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.

Exploring the effects of phenolic compounds to reduce intestinal damage and improve the intestinal barrier integrity: A systematic review of in vivo animal studies.

BACKGROUND & AIMS: The integrity of the intestinal barrier in the diseased is key to prevent further complications and disease such as sepsis and death, whereas, the role of food bioactive molecules (i. e. phenolic compounds (PCs) on the intestinal barrier, is still unknown. The current aim was to explore the benefits of the oral PC administration on the intestinal barrier integrity in animals. METHODS: The effects of PCs on the intestinal barrier integrity in in vivo animal models of intestinal inflammation were assessed up-to August 2020 from the PubMed, SCOPUS, and Cochrane Library databases under the PRISMA methodology. The risk of bias was assessed from ARRAY and SCYRCLE tools. RESULTS: From 1241 articles, 14 studies were included. In animals, oral resveratrol (n = 6) improves the intestinal barrier integrity and reduces intestinal damage. Additionally, grape seed extract (n = 2), curcumin (n = 1), genistein (n = 1), chlorogenic acid (n = 1), grape pomace (n = 1), olive leaf (n = 1) or cranberry extract (n = 1) improve the intestinal barrier integrity downregulating various inflammatory molecules (TNF-α, and other interleukins), and increasing the antioxidant enzymes in animals. Furthermore, resveratrol, quercetin, epigallocatechin, and other PCs improve the epithelial barrier integrity and pro-inflammatory molecule expression in the intestinal epithelia. CONCLUSIONS: The oral PC administration in animals improves the intestinal barrier integrity and function from three main mechanisms: 1) The reduction of pro-inflammatory molecules, 2) the improvement in tight-junction protein expression, and 3) the improvement of the antioxidant intracellular activity suggesting the potential use of PCs in the management of intestinal injury in humans, particularly for resveratrol, the most studied PC.

Proteomic Analysis of Heart and Kidney Tissues in Healthy and Metabolic Syndrome Rats after Hesperidin Supplementation.

SCOPE: Proteomics has provided new strategies to elucidate the mechanistic action of hesperidin, a flavonoid present in citrus fruits. Thus, the aim of the present study is to determine the effects of hesperidin supplementation (HS) on the proteomic profiles of heart and kidney tissue samples from healthy and metabolic syndrome (MS) rats. METHODS AND RESULTS: 24 Sprague Dawley rats are randomized into four groups: healthy rats fed with a standard diet without HS, healthy rats administered with HS (100 mg kg-1 day-1 ), MS rats without HS, and MS rats administered with HS (100 mg kg-1 day-1 ) for eight weeks. Heart and kidney samples are obtained, and proteomic analysis is performed by mass spectrometry. Multivariate, univariate, and ingenuity pathways analyses are performed. Comparative and semiquantitative proteomic analyses of heart and kidney tissues reveal differential protein expression between MS rats with and without HS. The top diseases and functions implicated are related to the cardiovascular system, free radical scavenging, lipid metabolism, glucose metabolism, and renal and urological diseases. CONCLUSION: This study is the first to demonstrate the protective capacity of hesperidin to change to the proteomic profiles in relation to different cardiovascular risk biomarkers in the heart and kidney tissues of MS rats.

Anthocyanin Tissue Bioavailability in Animals: Possible Implications for Human Health. A Systematic Review.

Anthocyanins (ACNs) are promising health-enhancing phenolic compounds. We focus on ACN animal tissue bioavailability to provide an evidentiary link between tissue ACNs and their associated health properties. We performed a systematic review of electronic libraries; 279 results were retrieved, and 13 publications met inclusion criteria. Extracted information included animal model employed, administration route, doses, analysis method, and ACN concentration values in tissues. Total ACN concentrations were detected in mice kidney (2.17 × 105 pmol/g), liver (1.73 × 105 pmol/g), heart (3.6 × 103 pmol/g), and lung (1.16 × 105 pmol/g); and in pig brain (6.08 × 103 pmol/g). ACNs showed a predominance of parent ACNs in long-term experiments versus an ACN metabolite predominance in short-term experiments. ACNs detected in animal tissues, such as cyanidin-3-glucoside, suggest it may have an important role in human health. This information could be useful to determine proper ACN-intake biomarkers in biological samples in futures studies.

Phenol-enriched olive oils improve HDL antioxidant content in hypercholesterolemic subjects. A randomized, double-blind, cross-over, controlled trial.

At present, high-density lipoprotein (HDL) function is thought to be more relevant than HDL cholesterol quantity. Consumption of olive oil phenolic compounds (PCs) has beneficial effects on HDL-related markers. Enriched food with complementary antioxidants could be a suitable option to obtain additional protective effects. Our aim was to ascertain whether virgin olive oils (VOOs) enriched with (a) their own PC (FVOO) and (b) their own PC plus complementary ones from thyme (FVOOT) could improve HDL status and function. Thirty-three hypercholesterolemic individuals ingested (25 ml/day, 3 weeks) (a) VOO (80 ppm), (b) FVOO (500 ppm) and (c) FVOOT (500 ppm) in a randomized, double-blind, controlled, crossover trial. A rise in HDL antioxidant compounds was observed after both functional olive oil interventions. Nevertheless, α-tocopherol, the main HDL antioxidant, was only augmented after FVOOT versus its baseline. In conclusion, long-term consumption of phenol-enriched olive oils induced a better HDL antioxidant content, the complementary phenol-enriched olive oil being the one which increased the main HDL antioxidant, α-tocopherol. Complementary phenol-enriched olive oil could be a useful dietary tool for improving HDL richness in antioxidants.

Hydroxytyrosol and its complex forms (secoiridoids) modulate aorta and heart proteome in healthy rats: Potential cardio-protective effects.

SCOPE: Hydroxytyrosol (HT) is the major phenolic compound in virgin olive oil (VOO) in both free and complex forms (secoiridoids; SEC). Proteomics of cardiovascular tissues such as aorta or heart represents a promising tool to uncover the mechanisms of action of phenolic compounds in healthy animals. METHODS AND RESULTS: Twelve female Wistar rats were separated into three groups: a standard diet and two diets supplemented in phenolic compounds (HT and SEC) adjusted to 5 mg/kg/day during 21 days. Proteomic analyses of aorta and heart tissues were performed by nano-LC and MS. Ingenuity Pathway Analysis was used to generate interaction networks. HT or SEC modulated aorta and heart proteome compared to the standard diet. The top-scored networks were related to Cardiovascular System. HT and SEC downregulated proteins related to proliferation and migration of endothelial cells and occlusion of blood vessels in aorta and proteins related to heart failure in heart tissue. SEC showed higher fold change values compared to HT, attributed to higher concentration of HT detected in heart tissue. CONCLUSION: Changes at proteomic level in cardiovascular tissues may partially account for the underlying mechanisms of VOO phenols cardiovascular protection being the SEC effects higher than free HT.

Virgin Olive Oil Enriched with Its Own Phenols or Complemented with Thyme Phenols Improves DNA Protection against Oxidation and Antioxidant Enzyme Activity in Hyperlipidemic Subjects.

The effects of virgin olive oil (VOO) enriched with its own phenolic compounds (PC) and/or thyme PC on the protection against oxidative DNA damage and antioxidant endogenous enzymatic system (AEES) were estimated in 33 hyperlipidemic subjects after the consumption of VOO, VOO enriched with its own PC (FVOO), or VOO complemented with thyme PC (FVOOT). Compared to pre-intervention, 8-hydroxy-2'-deoxyguanosine (a marker for DNA damage) decreased in the FVOO intervention and to a greater extent in the FVOOT with a parallel significant increase in olive and thyme phenolic metabolites. Superoxide dismutase (AEES enzyme) significantly increased in the FVOO intervention and to a greater extent in the FVOOT with a parallel significant increase in thyme phenolic metabolites. When all three oils were compared, FVOOT appeared to have the greatest effect in protecting against oxidative DNA damage and improving AEES. The sustained intake of a FVOOT improves DNA protection against oxidation and AEES probably due to a greater bioavailability of thyme PC in hyperlipidemic subjects.

Study of the catabolism of thyme phenols combining in vitro fermentation and human intervention.

The gut metabolism of four thyme phenolics (monoterpenes thymol and carvacrol, rosmarinic acid, and eriodictyol) was evaluated in vitro. After the in vitro transformations of the individual phenols had been studied, the presence of their microbial metabolites was investigated in human feces collected before and after a sustained intake (3 weeks) of 25 mL/day of a thyme phenol-enriched olive oil. Results of in vitro fermentation showed low degradation of thymol and carvacrol. By contrast, large catabolism was noted when rosmarinic acid and eriodictyol were fermented, yielding hydroxyphenylpropionic acid as the main metabolite. In accordance with these results, after the in vivo intervention with thyme phenol-enriched olive oil, an increase in the concentration of hydroxyphenylpropionic and phenylpropionic acids was observed in human feces, confirming the effective in vivo microbial degradation of rosmarinic acid and eriodictyol. Carvacrol was detected in fecal samples at trace levels, suggesting that monoterpenes are well absorbed in the upper part of the gastrointestinal tract.

Faecal microbial metabolism of olive oil phenolic compounds: in vitro and in vivo approaches.

SCOPE: In the present study, the individual colonic metabolism of the main components of the virgin olive oil phenolic fraction was evaluated by an in vitro model using human faecal microbiota. To assess differences in metabolism related to the molecular structure, four phenolic standards were selected, tyrosol, hydroxytyrosol, hydroxytyrosol acetate and oleuropein. After studying the in vitro colonic metabolism pathways of the individual phenols, the presence of their colonic metabolites was investigated in human faecal samples obtained before and after the sustained intake (3 weeks) of a daily dose of 25 mL of a phenol-enriched olive oil. METHODS AND RESULTS: The in vitro colon fermentation of the four individual phenolic compounds revealed (i) an increase in phenolic acids, (ii) the stability of hydroxytyrosol and tyrosol and (iii) the high degradation of hydroxytyrosol acetate and oleuropein. Additionally, a moderate intake of a phenol-rich olive oil raised the concentration in human faeces of free hydroxytyrosol and phenylacetic and phenylpropionic acids. CONCLUSION: The products of colonic catabolism of olive oil phenolic compounds could be good candidates for novel preventive strategies and open a promising line of research into the preventive action of olive oil phenols in colon and other bowel diseases.

Olive oil polyphenols enhance high-density lipoprotein function in humans: a randomized controlled trial.

OBJECTIVE: Olive oil polyphenols have shown beneficial properties against cardiovascular risk factors. Their consumption has been associated with higher cholesterol content in high-density lipoproteins (HDL). However, data on polyphenol effects on HDL quality are scarce. We, therefore, assessed whether polyphenol-rich olive oil consumption could enhance the HDL main function, its cholesterol efflux capacity, and some of its quality-related properties, such HDL polyphenol content, size, and composition. APPROACH AND RESULTS: A randomized, crossover, controlled trial with 47 healthy European male volunteers was performed. Participants ingested 25 mL/d of polyphenol-poor (2.7 mg/kg) or polyphenol-rich (366 mg/kg) raw olive oil in 3-week intervention periods, preceded by 2-week washout periods. HDL cholesterol efflux capacity significantly improved after polyphenol-rich intervention versus the polyphenol-poor one (+3.05% and -2.34%, respectively; P=0.042). Incorporation of olive oil polyphenol biological metabolites to HDL, as well as large HDL (HDL2) levels, was higher after the polyphenol-rich olive oil intervention, compared with the polyphenol-poor one. Small HDL (HDL3) levels decreased, the HDL core became triglyceride-poor, and HDL fluidity increased after the polyphenol-rich intervention. CONCLUSIONS: Olive oil polyphenols promote the main HDL antiatherogenic function, its cholesterol efflux capacity. These polyphenols increased HDL size, promoted a greater HDL stability reflected as a triglyceride-poor core, and enhanced the HDL oxidative status, through an increase in the olive oil polyphenol metabolites content in the lipoprotein. Our results provide for the first time a first-level evidence of an enhancement in HDL function by polyphenol-rich olive oil.

Application of dried spot cards as a rapid sample treatment method for determining hydroxytyrosol metabolites in human urine samples. Comparison with microelution solid-phase extraction.

Two different rapid sample pretreatment strategies, dried spot cards, and microelution solid-phase extraction plates (µSPE), with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) have been developed and validated for the determination of hydroxytyrosol and its metabolites in spiked human urine samples. Hydroxytyrosol, hydroxytyrosol-3'-O-glucuronide, hydroxytyrosol-4'-O-glucuronide, hydroxytyrosol-3-O-sulphate, and homovanillic alcohol-4'-O-glucuronide were used as the target compounds. Using the FTA DMPK-A dried urine spot card under optimum conditions, with 5 µL of preconcentrated urine volume and 100 µL of methanol/water (50/50, v/v) as the elution solvent, the extraction recovery (%R) of the compounds studied was higher than 80%, and the matrix effect (%ME) was less than 8%. The stability of these cards and punching at the centre or side of the card were also studied, obtaining an excellent stability after 7 days of storage and complete homogeneity across the surface of the dried drop. The different µSPE parameters that affect the efficiency were also studied, and under optimum conditions, the %R and the %ME were higher than 70% and lower than 17%, respectively. The linearity range in dried urine spot cards was 2.5-20 µM for all the metabolites, with the exception of hydroxytyrosol-3-O-sulphate and hydroxytyrosol, which were 0.3-70 µM and 2.5-50 µM respectively. With regards to µSPE, the linearity range was 0.5-5 µM for all the studied compounds, except for hydroxytyrosol-3-O-sulphate, which was 0.08-5 µM. The quantification limits (LOQs) were 0.3-2.5 µM and 0.08-0.5 µM in dried spot cards and in µSPE, respectively. The two developed methods were then applied and compared for determining hydroxytyrosol and its metabolites in human 24 h-urine samples after a sustained consumption (21 days) of a phenol-enriched virgin olive oil. The metabolites identified were hydroxytyrosol in its glucuronide and sulphate forms, homovanillic alcohol in its glucuronide and sulphate forms, homovanillic acid sulphate and hydroxytyrosol acetate sulphate.

Biomarkers of food intake and metabolite differences between plasma and red blood cell matrices; a human metabolomic profile approach.

Untargeted metabolomic analyses of plasma and red blood cells (RBCs) can provide complementary information on biomarkers of food consumption. To assess blood collection differences in biomarkers, fasting blood was drawn from 10 healthy individuals using sodium citrate and lithium heparin as anticoagulants. Plasma and RBCs were separated into aqueous and lipid fractions to be analyzed using 1D and 2D (1)H NMR spectroscopy. Fatty acids were analyzed using gas chromatography-mass spectrometry (GC-MS). Polyphenols were extracted from plasma and RBCs by micro-elution solid-phase extraction and analyzed by ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). (1)H NMR demonstrated higher aqueous metabolites such as glucose in plasma compared to RBCs, while RBCs contained higher ADP-ATP, creatine and acetone than plasma. Lipoproteins and their subclasses were higher in plasma than in RBCs. Percentages of saturated fatty acids (SFA) 16 : 0, 17 : 0, 20 : 0, 24 : 0 and polyunsaturated fatty acids (PUFA) 22 : 6 n-3 (docosahexaenoic acid) and 20 : 4 n-6 (arachidonic acid) were higher in RBCs than in plasma (p < 0.05), while SFA 14 : 0, monounsaturated fatty acids (MUFA) 14 : 1 n-5, 16 : 1 n-7, 17 : 1 n-7 and 18 : 1 n-9 and PUFA 18 : 3 n-3, 18 : 2 n-6, 18 : 3 n-6 and 20 : 3 n-6 were higher in plasma than in RBCs (p < 0.05). Polyphenols differed in plasma from those of RBCs. Biomarker concentrations were lower in sodium citrate compared to lithium heparin plasma. In conclusion, metabolomic profiles generated by NMR spectroscopy, GC-MS and UPLC-MS/MS analyses of RBCs versus plasma show complementary information on several specific molecular biomarkers that could be applied in nutritional assessment.

Inhibition of the transcription factor c-Jun by the MAPK family, and not the NF-κB pathway, suggests that peanut extract has anti-inflammatory properties.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is involved in inflammatory responses in atherosclerosis. We propose an in vitro cellular assay to evaluate the anti-inflammatory mechanisms of potential modifiers such as food extracts. In the current model we assessed an anti-inflammatory effect of polyphenol-rich peanut extract in lipopolysaccharide (LPS)-induced THP-1 monocytes. METHODS: THP-1 monocytes were incubated with peanut extract (5, 25, 50 and 100 µg/mL) consisting of 39% flavonols, 37% flavanols and 24% phenolic acid (or BAY 11-7082 (5 µM) as experiment control) for 1 h and then stimulated with LPS (500 ng/mL) for 4 h. Cytotoxicity was measured as lactate dehydrogenase (LDH) activity release. NF-κB and MAPK family were determined by TransAm kit while TNF-α mRNA levels and its mRNA stability by RT-PCR. Intra- and extracellular TNF-α protein was measured by ELISA, and TNF-α converting enzyme (TACE) activity by a fluorimetric assay. RESULTS: Peanut extract inhibited the maximal LPS-induced extracellular TNF-α protein secretion by 18%, 29% and 47% at 25, 50 and 100 µg/mL, respectively (P<0.05). LPS stimulation revealed that 85% of TNF-α was released extracellularly while 15% remained intracellular. Peanut extract did not modify NF-κB but, instead, reduced c-Jun transcription factor activity (P<0.05), decreased TNF-α mRNA (albeit non-significantly) and had no effect on mRNA stability and TACE activity. CONCLUSION: Polyphenol-rich peanut extract reduces extracellular TNF-α protein by inhibiting c-Jun transcription factor from MAPK family, suggesting an anti-inflammatory effect. The proposed THP-1 monocyte model could be used to assess food extract impact (site and size effects) on the inflammation pathway.