García-Cordero J, Martinez A, Blanco-Valverde C, Pino A, Puertas-Martín V, San Román R, de Pascual-Teresa S. Regular consumption of cocoa and red berries as a strategy to improve cardiovascular biomarkers via modulation of microbiota metabolism in healthy aging adults. Nutrients. 2023;15(10):2299.
To evaluate the effects of red berry anthocyanins and cocoa flavanols, both separately and together, on biomarkers of cardiovascular health
Randomized, parallel-group study, single-blinded to the research team and lasting 12 weeks
Sixty adults, aged 45 to 85 years, were enrolled in the study. One participant dropped out, so the final analysis included a total of 59. One-fifth of the participants were chronic smokers.
The volunteers were divided into 3 groups:
- Group 1 consumed 5 g/day of a mixture of “red berries” containing 104 mg total polyphenols and 13.9 mg total anthocyanins.
- Group 2 consumed 2.5 g/day of a polyphenol-rich cocoa powder containing 9.6 mg flavanols, including 4.7 mg epicatechin.
- Group 3 consumed the same amounts of both cocoa and red berries, for a total of 7.5 g/day.
The product was prepared and supplied by Salengei®, a Spanish manufacturer of nutritional supplements that sells these cocoa and berry extracts as part of their regular product line. (The product is not commercially available in the United States.)
The cocoa powder was free of sugar, sweeteners, and emulsifiers and contained 7,899 mg total polyphenols, 668 mg theobromine, and 275 mg caffeine per 100 g cocoa powder. The cocoa powder, in the amount consumed by the volunteer, provided a daily intake of 197.5 mg total polyphenols. Additionally, the cocoa powder contained 383.6 mg total flavanols per 100 g, which provided volunteers with a daily intake of 9.6 mg flavanols, including 4.7 mg epicatechin.
Red berry mixture
The second product was a red berry mixture made of pure, dried redcurrants (33.3%), blackcurrants (33.3%), raspberries (16.7%), and blueberries (16.7%). The determination of total polyphenols and anthocyanins showed that the red berry mixture contained 2,079 mg total polyphenols and 277.7 mg total anthocyanins in 100 g of the product, which provided the volunteers with a daily intake of 104 mg total polyphenols and 13.9 mg total anthocyanins, including 4.8 mg delphinidin-3-O-rutinoside and 3.5 mg cyanidin-3-O-rutinoside.
Study Parameters Assessed
The cardiovascular biomarkers used in this study included homocysteine, angiotensin-converting enzyme (ACE), nitric oxide (NO), flow-mediated vasodilation (FMD), blood pressure, and lipid profile. Additionally, to assess the effect on the microbiome, investigators also measured certain metabolites, such as secondary bile acids (SBAs), short-chain fatty acids (SCFAs), and trimethylamine N-oxide (TMAO).
Each participant had 3 visits at the clinic. The first was to determine eligibility, the second for baseline measurements of the above parameters, and the final visit was at the end of the 12-week study. At each visit, investigators took an early morning urine sample, a 20-mL blood sample, weight, height, and waist circumference (anthropometric measurements), blood pressure, and 24-hour diet records. They tracked compliance by phone every 2 weeks throughout the study duration.
Changes compared to baseline in the following cardiovascular biomarkers:
- Nitric oxide
- Blood pressure
Secondary outcomes included changes in the microbiota-related metabolites, including:
The group that consumed cocoa showed a significant reduction in TMAO (P=0.03) and uric acid (P=0.01) levels in serum, accompanied by an increase in FMD values (P=0.03) and total polyphenols, corrected by creatinine (P=0.03) after the intervention. These latter values negatively correlated with the TMAO concentration (r=–0.57, P=0.02). Additionally, investigators observed an increase in carbohydrate fermentation in the groups that had consumed cocoa (P=0.04) and red berries (P=0.04) between the beginning and the end of the intervention. This increase in carbohydrate fermentation correlated with lower ratios of total cholesterol (TC) to high-density lipoprotein (HDL) (P= 0.01), systolic blood pressure (P=0.01), and diastolic blood pressure (P=0.01). The results showed a positive modulation of microbiota metabolism after a regular intake of cocoa flavanols and red berry anthocyanins, which led to an improvement in cardiovascular function, especially in the group that consumed cocoa.
This trial was registered at ClinicalTrials.gov, NCT04348162. The authors declared no conflict of interest. Funding was through the Spanish National Research Council.
We often speak of synergy and how the whole is more than the sum of its parts, and yet we are usually constrained by research that examines natural agents in isolation, especially in studies on specific foods or nutraceuticals. This study is a refreshing exception: It looks at the combined effects of both cocoa and berries.
We should point out what wasn’t seen in this study’s results. The authors report, “We found no significant difference between the groups at baseline in each of the parameters analyzed. Additionally, we found no significant differences between groups at the end of the intervention in the concentrations of total serum protein, NO, homocysteine, TMAO and ACE activity corrected by total protein ... Between visits, we only observed a statistically significant increase for FMD values, in the cocoa powder group (P=0.03) and a decrease in TMAO levels after the 12-week intervention in the C group (P=0.03). We did not find any other significant change in any of the analyzed parameters between baseline and 12-weeks for any of the diets.” The berries on their own appear to have no benefit, and their addition to the cocoa appears to have negated its effect on FMD and TMAO.
Keep in mind that the participants were only assigned to 3 possible interventions: cocoa, berries, or cocoa plus berries. There was no placebo/control group. The impact on FMD and TMAO was significant only in the cocoa group. It appears that the red berry powder did not add to the benefit.
Improvements in biomarkers of cardiovascular disease with cocoa is in keeping with recent outcomes in a large, randomized, double-blind, placebo-controlled trial. In a study involving 21,442 older Americans (12,666 women) between 2015 and 2020, Sesso et al looked at the consumption of cocoa extract for the prevention of cardiovascular disease (CVD).1 Participants were randomly assigned to cocoa extracts (500 mg flavanols/d, including 80 mg (-)-epicatechin) or placebo. In Sesso’s study, during a median follow-up of 3.6 years, 410 participants taking cocoa extract and 456 taking placebo had confirmed total cardiovascular events (HR: 0.90; 95% CI: 0.78, 1.02; P=0.11). For secondary endpoints, HRs were 0.73 (95% CI: 0.54, 0.98) for CVD death, 0.87 (95% CI: 0.66, 1.16) for MI, and 0.91 (95% CI: 0.70, 1.17) for stroke. Although cocoa supplementation did not achieve statistical significance in reducing total cardiovascular events among these older adults, it did reduce CVD death by 27%. That should still be adequate to encourage most adults to consume a bit more cocoa. It also is a solid argument for the protocol in this current (or dare I write currant?) paper under review that combined cocoa with berry extracts.
While the cocoa findings in this study are in keeping with what we expect, results of the mixed berry group conflict with some of the data found in prior studies. For example, earlier publications have suggested that flavanols and anthocyanins lower homocysteine in those with insulin resistance.2 However, the lack of change in these parameters in this study is not unexpected as the participants were healthy.
The mixed berry powder in this study was unique. The berry powder contained 2/3 currants (dried redcurrants [33.3%], blackcurrants [33.3%], raspberries [16.7%], and blueberries [16.7%]). Currants are native to western Europe, and though they are grown in the United States, they are relatively uncommon in the American diet. Dried currants are available, but those too are rarely eaten here. When a baking recipe calls for dried currants, I substitute raisins. Admittedly, they are not nutritional equivalents.
The product used in this trial was not inexpensive. This study did not require study participants to consume large quantities of these powders. Those who received both cocoa and fruit powders were asked to consume just 7.5 grams per day. That’s about a quarter of an ounce. If you try to consume plain cocoa, you will quickly discover that its flavor strongly asks to have some sweetener added. Thus, the addition of dried fruit powders might be appreciated.
The results from this study do suggest that older adults should be advised to add a spoonful of powdered cocoa daily. Nevertheless, we might expect less-than-complete compliance to even this suggestion, so instead we might wonder if there is some food stuff that might be substituted, some sort of fruit-and-chocolate candy or cookie-like morsel, that might be rationed out daily?
Cardiovascular disease remains the second leading cause of death worldwide, second only to cancer. This is a disease of ageing; the vast majority of those afflicted are over the age of 60. This disease progresses with age and involves increasing dysfunction of the vascular endothelium, along with increased arterial thickening and stiffness. Dietary polyphenols appear to lessen the effects of ageing, in particular preventing decline of cognitive function3 and the development of CVD.5-7
Consumption of foods high in flavanols—for example tea,8 apples,9 red wine,10 and chocolate11—is negatively associated with CVD risk. This is seen even in those who have already suffered a cardiovascular event.12
A 2016 meta-analysis conducted by Lin et al reported that cocoa flavanol intake significantly improved various cardiovascular biomarkers, such as fasting insulin, insulin resistance, triglycerides, HDL-C, C-reactive protein, and vascular cell adhesion molecule 1 (VCAM-1).13 A separate meta-analysis by Arab et al reported that tea consumption (3 cups vs less than 1 cup per day) reduced the risk of suffering a heart attack by 21%.14
It is thought that consuming flavanols and anthocyanins shifts the gut microbiome in positive ways.15,16 Even chocolate’s positive effect on mood and emotions is attributed to shifts in bacterial populations.17 The results of this current clinical trial serve to further support these theories.
Attention has shifted to the impact these foods have on the intestinal microbiome; the thinking is that changes in the gut flora are responsible for some of the benefits associated with consumption.18 For example, trimethylamine N-oxide (TMAO) is formed by gut bacteria from choline-rich foods, and TMAO blood levels have been linked with CVD risk in large-scale clinical studies.19 In years past, our focus was on dietary choline levels, but that has shifted to whether TMAO-producing gut bacteria are present or not. Thus, this study is notable in that it may be the first to show that cocoa lowers TMAO levels in humans. Cocoa’s effects on gut bacteria, reducing TMAO production, may explain its well-documented benefit against CVD. This may be the most important finding of this study.
The study authors suggest that another way that the gut microbiome may change CVD risk is through production of secondary bile acids and short-chain fatty acids. Both may act as hormone-like molecules affecting inflammation and glucose and fat metabolism. Only those consuming red berries exhibited a significant difference in bile acids after 12 weeks, so this does not appear to explain the changes in TMAO levels. There were slight increases in most of the short-chain fatty acids in all the intervention groups. A significant negative correlation was seen between total polyphenol levels and TMAO levels (P=0.02), so high polyphenol intake was associated with lower TMAO level.
The study authors list a number of known pathways by which flavanols have been theorized to protect against CVD including:
- Antioxidant and chelating properties (ie, inactivating ROS and preventing the oxidation of LDL)
- Reducing inflammation of the blood vessel wall by inhibiting the influx of leucocytes
- Decreasing the activity of enzymes related to increased ROS production and oxidative damage (eg, xanthine oxidase, NADPH oxidase) and inflammation (eg, 15-lipoxygenase, COX-2)
- Reducing the synthesis of pro-inflammatory molecules
- Suppressing the activity of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA), which plays a key role in the synthesis of cholesterol
- Possessing antiobesity properties, improving lipid profile, and decreasing insulin resistance
- Helping seal and reinforce blood vessel walls by enhancing collagen synthesis
This study certainly suggests that the key action in cocoa’s protective effect may be shifting the gut microbiome to decrease TMAO levels.
What the study’s authors don’t mention is this possible explanation. Cocoa is a fermented food product. Bacterial or fungal metabolites may remain from that initial fermentation of the cocoa beans that are still active, and these may act to hinder or enhance growth of specific gut bacteria in the gut microbiome of those who consume the finished product.
A recent analysis of the microorganisms involved in cocoa bean fermentation suggests a complex ecosystem of 99 separate organisms that vary by geographical growing region.20
It might be that somewhere in this mix of microbial participants is an organism that hinders TMAO-producing bacteria or that enhances the growth of other organisms that do so.
Admittedly, this is a new thought on my part, and there are no citations yet that I might share from others thinking along the same line and publishing their research.
A recent analysis of the microorganisms involved in cocoa bean fermentation suggests a complex ecosystem of 99 separate organisms that vary by geographical growing region.
Some of the key bacteria responsible for this fermentation may someday be proven to have specific value, even if at this point we see no hint in the literature that they are being investigated to prevent CVD. We must remember, though, that the major chocolate companies tend to do proprietary research. For example, both the Mars and Callebaut companies market proprietary chocolates that they claim are manufactured in a way to enhance health benefit.
The other question I find myself chewing on is what to do with this information when it comes to clinical practice, or even my own eating habits. The idea of routine consumption of chocolate polyphenols has been encountered before. There has been controversy in the past as we have sought a standardized dose of polyphenols to recommend. Perhaps we have misattributed benefit to these polyphenols, and chocolate doses should be standardized to some still-unidentified fermentation metabolite? This study’s results suggest that the berry-derived anthocyanins may be less crucial than previously thought in preventing CVD. That thought should be treated with caution as results have varied so much between studies that data from this single study should not be considered definitive. The recommendation that people, particularly those at risk for heart disease because of age, should consume with some regularity cocoa or products derived from cocoa continues to be reasonable.
The idea that, since we consider cocoa and chocolate fermented foods, at least some of their benefits may be due to their influence on the gut microbiome is probably the greatest take-home from this study.