4-Week consumption of anthocyanin-rich blood orange juice does not affect LDL-cholesterol or other biomarkers of CVD risk and glycaemia compared with standard orange juice: a randomised controlled trial.

Elevated circulating cholesterol levels are a risk factor for CVD which is also associated with sub-optimal vascular function. There is emerging evidence that anthocyanins can cause beneficial cardio-protective effects by favourably modulating lipoprotein profiles. We compared the effects of blood orange juice which is rich in anthocyanins and blonde orange juice without anthocyanins on LDL-cholesterol and other biomarkers of CVD risk, vascular function and glycaemia. In all, forty-one participants (aged 25-84 years) with a waist circumference >94 cm (men) and >80 cm (women) completed a randomised, open label, two-arm cross-over trial. For 28 d participants ingested (i) 500 ml blood orange juice providing 50 mg anthocyanins/d and (ii) 500 ml blonde orange juice without anthocyanins. There was a minimum 3-week washout period between treatments. LDL-cholesterol and other biomarkers associated with CVD risk and glycaemia were assessed at the start and end of each treatment period. No significant differences were observed in total, HDL- and LDL-cholesterol, TAG, glucose, fructosamine, nitric oxide, C-reactive protein, aortic systolic blood pressure and diastolic blood pressure or carotid-femoral and brachial-ankle pulse wave velocity after 28 d ingestion of blood orange juice compared with standard orange juice. The lack of effect on LDL-cholesterol may be due to the modest concentration of anthocyanins in the blood orange juice.

Meta-Analysis of the Effects of Foods and Derived Products Containing Ellagitannins and Anthocyanins on Cardiometabolic Biomarkers: Analysis of Factors Influencing Variability of the Individual Responses.

Understanding interindividual variability in response to dietary polyphenols remains essential to elucidate their effects on cardiometabolic disease development. A meta-analysis of 128 randomized clinical trials was conducted to investigate the effects of berries and red grapes/wine as sources of anthocyanins and of nuts and pomegranate as sources of ellagitannins on a range of cardiometabolic risk biomarkers. The potential influence of various demographic and lifestyle factors on the variability in the response to these products were explored. Both anthocyanin- and ellagitannin-containing products reduced total-cholesterol with nuts and berries yielding more significant effects than pomegranate and grapes. Blood pressure was significantly reduced by the two main sources of anthocyanins, berries and red grapes/wine, whereas waist circumference, LDL-cholesterol, triglycerides, and glucose were most significantly lowered by the ellagitannin-products, particularly nuts. Additionally, we found an indication of a small increase in HDL-cholesterol most significant with nuts and, in flow-mediated dilation by nuts and berries. Most of these effects were detected in obese/overweight people but we found limited or non-evidence in normoweight individuals or of the influence of sex or smoking status. The effects of other factors, i.e., habitual diet, health status or country where the study was conducted, were inconsistent and require further investigation.

Development, validation and evaluation of an analytical method for the determination of monomeric and oligomeric procyanidins in apple extracts.

There is a lack of data for individual oligomeric procyanidins in apples and apple extracts. Our aim was to develop, validate and evaluate an analytical method for the separation, identification and quantification of monomeric and oligomeric flavanols in apple extracts. To achieve this, we prepared two types of flavanol extracts from freeze-dried apples; one was an epicatechin-rich extract containing ∼30% (w/w) monomeric (-)-epicatechin which also contained oligomeric procyanidins (Extract A), the second was an oligomeric procyanidin-rich extract depleted of epicatechin (Extract B). The parameters considered for method optimisation were HPLC columns and conditions, sample heating, mass of extract and dilution volumes. The performance characteristics considered for method validation included standard linearity, method sensitivity, precision and trueness. Eight laboratories participated in the method evaluation. Chromatographic separation of the analytes was best achieved utilizing a Hilic column with a binary mobile phase consisting of acidic acetonitrile and acidic aqueous methanol. The final method showed linearity for epicatechin in the range 5-100µg/mL with a correlation co-efficient >0.999. Intra-day and inter-day precision of the analytes ranged from 2 to 6% and 2 to 13% respectively. Up to dp3, trueness of the method was >95% but decreased with increasing dp. Within laboratory precision showed RSD values <5 and 10% for monomers and oligomers, respectively. Between laboratory precision was 4 and 15% (Extract A) and 7 and 30% (Extract B) for monomers and oligomers, respectively. An analytical method for the separation, identification and quantification of procyanidins in an apple extract was developed, validated and assessed. The results of the inter-laboratory evaluation indicate that the method is reliable and reproducible.

Potent inhibition of VEGFR-2 activation by tight binding of green tea epigallocatechin gallate and apple procyanidins to VEGF: relevance to angiogenesis.

SCOPE: Excessive concentrations of vascular endothelial growth factor (VEGF) drive angiogenesis and cause complications such as increased growth of tumours and atherosclerotic plaques. The aim of this study was to determine the molecular mechanism underlying the potent inhibition of VEGF signalling by polyphenols. METHODS AND RESULTS: We show that the polyphenols epigallocatechin gallate from green tea and procyanidin oligomers from apples potently inhibit VEGF-induced VEGF receptor-2 (VEGFR-2) signalling in human umbilical vein endothelial cells by directly interacting with VEGF. The polyphenol-induced inhibition of VEGF-induced VEGFR-2 activation occurred at nanomolar polyphenol concentrations and followed bi-phasic inhibition kinetics. VEGF activity could not be recovered by dialysing VEGF-polyphenol complexes. Exposure of VEGF to epigallocatechin gallate or procyanidin oligomers strongly inhibited subsequent binding of VEGF to human umbilical vein endothelial cells expressing VEGFR-2. Remarkably, even though VEGFR-2 signalling was completely inhibited at 1 µM concentrations of polyphenols, endothelial nitric oxide synthase was shown to still be activated via the PI3K/Akt signalling pathway which is downstream of VEGFR-2. CONCLUSION: These data demonstrate for the first time that VEGF is a key molecular target for specific polyphenols found in tea, apples and cocoa which potently inhibit VEGF signalling and angiogenesis at physiological concentrations. These data provide a plausible mechanism which links bioactive compounds in food with their beneficial effects.

Effects of bioactive-rich extracts of pomegranate, persimmon, nettle, dill, kale and Sideritis and isolated bioactives on arachidonic acid induced markers of platelet activation and aggregation.

BACKGROUND: The beneficial effect of fruit- and vegetable-rich diets on cardiovascular health is partly attributed to the effect of their bioactive compounds on platelet function. The aim of this study was to investigate the effects of bioactive-rich plant extracts and isolated bioactive metabolites on platelet function. Blood samples from healthy subjects (n = 4) and subjects with metabolic syndrome (n = 4) were treated with six extracts of bioactive-rich plants consumed as traditional foods in the Black Sea region, or with human metabolites of the bioactives quercetin and sulforaphane. Markers of arachidonic acid induced platelet activation and platelet-leucocyte aggregation were assessed using flow cytometry. RESULTS: In subjects with metabolic syndrome, kale extract significantly inhibited agonist induced P-selectin expression (P = 0.004). Sulforaphane-cysteine-glycine, a human plasma metabolite of the related glucosinolate, glucoraphanin, significantly inhibited P-selectin and GPIIb-IIIa expression (P = 0.020 and 0.024, respectively) and platelet-neutrophil aggregation (P = 0.027). Additionally, pomegranate extract significantly inhibited GPIIb-IIIa expression (P = 0.046) in subjects with metabolic syndrome. In healthy subjects only dill extract significantly inhibited agonist induced P-selectin expression (P = 0.025). CONCLUSION: These data show that bioactive-rich extracts of kale and pomegranate that are consumed as traditional plant foods of Black Sea area countries were effective in modulating platelet function.

Bioactive-rich extracts of persimmon, but not nettle, Sideritis, dill or kale, increase eNOS activation and NO bioavailability and decrease endothelin-1 secretion by human vascular endothelial cells.

BACKGROUND: There is increasing evidence that consumption of plant bioactives such as polyphenols and glucosinolates reduces cardiovascular disease risk and improves endothelial function. In the Black Sea area, a number of plants are consumed alone and as ingredients in traditional foods, and dill, nettle, kale, Sideritis and persimmon were identified as bioactive-rich traditional food plants. The present study investigated the effects of plant extracts on cellular markers of endothelial function (eNOS activation and expression and ET-1 secretion). RESULTS: Treatment of human umbilical vein endothelial cells with persimmon extract significantly increased Akt and eNOS phosphorylation and nitric oxide metabolites and significantly decreased secretion of ET-1 to the media after 24 h compared with a vehicle control (all P < 0.01). None of the other plant extracts significantly altered any markers of endothelial function. CONCLUSION: These findings suggest that persimmon fruit contains bioactives that can improve endothelial function via activation of eNOS and reduction in ET-1 secretion, but that dill, kale, Sideritis and nettle do not.

Lack of effect of bioactive-rich extracts of pomegranate, persimmon, nettle, dill, kale and Sideritis and isolated bioactives on platelet function.

BACKGROUND: The health benefits of fruit and vegetable-rich diets may be partly due to modulation of platelet activity by bioactive phytochemicals. The aim of this study was to investigate the effects of bioactive-rich plant extracts and isolated bioactive metabolites on platelet function. Blood samples (n =15 subjects) were treated with extracts of bioactive-rich plants consumed as traditional foods in the Black Sea region, or with human metabolites of the bioactives quercetin and sulforaphane. Platelet function was assessed using the PFA-100. RESULTS: None of the extracts containing various flavonoids, glucosinolates and other bioactives, or isolated bioactive metabolites of quercetin or sulforaphane, caused significant changes in PFA-100 closure time (CT). In contrast, the positive controls (aspirin and Abciximab) consistently caused significant increases in CT for the platelet agonists epinephrine and ADP, respectively. CONCLUSION: These data do not support the notion that these plant bioactives can improve human platelet function.

Bioavailability of epicatechin and effects on nitric oxide metabolites of an apple flavanol-rich extract supplemented beverage compared to a whole apple puree: a randomized, placebo-controlled, crossover trial.

SCOPE: Flavanol-rich foods are known to exert beneficial effects on cardiovascular health. The biological effects depend on bioavailability of flavanols which may be influenced by food matrix and dose ingested. We compared the bioavailability and dose-response of epicatechin from whole apple and an epicatechin-rich extract, and the effects on plasma and urinary nitric oxide (NO) metabolites. METHODS AND RESULTS: In a randomized, placebo-controlled, crossover trial, subjects consumed drinks containing 70 and 140 mg epicatechin from an apple extract and an apple puree containing 70 mg epicatechin. Blood and urine samples were collected for 24 h post ingestion. Maximum plasma concentration, AUC(0-24 h) , absorption and urinary excretion were all significantly higher after ingestion of both epicatechin drinks compared with apple puree (p < 0.05). Time to maximum plasma concentration was significantly later for the puree compared with the drinks (p < 0.01). Epicatechin bioavailability was >2-fold higher after ingestion of the 140 mg epicatechin drink compared to the 70 mg epicatechin drink (p < 0.05). Excretion of NO metabolites was higher for all test products compared with placebo, which was significant for the high dose drink (p = 0.016). CONCLUSIONS: Oral bioavailability of apple epicatechin increases at higher doses, is reduced by whole apple matrix and has the potential to increase NO bioavailability.

Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli.

SCOPE: Sulforaphane (a potent anticarcinogenic isothiocyanate derived from glucoraphanin) is widely considered responsible for the protective effects of broccoli consumption. Broccoli is typically purchased fresh or frozen and cooked before consumption. We compared the bioavailability and metabolism of sulforaphane from portions of lightly cooked fresh or frozen broccoli, and investigated the bioconversion of sulforaphane to erucin. METHODS AND RESULTS: Eighteen healthy volunteers consumed broccoli soups produced from fresh or frozen broccoli florets that had been lightly cooked and sulforaphane thio-conjugates quantified in plasma and urine. Sulforaphane bioavailability was about tenfold higher for the soups made from fresh compared to frozen broccoli, and the reduction was shown to be due to destruction of myrosinase activity by the commercial blanching-freezing process. Sulforaphane appeared in plasma and urine in its free form and as several thio-conjugates forms. Erucin N-acetyl-cysteine conjugate was a significant urinary metabolite, and it was shown that human gut microflora can produce sulforaphane, erucin, and their nitriles from glucoraphanin. CONCLUSION: The short period of blanching used to produce commercial frozen broccoli destroys myrosinase and substantially reduces sulforaphane bioavailability. Sulforaphane was converted to erucin and excreted in urine, and it was shown that human colonic flora were capable of this conversion.

Cardiovascular disease risk biomarkers and liver and kidney function are not altered in postmenopausal women after ingesting an elderberry extract rich in anthocyanins for 12 weeks.

Growing evidence supports a cardio-protective role for anthocyanins; however, there is limited evidence on their efficacy and safety following the consumption of relatively high but dietarily achievable doses in humans. We conducted a parallel-designed, randomized, placebo-controlled study to examine the effect of chronic consumption of anthocyanins on biomarkers of cardiovascular disease (CVD) risk and liver and kidney function in 52 healthy postmenopausal women (n = 26 in treatment and placebo groups). Volunteers (BMI, 24.7 +/- 3.6 kg/m(2); age, 58.2 +/- 5.6 y) consumed 500 mg/d anthocyanins as cyanidin glycosides (from elderberry) or placebo for 12 wk (2 capsules twice/d). At the beginning (wk 0) and end of the 12-wk intervention, levels of anthocyanins and biomarkers of CVD (inflammatory biomarkers, platelet reactivity, lipids, and glucose) and liver and kidney function (total bilirubin, albumin, urea, creatinine, alkaline phosphatase, alanine aminotransferase, and gamma-glutyl transferase) were assessed in fasted blood. Anthropometric, blood pressure, and pulse measurements were also taken. In addition, postprandial plasma anthocyanins were measured (t = 1, 2, 3 h) following a 500-mg oral bolus dose. After 12 wk of chronic exposure to anthocyanins, there was no significant change in biomarkers of CVD risk and liver and kidney function remained within clinically acceptable ranges. We observed no plasma accumulation of anthocyanins; however, postprandial metabolism increased (P = 0.02). In conclusion, these data suggest that chronic consumption of 500 mg/d of elderberry extract for 12 wk is apparently safe, but ineffective in altering biomarkers of CVD risk in healthy postmenopausal women.

Processing blackcurrants dramatically reduces the content and does not enhance the urinary yield of anthocyanins in human subjects.

Blackcurrant (BC) fruits are a rich source of biologically active anthocyanins but little is known of the anthocyanin content of commonly consumed BC products or how processing affects the absorption. We report that whereas fresh and frozen whole BC were a rich source of anthocyanins, containing 897 and 642mg (100gFW)(-1) of total anthocyanins the levels in all other products were substantially lower (0.05-10.3% of the levels in fresh fruit). Further, when the absorption and excretion of BC was assessed in volunteers consuming a portion (100g) of frozen whole BC (642mg total anthocyanins) and, 300g of a BC drink made by diluting concentrated syrup (33.6mg total anthocyanins), only small quantities of BC anthocyanins were excreted in urine (fruit, 0.053±0.022%; drink, 0.036±0.043%; mean percent urinary yield±SD) and they were not detected in plasma. These data indicate that fresh and frozen BC, but not processed products, are rich sources of anthocyanins but, regardless of the food source, these anthocyanins are poorly bioavailable.

Effect of high-dose iron supplements on fractional zinc absorption and status in pregnant women.

BACKGROUND: Women have an increased risk of iron deficiency during pregnancy because of the demands of the developing fetus. Iron supplements are commonly advocated as a prophylactic treatment and are generally taken with meals to reduce side effects, but iron can interfere with the absorption of zinc. OBJECTIVE: The aim was to determine the effect of consuming an iron supplement (100 mg Fe/d as ferrous gluconate) with meals from 16 wk gestation to term on zinc status and absorption. DESIGN: Stable-isotope techniques were used to measure zinc status (exchangeable zinc pool, EZP) and fractional zinc absorption (FZA) in early and late pregnancy from a meal consumed at a different time from that of iron supplement or placebo consumption in 6 women given iron supplements and 7 given a placebo. RESULTS: FZA increased during pregnancy, independent of iron supplementation. FZA was significantly higher (P < 0.001) at week 34 than at weeks 16 and 24, and urinary zinc excretion was higher at week 34 than at week 16 (P = 0.02). The size of the EZP remained unchanged throughout pregnancy and was unaffected by iron supplementation. The iron status of iron-supplemented women was higher than that of the placebo group. CONCLUSIONS: In iron-replete pregnant women who consumed a Western diet, no detectable adverse effects on zinc metabolism were observed after ingestion of 100 mg Fe/d. An increase in the efficiency of zinc absorption was observed during late pregnancy.