and intraperitoneal administration. RESULTS: Oral treatment with resveratrol had no apparent systemic protective effects. However, resveratrol reduced the levels of lipid peroxidation in the small intestine and colon. Importantly, the administration of LPS caused a decrease in resveratrol absorption. When resveratrol bioavailability after i.p. administration was compared to that observed after oral administration, a different profile of resveratrol metabolites was found in plasma. CONCLUSION: These results highlight the importance of studying the bioavailability of the assayed compounds in the experimental models used to be able to choose the best route of administration depending on the target organ and to determine which compounds or derived metabolites are effective treating the studied disease.

Re-examining the role of the gut microbiota in the conversion of the lipid-lowering statin monacolin K (lovastatin) into its active ß-hydroxy acid metabolite.

Monacolin K (MK, lovastatin), a naturally occurring statin, only exerts lipid-lowering effects in its active ß-hydroxy acid form (MKA). This activation was thought to be mediated by the gut microbiota (GM). We report here for the first time that the GM does not convert MK into MKA (a spontaneous pH-dependent conversion) but catabolises MKA. The GM might hamper the lipid-lowering effects by degrading the active metabolite MKA.

Tissue deconjugation of urolithin A glucuronide to free urolithin A in systemic inflammation.

Urolithin A (Uro-A) is an anti-inflammatory and cancer chemopreventive metabolite produced by the gut microbiota from the polyphenol ellagic acid. However, in vivo conjugation of Uro-A to Uro-A glucuronide (Uro-A glur) dramatically hampers its activity. We describe here for the first time the tissue deconjugation of Uro-A glur to Uro-A after lipopolysaccharide (LPS)-induced inflammation, which could explain the systemic in vivo activity of free Uro-A in microenvironments subjected to inflammatory stimuli.

The gut microbiota urolithin metabotypes revisited: the human metabolism of ellagic acid is mainly determined by aging.

Understanding individuals' response to dietary bioactives is crucial for personalized nutrition. We report here for the first time in a Caucasian cohort (5-90 years, n = 839) that aging is the main factor that determines the gut microbiota involved in the ellagic acid-ellagitannin metabolism (urolithin metabotypes), with potential consequences for human health.

Consumption of pomegranate decreases plasma lipopolysaccharide-binding protein levels, a marker of metabolic endotoxemia, in patients with newly diagnosed colorectal cancer: a randomized controlled clinical trial.

Gut microbiota dysbiosis alters the intestinal barrier function, increases plasma lipopolysaccharide (LPS) levels, which promotes endotoxemia, and contributes to the onset and development of colorectal cancer (CRC). We report here for the first time the reduction of plasma LPS-binding protein (LBP) levels, a marker of endotoxemia, after pomegranate consumption in newly diagnosed CRC patients.

Effect of mediterranean forest parasite with Curculio sp. on nutritional value of acorn for Iberian pig feeding and fat characteristics.

Sixteen Iberian barrows of the same age with an average initial live weight of 100.1kg were randomly distributed in two groups of eight pigs each. One group was fed healthy acorns and the other group received acorns infested of Curculio sp. The subcutaneous backfat from pigs fed healthy acorns had higher C18:1n-9, MUFA and C20:5n-3 and lower C18:0 and SFA proportions than that from the pigs fed acorns infested with Curculio. The consumption of acorns infested with Curculio sp. led to a reduction of C18:1n-9, MUFA, C18:2n-6, C18:3n-3, C22:5n-3 and PUFA proportions in neutral lipids from Longissimus dorsi muscle with respect to consumption of healthy acorns, whereas in polar lipids it produced a reduction in C18:1n-9, MUFA and C18:4n-3 proportions and an increase in C18:2n-6, C20:4n-6, n-6 and C20:5n-3 proportions and of n-6/n-3 ratio with respect to the healthy acorns consumption. The pigs fed healthy acorns had higher intramuscular fat percentage in Longissimus dorsi than pigs fed with acorns infested with Curculio (9.95 vs 7.09% SEM=0.60).

Pomegranate juice supplementation in chronic obstructive pulmonary disease: a 5-week randomized, double-blind, placebo-controlled trial.

OBJECTIVE: The aim of the present study is to investigate the effect of antioxidant polyphenol-rich pomegranate juice (PJ) supplementation for 5 weeks on patients with stable chronic obstructive pulmonary disease (COPD), since the oxidative stress plays a major role in the evolution and pathophysiology of COPD. DESIGN: A randomized, double-blind, placebo-controlled trial was conducted. SUBJECTS: A total of 30 patients with stable COPD were randomly distributed in two groups (15 patients each). INTERVENTIONS: Both groups consumed either 400 ml PJ daily or matched placebo (synthetic orange-flavoured drink) for 5 weeks. Trolox Equivalent Antioxidant Capacity (TEAC) of PJ, blood parameters (14 haematological and 18 serobiochemical), respiratory function variables, bioavailability of PJ polyphenols (plasma and urine) and urinary isoprostane (8-iso-PGF(2alpha)) were evaluated. RESULTS: The daily dose of PJ (containing 2.66 g polyphenols) provided 4 mmol/l TEAC. None of the polyphenols present in PJ were detected in plasma or in urine of volunteers. The most abundant PJ polyphenols, ellagitannins, were metabolized by the colonic microflora of COPD patients to yield two major metabolites in both plasma and urine (dibenzopyranone derivatives) with no TEAC. No differences were found (P > 0.05) between PJ and placebo groups for any of the parameters evaluated (serobiochemical and haematological), urinary 8-iso-PGF(2alpha), respiratory function variables and clinical symptoms of COPD patients. CONCLUSIONS: Our results suggest that PJ supplementation adds no benefit to the current standard therapy in patients with stable COPD. The high TEAC of PJ cannot be extrapolated in vivo probably due to the metabolism of its polyphenols by the colonic microflora. The understanding of the different bioavailability of dietary polyphenols is critical before claiming any antioxidant-related health benefit. SPONSORSHIP: 'Fundación Séneca' (Murcia, Spain), Project PB/18/FS/02 and Spanish CICYT, Project AGL2003-02195.

Effect of bovine ABCG2 polymorphism Y581S SNP on secretion into milk of enterolactone, riboflavin and uric acid.

The ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) is an efflux protein involved in the bioavailability and milk secretion of endogenous and exogenous compounds, actively affecting milk composition. A limited number of physiological substrates have been identified. However, no studies have reported the specific effect of this polymorphism on the secretion into milk of compounds implicated in milk quality such as vitamins or endogenous compounds. The bovine ABCG2 Y581S polymorphism is described as a gain-of-function polymorphism that increases milk secretion and decreases plasma levels of its substrates. This work aims to study the impact of Y581S polymorphism on plasma disposition and milk secretion of compounds such as riboflavin (vitamin B2), enterolactone, a microbiota-derived metabolite from the dietary lignan secoisolariciresinol and uric acid. In vitro transport of these compounds was assessed in MDCK-II cells overexpressing the bovine ABCG2 (WT-bABCG2) and its Y581S variant (Y581S-bABCG2). Plasma and milk levels were obtained from Y/Y homozygous and Y/S heterozygous cows. The results show that riboflavin was more efficiently transported in vitro by the Y581S variant, although no differences were noted in vivo. Both uric acid and enterolactone were substrates in vitro of the bovine ABCG2 variants and were actively secreted into milk with a two-fold increase in the milk/plasma ratio for Y/S with respect to Y/Y cows. The in vitro ABCG2-mediated transport of the drug mitoxantrone, as a model substrate, was inhibited by enterolactone in both variants, suggesting the possible in vivo use of this enterolignan to reduce ABCG2-mediated milk drug transfer in cows. The Y581S variant was inhibited to a lesser extent probably due to its higher transport capacity. All these findings point to a significant role of the ABCG2 Y581S polymorphism in the milk disposition of enterolactone and the endogenous molecules riboflavin and uric acid, which could affect both milk quality and functionality.

Effect of captopril on mushroom tyrosinase activity in vitro.

The study presented here demonstrates that the antihypertensive drug captopril ([2S]-N-[3-mercapto-2-methylpropionyl]-L-proline) is an irreversible non-competitive inhibitor and an irreversible competitive inhibitor of the monophenolase and diphenolase activities of mushroom tyrosinase when L-tyrosine and L-DOPA were assayed spectrophotometrically in vitro, respectively. Captopril was rendered unstable by tyrosinase catalysis because of the interaction between the enzymatic-generated product (o-quinone) and captopril to give rise to a colourless conjugate. Therefore, captopril was able to prevent melanin formation. The spectrophotometric recordings of the inhibition of tyrosinase by captopril were characterised by the presence of a lag period prior to the attainment of an inhibited steady state rate. The lag period corresponded to the time in which captopril was reacting with the enzymatically generated o-quinone. Increasing captopril concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady state rate were dependent of captopril, substrate and tyrosinase concentrations. The inhibition of both monophenolase and diphenolase activities of tyrosinase by captopril showed positive kinetic co-operativity which arose from the protection of both substrate and o-quinone against inhibition by captopril. Inhibition experiments carried out using a latent mushroom tyrosinase demonstrated that captopril only bound the enzyme at its active site. The presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition. This could be due to the formation of both copper-captopril complex and disulphide interchange reactions between captopril and cysteine rich domains at the active site of the enzyme.

Slow-binding inhibition of mushroom (Agaricus bisporus) tyrosinase isoforms by tropolone.

A kinetic study of the inhibition of mushroom tyrosinase by tropolone has been made. Three tyrosinase isoforms were used: two commercial tyrosinases from Fluka and Sigma (isoelectric points of 4. 3 and 4.1, respectively) and one purified isoform from mushroom strain U1 (isoelectric point of 4.5). Tropolone is a slow-binding inhibitor of these mushroom tyrosinase isoforms. Increasing tropolone concentrations provoked a progressive decrease in both the initial velocity and the final (inhibited) steady-state rate in the progress curves of product accumulation. A rapid formation of an enzyme-inhibitor complex, which further undergoes a slow reversible reaction, could take place since the inhibition of the different isoforms was partially reversed by the addition of CuSO(4). The kinetic parameters that described the inhibition by tropolone were evaluated by nonlinear regression fits. Incubation experiments of the different isoforms with tropolone demonstrated that this inhibitor only could bind to the "oxy" form of tyrosinase which justifies a mechanism previously proposed to explain the inhibition of tyrosinase by slow-binding inhibitors.

Kinetics of activation of latent mushroom (Agaricus bisporus) tyrosinase by benzyl alcohol.

A latent isoform of Agaricus bisporus tyrosinase has been isolated and activated by benzyl alcohol, one of the major volatile compounds in mushrooms of this genus. The progress curve that describes the activation process reached the steady-state rate (V(ss)) after a lag period (tau). The rate of active tyrosinase formation was calculated by coupling the oxidation of o-diphenols to the activation process. V(ss) depended on benzyl alcohol, o-diphenol, and latent tyrosinase concentrations. The lag period depended on benzyl alcohol concentrations but not on o-diphenol and enzyme concentrations. The size of the latent mushroom tyrosinase was 67 kDa, determined by SDS-PAGE and Western blotting assays. This size was not modified after activation by benzyl alcohol. The presence of a lag period and the lack of change of the molecular mass of the protein after activation could indicate a slow conformational change of the protein to render the final active form. The values of the kinetic constants V(max) and K(m) on the o-diphenols 4-tert-butylcatechol, L-DOPA, and dopamine were different between the latent tyrosinase activated by benzyl alcohol and the commercial tyrosinase. They might indicate that a different final active tyrosinase, depending on the activator used, could arise.

Activation of a latent mushroom (Agaricus bisporus) tyrosinase isoform by sodium dodecyl sulfate (SDS). Kinetic properties of the SDS-activated isoform.

This study reports the activation of a latent mushroom tyrosinase isoform by sodium dodecyl sulfate (SDS). The activation process of latent mushroom tyrosinase by SDS is characterized by the presence of a lag period (tau) prior to the attainment of a steady-state rate (V(ss)). This could be related to a slow conformational change of the latent enzyme to render the active isoform. The molecular size of the latent isoform was 67 kDa as determined by SDS-PAGE and western-blotting assays. This size did not change after activation by SDS. The molecular size of the protease-activated isoform was 43 kDa. tau and V(ss) displayed a sigmoidal relationship to the concentration of SDS, but tau was not dependent on o-diphenol or enzyme concentration. Increasing SDS concentrations decreased tau, but then lower V(ss) values were detected because of a possible excess of unfolding and subsequent denaturation of the protein. The same reaction mechanism operated in both SDS-activated and protease-activated tyrosinase isoforms despite their different kinetic features. A possible mechanism for the activation of this latent tyrosinase by SDS is proposed.

Kinetic study of the oxidation of gamma-L-glutaminyl-4-hydroxybenzene catalyzed by mushroom (Agaricus bisporus) tyrosinase.

Despite the importance of the substrate gamma-L-glutaminyl-4-hydroxybenzene (GHB) in the melanin biosynthesis pathway in mushrooms Agaricus bisporus, the kinetics of its oxidation catalyzed by tyrosinase has never been properly characterized. For this purpose GHB and its corresponding o-diphenol (GDHB) were isolated and purified from A. bisporus mushrooms. The kinetic constants that characterize the action of tyrosinase on GHB and GDHB are = 2.10 +/- 0.10 microM/min, = 0.30 +/- 0.03 mM, = 210.0 +/- 7.3 microM/min, and = 7.80 +/- 0.41 mM. The oxygen kinetic constants for tyrosinase in the presence of these compounds are = 3. 20 +/- 0.21 microM/min, = 1.50 +/- 0.12 microM, = 200.2 +/- 8.1 microM/min, and = 100.2 +/- 8.2 microM. These values were compared to those obtained for the pair L-tyrosine/L-DOPA. The kinetic and structural reaction mechanisms of tyrosinase were corroborated for these physiological phenolic compounds.

Effect of wounding on phenolic enzymes in six minimally processed lettuce cultivars upon storage.

The effect of wounding on polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine ammonia-lyase (PAL) was studied in six minimally processed lettuce (Lactuca sativa L.) cultivars upon storage for 7 days at 5 degrees C (Iceberg Mikonos (IM), I. Green Queen (IGQ), I. Asdrúbal (IA), Little Gem Sandra (LGS), Romaine Cazorla (RC), and R. Modelo (RM)). Wounding of lettuce tissue midribs (because of minimal processing) caused an exponential increase in PPO activity due to the activation process from latent to fully active PPO by following first order kinetics in the time range from 3.7 days (LGS) to 6.3 days (RC). However, total PPO activity (active plus latent) remained constant. Isoform pattern of PPO changed upon storage probably because of posttranslational processes. POD activity linearly increased with induction of new POD isoenzymes. PAL activity presented a typical bell-shaped induction pattern in four cultivars. Only IM and IGQ showed a second induction response which has not been previously described in the literature. IM was the cultivar most susceptible to browning and RC was the cultivar least susceptible. However, no clear correlation was observed between browning and any of the biochemical and physiological attributes investigated (PPO, PAL, and POD activities, total and individual phenols accumulation, and ascorbic acid content).

Postharvest induction modeling method using UV irradiation pulses for obtaining resveratrol-enriched table grapes: a new "functional" fruit?

A modeling method for the induction of resveratrol synthesis by UV irradiation pulses in Napoleon table grapes is proposed. The method is based on the combination of four main parameters: irradiation power (IW), irradiation time (IT), irradiation distance (ID), and number of elapsed days to achieve the highest resveratrol accumulation (D(m)). Maximum resveratrol content (11-fold higher than untreated grapes) was achieved using the combination: IW = 510 W, IT = 30 s, ID = 40 cm, and D(m) = 3 days. Sensory characteristics and main features of irradiated grapes (color, weight, firmness, flavor, size, ripening index and vitamin C content) remained unaltered after 1 week of storage. UV induction signal migrated to the hidden side of the grape skin with a delay of 3 days as compared to the directly irradiated side. Phenolic compounds were not detected in Napoleon grape flesh. Resveratrol content per standard serving (200 g) of irradiated grape was about 3 mg, an amount more than 10-fold higher than that of untreated Napoleon grapes. This means that a serving of irradiated grape (unpeeled) could supply the resveratrol content equivalent to 3 glasses of a red wine with high resveratrol content ( approximately 1 mg/glass). Therefore, controlled UV irradiation pulses are useful as a simple postharvest treatment (and alternative to genetic engineering) to obtain possible "functional" grapes (with enhanced health-promoting properties) as a dietary source of high resveratrol content.

Characterization of the total free radical scavenger capacity of vegetable oils and oil fractions using 2,2-diphenyl-1-picrylhydrazyl radical.

The total free radical scavenger capacity (RSC) of 57 edible oils from different sources was studied: olive (24 brands of oils), sunflower (6), safflower (2), rapeseed (3), soybean (3), linseed (2), corn (3), hazelnut (2), walnut (2), sesame (2), almond (2), mixture of oils for salad (2), "dietetic" oil (2), and peanut (2). Olive oils were also studied according to their geographical origins (France, Greece, Italy, Morocco, Spain, and Turkey). RSC was determined spectrophotometrically by measuring the disappearance of the radical 2,2-diphenyl-1-picrylhydrazyl radical (DPPH(*)) at 515 nm. The disappearance of the radical followed a double-exponential equation in the presence of oils and oil fractions, which suggested the presence of two (fast and slow) groups of antioxidants. RSC was studied for the methanol-soluble phase ("methanolic fraction", MF) of the oil, the fraction nonsoluble in methanol ("lipidic fraction", LF), and the nonfractionated oil ("total oil"; TF = MF + LF). Only olive, linseed, rapeseed, safflower, sesame, and walnut oils showed significant RSC in the MF due to the presence of phenolic compounds. No significant differences were found in the RSC of olive oils from different geographical origins. Upon heating at 180 degrees C the apparent constant for the disappearance of RSC (k(T)) and the half-life (t1/2) of RSC for MF, LF, and TF were calculated. The second-order rate constants (k2) for the antiradical activity of some phenolic compounds present in oils are also reported.

Kinetic study of the activation process of a latent mushroom (Agaricus bisporus) tyrosinase by serine proteases.

Latent mushroom tyrosinase can be considered as a zymogen when activated by proteases because the activation process fulfilled all of the kinetic dependencies predicted by a theoretical zymogen activation model previously reported. The activation was studied under two assay conditions: high and low ratio of latent tyrosinase/serine protease (trypsin and subtilisin Carlsberg) concentrations, in the presence and in the absence of a serine protease inhibitor (aprotinin). The size of the latent enzyme was 67 kDa, determined by denaturing SDS-PAGE electrophoresis and Western blot assays. After proteolytic activation, the size was 43 kDa, with an intermediate band of 58 kDa. The values of the catalytic () and Michaelis () constants for the active forms of tyrosinase resulting from the activation by subtilisin, trypsin, or sodium dodecyl sulfate on the substrate tert-butylcatechol were slightly different, which could support the idea of "one activator-one different active tyrosinase". Vacuum infiltration experiments tried to reproduce in vivo the role of mushroom serine proteases in the activation of latent tyrosinase. The use of serine protease inhibitors is proposed as a new alternative tool to prevent melanin formation.