Colin D. Kay

Effects of chocolate, cocoa, and flavan-3-ols on cardiovascular health: a systematic review and meta-analysis of randomized trials

BACKGROUND There is substantial interest in chocolate and flavan-3-ols for the prevention of cardiovascular disease (CVD). OBJECTIVE The objective was to systematically review the effects of chocolate, cocoa, and flavan-3-ols on major CVD risk factors. DESIGN We searched Medline, EMBASE, and Cochrane databases for randomized controlled trials (RCTs) of chocolate, cocoa, or flavan-3-ols. We contacted authors for additional data and conducted duplicate assessment of study inclusion, data extraction, validity, and random-effects meta-analyses. RESULTS We included 42 acute or short-term chronic (≤18 wk) RCTs that comprised 1297 participants. Insulin resistance (HOMA-IR: -0.67; 95% CI: -0.98, -0.36) was improved by chocolate or cocoa due to significant reductions in serum insulin. Flow-mediated dilatation (FMD) improved after chronic (1.34%; 95% CI: 1.00%, 1.68%) and acute (3.19%; 95% CI: 2.04%, 4.33%) intakes. Effects on HOMA-IR and FMD remained stable to sensitivity analyses. We observed reductions in diastolic blood pressure (BP; -1.60 mm Hg; 95% CI: -2.77, -0.43 mm Hg) and mean arterial pressure (-1.64 mm Hg; 95% CI: -3.27, -0.01 mm Hg) and marginally significant effects on LDL (-0.07 mmol/L; 95% CI: -0.13, 0.00 mmol/L) and HDL (0.03 mmol/L; 95% CI: 0.00, 0.06 mmol/L) cholesterol. Chocolate or cocoa improved FMD regardless of the dose consumed, whereas doses >50 mg epicatechin/d resulted in greater effects on systolic and diastolic BP. GRADE (Grading of Recommendations, Assessment, Development and Evaluation, a tool to assess quality of evidence and strength of recommendations) suggested low- to moderate-quality evidence of beneficial effects, with no suggestion of negative effects. The strength of evidence was lowered due to unclear reporting for allocation concealment, dropouts, missing data on outcomes, and heterogeneity in biomarker results in some studies. CONCLUSIONS We found consistent acute and chronic benefits of chocolate or cocoa on FMD and previously unreported promising effects on insulin and HOMA-IR. Larger, longer-duration, and independently funded trials are required to confirm the potential cardiovascular benefits of cocoa flavan-3-ols.

Anthocyanin metabolites in human urine and serum

In the present study we investigated the metabolic conversion of cyanidin glycosides in human subjects using solid-phase extraction,HPLC-diode array detector, MS, GC, and enzymic techniques. Volunteers consumed approximately 20 g chokeberry extract containing 1.3 g cyanidin 3-glycosides (899 mg cyanidin 3-galactoside, 321 mg cyanidin 3-arabinoside, 51 mg cyanidin 3-xyloside and 50 mg cyanidin 3-glucoside). Blood samples were drawn at 0, 0.5, 1, and 2 h post-consumption of the extract. Urine samples were also collected at 0, 4-5,and 22-24h. We have confirmed that human subjects have the capacity to metabolise cyanidin 3-glycosides, as we observed at least ten individual anthocyanin metabolites in the urine and serum. Average concentrations of anthocyanins and anthocyanin metabolites in the urine reached levels of 17.9 (range 14.9-20.9) l.mol/l within 5 h post-consumption and persisted in 24h urine samples at levels of 12.1 (range 11.1-13.0) nmol/l. In addition, average total levels of anthocyanins and anthocyanin metabolites detected in the serum were observed at 5917 (range 197.3-986.1) nmol/ within 2h post-consumption. Cyanidin 3-galactoside accounted for 55.4% (9.9(range 7-2-12-6) l.mol/) and 66.0% (390.6 (range 119.4-661-9) nmol V) of the detected anthocyanins in the urine and serum samples,respectively. The metabolites were identified as glucuronide conjugates, as well as methylated and oxidised derivatives of cyanidin 3-galactoside and cyanidin glucuronide. Conjugation probably affects the biological activity of anthocyanins and these metabolic products are likely in part responsible for the reported health benefits associated with the consumption of anthocyanins.

THE EFFECT OF WILD BLUEBERRY (VACCINIUM ANGUSTIFOLIUM) CONSUMPTION ON POSTPRANDIAL SERUM ANTIOXIDANT STATUS IN HUMAN SUBJECTS

The aim of the present study was to determine whether the consumption of wild blueberries (Vaccinium angustifolium), a concentrated source of non-nutritive antioxidant phytochemicals, would enhance postprandial serum antioxidant status in healthy human subjects. A single-blinded crossover study was performed in a group of eight middle-aged male subjects (38-54 years). Subjects consumed a high-fat meal and a control supplement followed 1 week later by the same high-fat meal supplemented with 100.0 g freeze-dried wild blueberry powder. Upon brachial vein catheterization, fasting and postprandial serum samples were taken sequentially and analysed for lipids and glucose and for serum antioxidant status. Serum antioxidant status was determined using the oxygen radical absorbance capacity (ORAC) assay and the total antioxidant status (TAS) assay. The wild-blueberry treatment was associated with a significant treatment effect as determined by the ORAC assay (water-soluble fraction ORAC(perchloric acid (PCA)), P=0.04). Significant increases in serum antioxidant status above the controls were observed at 1 h (ORAC(PCA) (8.5 % greater), P=0.02; TAS (4.5 % greater), P=0.05), and 4 h (ORAC(total) (15.0 % greater), P=0.009; ORAC(acetone) (16.0 % greater), P=0.007) post-consumption of the high-fat meal. In conclusion, the consumption of wild blueberries, a food source with high in vitro antioxidant properties, is associated with a diet-induced increase in ex vivo serum antioxidant status. It has been suggested that increasing the antioxidant status of serum may result in the reduced risk of many chronic degenerative diseases.

The pharmacokinetics of anthocyanins and their metabolites in humans.

Anthocyanins are phytochemicals with reported vasoactive bioactivity. However, given their instability at neutral pH, they are presumed to undergo significant degradation and subsequent biotransformation. The aim of the present study was to establish the pharmacokinetics of the metabolites of cyanidin‐3‐glucoside (C3G), a widely consumed dietary phytochemical with potential cardioprotective properties.

Anthocyanin stability and recovery: implications for the analysis of clinical and experimental samples.

The proportion of ingested anthocyanins to reach the systemic circulation is reported to be a small percentage of their ingested dose. This may be due to physiochemical degradation in vivo or following routine sample treatment. Therefore, this study aimed to quantitatively investigate the effect of anthocyanin structure on their stability under simulated (in vitro) physiological conditions and to assess their degradation and recovery following routine preanalytical sample extraction and storage. It was demonstrated that B-ring hydroxylation mediated the degradation of anthocyanins to their phenolic acid and aldehyde constituents, successful anthocyanin extraction is dependent on both sample preparation technique and anthocyanin structure, and anthocyanins are stable through multiple freeze-thaw cycles. These data indicate that significant portions of ingested anthocyanins are likely to degrade to phenolic acids and aldehyde in vivo. Consequently, these compounds should be the target of future bioavailability and bioactivity studies to establish the true occurrence and impact of anthocyanins on human health.

The bioactivity of dietary anthocyanins is likely to be mediated by their degradation products

To date the in vitro mechanistic bioactivity of anthocyanins has been exclusively explored using aglycones and glycoside conjugates, despite a lack of evidence establishing these as the biologically available forms. We conducted intestinal epithelial cell (Caco-2 cells) culture experiments, which indicated that after a 4 h incubation of anthocyanins in cell-free culture media (DMEM), 57% of the initial cyanidin-3-glucoside (C3G) and 96% of cyanidin had degraded. The level of degradation was not statistically different from that of cultured cell incubations, suggesting that degradation was spontaneous. Degradation products were identified as protocatechuic acid (PCA) and phloroglucinaldehyde (PGA), and were confirmed in two other buffer matrices (phosphate and Hanks buffers). In cultured cell media the degradation products PCA and PGA were metabolised to glucuronide and sulphate conjugates, as indicated by both enzyme hydrolysis (sulphatase and glucuronidase treatments) and MS (PCA and PGA m/z = 155; sulphate = 235; glucuronide = 331). These data suggest a significant proportion of intestinal metabolites of anthocyanins are likely to be conjugates of their degradation products. Future efforts to establish the biological activities of anthocyanins should therefore include the investigation of phenolic acid and aldehyde products of degradation, along with their respective metabolites.

Relative impact of flavonoid composition, dose and structure on vascular function: A systematic review of randomised controlled trials of flavonoid‐rich food products

SCOPE Previous systematic reviews suggest beneficial effects of flavonoids on biomarkers of cardiovascular disease (CVD) risk, but have overlooked the impact of dose response or food complexity. The aim of the present study was to examine the relative impact of composition, flavonoid structure and dose. METHODS AND RESULTS MEDLINE, EMBASE and Cochrane were searched for randomised controlled trials (RCTs) of flavonoids or flavonoid-rich foods/extracts. Flavonoid composition was established using United States Department of Agriculture (USDA) and Phenol-Explorer databases. Effects of six flavonoid subgroups on endothelial function (flow-mediated dilation; FMD), and systolic and diastolic blood pressures were assessed by random effects meta-analyses and regression analyses. Meta-analyses of combined flavonoid subclasses showed significant improvements in FMD (chronic, 0.73% (0.17, 1.30) 14 RCTs; acute, 2.33% (1.58, 3.08) 18 RCTs) and blood pressures (systolic, -1.46 mmHg (-2.38, -0.53) 63 RCTs; diastolic, -1.25 mmHg (-1.82, -0.67) 63 RCTs). Similar benefits were observed for the flavan-3-ol, catechol flavonoids (catechins, quercetin, cyanidin etc.), procyanidins, epicatechin and catechin subgroups. Dose-response relationships were non-linear for FMD (R(2) ≤ 0.30), with greater associations observed when applying polynomial regression analyses (R(2) ≤ 0.72); there was no indication of a dose response for blood pressure. CONCLUSION The present analysis suggests that flavonoid bioactivity does not follow a classical linear dose-response association and this may have important biological implications.

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.

Effects of sugar-sweetened and sugar-free cocoa on endothelial function in overweight adults.

BACKGROUND Studies of cocoa suggest an array of cardiovascular benefits; however, the effects of daily intake of sugar-free and sugar-sweetened cocoa beverages on endothelial function (EF) have yet to be established. METHODS 44 adults (BMI 25-35 kg/m2) participated in a randomized, controlled, crossover trial. Participants were randomly assigned to a treatment sequence: sugar-free cocoa beverage, sugar-sweetened cocoa beverage, and sugar-sweetened cocoa-free placebo. Treatments were administered daily for 6 weeks, with a 4-week washout period. RESULTS Cocoa ingestion improved EF measured as flow-mediated dilation (FMD) compared to placebo (sugar-free cocoa: change, 2.4% [95% CI, 1.5 to 3.2] vs. -0.8% [95% CI, -1.9 to 0.3]; difference, 3.2% [95% CI, 1.8 to 4.6]; p<0.001 and sugar-sweetened cocoa: change, 1.5% [95% CI, 0.6 to 2.4] vs. -0.8% [95% CI, -1.9 to 0.3]; difference, 2.3% [95% CI, 0.9 to 3.7]; p=0.002). The magnitude of improvement in FMD after consumption of sugar-free versus sugar-sweetened cocoa was greater, but not significantly. Other biomarkers of cardiac risk did not change appreciably from baseline. BMI remained stable throughout the study. CONCLUSIONS Daily cocoa ingestion improves EF independently of other biomarkers of cardiac risk, and does not cause weight gain. Sugar-free preparations may further augment endothelial function.

The future of flavonoid research

There are insufficient data to provide conclusive evidence on the health effects of most flavonoid subclasses. Future research of polyphenol bioactivity requires a more complete understanding of their intake, bioavailability and metabolism. The following summarises the limitations of polyphenol research as described across various reviews throughout the literature and presents the key requirements for future research. These include establishing the effects of processing, bioavailability and metabolism, developing physiologically appropriate in vitro models, standard methods of analysis and appropriate clinical biomarkers. The future of flavonoid research will undoubtedly depend upon the resolve of these issues, and although the field has shown continuous progress for many years, progress will likely slow if these challenges are not met.