Cremonini E, Daveri E, Iglesias DE, et al. A randomized placebo-controlled cross-over study on the effects of anthocyanins on inflammatory and metabolic responses to a high-fat meal in healthy subjects. Redox Biol. 2022;51:102273.
To assess the effects of a supplement rich in anthocyanins (the water-soluble forms of anthocyanidins) on parameters of inflammation (ie, endotoxemia) as well as lipid and glucose metabolism following a high-fat meal
Anthocyanins lessened endotoxemia and several postprandial blood parameters associated with dysmetabolism after high-fat meal consumption in healthy adults.
Double-blind, randomized, placebo-controlled crossover trial
A total of 25 healthy volunteers (11 male; 14 female), aged 19 to 35 years with a body mass index (BMI) between 21 and 29.9 kg/m2, participated in this study.
Since this study is on “healthy” participants, the exclusion criteria were quite extensive: high blood pressure (systolic blood pressure ≥160 mm Hg or diastolic blood pressure ≥95 mm Hg), abnormal fasting glucose (<50 mg/dL or >100 mg/dL), high fasting triglycerides (>150 mg/dL), severe or incompatible dietary restrictions (eg, vegetarianism), current use of agents that may interfere with the blood parameters (herbal supplements, anti-inflammatory medications, or medications that interfere with insulin metabolism), regular participation in endurance exercise activities, tobacco use of any kind within the previous year, heavy alcohol daily intake, or any substance abuse or dependence.
Many medical conditions were also exclusionary: history of stroke, hepatic, kidney, or thyroid disease or cancer; gastrointestinal (GI) tract disorders (including malabsorption) or GI surgery; severe eating disorders; psychiatric conditions (depression, anxiety, or other); diarrhea or oral antibiotic intake within the last 4 weeks; weight change (>5%) in the last 8 weeks; allergy or sensitivity to components in the study’s anthocyanin formula (CDRE) or high-fat meal (HFM).
This is a crossover trial in which each subject ingests a high-fat meal on 2 separate occasions, separated by a washout period of at least 7 days but less than 30 days. On each of these occasions, participants ingested either the interventional agent or a placebo during the HFM.
The HFM consisted of an English muffin, sausage, egg, and cheese, with carotenoid-free palm oil added to bring the total dietary fat to the desired level. Total caloric composition per meal was 1,026 kcal (70.5 g total fat with 29.8 g saturated fat, 270 mg cholesterol, 70.2 g carbohydrate, and 33 g protein.) The percentage of calories for each macronutrient was 62% from fat, 25% from carbohydrates, and 13% from protein.
The intervention was a cyanidin- and delphinidin-rich extract (CDRE) powder (4 g): 1 g of AC-rich extracts (150 mg bilberry extract, 230 mg black currant extract, and 620 mg black rice extract) and 3 g of a mix of maltodextrins. The CDRE powder contained 320.4 mg/g anthocyanidin content with 52% cyanidin and 38% delphinidin glucosides, for a total of 90%. (Other anthocyanins made up the remaining 10%)
The placebo powder (4 g) consisted of 3.85 g of the same maltodextrins included in the CDRE, 125 mg of Red Dye No. 40, and 25 mg of Blue Dye No. 1.
Participants were asked to fast 12 hours overnight before each visit, to not consume polyphenol-rich foods for 24 hours before each study visit, to consume a low-fat meal at dinner the night before the HFM visit, and to complete a 3-day food record before visits 1 and 2.
Blood was collected before consuming the HFM to obtain baseline (T0) and ensure each participant was fasting. Blood was then collected at 0.5, 1, 2, 3, and 5 hours postprandially.
Study Parameters Assessed
The following parameters were assessed:
- lipopolysaccharides (LPS)/LPS-binding protein (LBP)
- free fatty acid (FFA)
- gastric inhibitory polypeptide (GIP)
- glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2)
- insulin, leptin, adiponectin, and ghrelin
- lipids (triglycerides, total, high-density lipoprotein [HDL], and calculated low-density lipoprotein [LDL])
- peripheral blood mononuclear cells (PMBCs), used to determine expression of inflammation/oxidation intermediaries (tumor necrosis factor alpha [TNF-α], NOX4, JNK [c-Jun N-terminal kinases] phosphorylation)
- metabolites of anthocyanins with mass spectrometry (MS)
The primary endpoint of this study was to assess whether CDRE could attenuate postprandial endotoxemia induced by consumption of a HFM.
Not surprisingly, metabolites of anthocyanidins were higher when participants consumed the CDRE vs placebo. Overall, the peak in concentration was 0.5 to 1.0 hour after CDRE ingestion with elevated levels for 2 to 3 hours and resumption to baseline at 5 hours with little exception.
LPS and its binding protein (LBP) were the markers of endotoxemia. Area under the curve (AUC) values for LPS over the 5-hour postprandial period were 44% lower (0.38 ± 0.15 vs 0.68 ± 0.12 EU/mL x 5 h) when participants received CDRE vs when they received placebo (P=0.04).
The CDRE effect on blood glucose AUC was significant (P<0.02) with the mean glucose concentrations for the CDRE meal 42% lower than for the placebo meal across the 5-h post-HFM period (23.0 ± 4.4 vs 39.6 ± 8.0 mg/dL, respectively).
Unlike plasma glucose, there was no indication of a CDRE effect for the insulin AUC across the 5-h post-HFM period (73.1 ± 9.6 and 79.1 ± 9.4 mU/L, for the CDRE and placebo groups respectively).
Triglycerides: There was a significant CDRE effect for AUC (P=0.03). The concentration of plasma triglycerides was lower for the CDRE than for the placebo (161.5 ± 18.5 vs 203.5 ± 29.6 mg/dL for 5 h).
Total cholesterol: The AUC for total cholesterol was lower with the CDRE meal than for the placebo (31.2 ± 7.0 vs 57.0 ± 10.0 mg/dL for 5 h, respectively, P<0.06).
There were no differences in AUC between the 2 meals for LDL- and HDL-cholesterol.
Interestingly, free fatty acids decreased between baseline and 30-min post-HFM in both groups (P<0.05) and remained low, not returning to baseline after 5 hours. There was no effect of CDRE on the values of free fatty acids at the different times.
Of the gut-secreted hormones, only GIP showed a significant difference with CDRE. For GIP, the AUC for the CDRE group was 13% lower than for the placebo group (0.77 ± 0.06 vs 0.89 ± 0.08 μM; P=0.05). No differences in AUC were observed for GLP-1 or GLP-2 between groups. AUC were similar for CDRE and placebo for leptin, ghrelin, and adiponectin.
Inflammation & oxidative stress markers (PBMCs)
The consumption of the HFM and placebo led to increases in interleukin 8 (IL-8, 51%), TNF-α (45%), and NOX4 (74%, P<0.04) mRNA levels. With CDRE consumed, there was no significant HFM-mediated increases in TNF-α or NOX4, while the increase in IL-8 was also not affected. IL-18, IL-1β, TLR4, and NOX2 did not differ between CDRE and placebo.
Disclosures as stated in the publication were “SNH, SMW, and RG are, and AM was employed by Pharmanex Research, NSE Products Inc., Provo, UT, USA. CGF and PIO have received research grants from NSE Products Inc. as well as from other food companies and government agencies with an interest in health and nutrition. CGF and PIO are members of the NSE Products Inc. Scientific Advisory Board.” The clinicaltrials.gov identifier is NCT03309982.
Practice Implications & Limitations
In this study, the addition of anthocyanidins lessened the detrimental metabolic effects of a high-fat meal (HFM) in healthy volunteers. Of note, the quantity of anthocyanidins ingested during the test meal is feasible with some focused effort on choosing high-anthocyanin foods. Given the simplicity of this “intervention” and social pressures to eat less than ideally at times, this study has real-world application for mitigating the dysmetabolism of occasional dietary indiscretions.
This study used postprandial metabolic parameters, which may be a more accurate assessment of dysmetabolism than the routine use of many of these parameters in the fasting state. Postprandial dysmetabolism is a risk factor for many diseases, especially cardiovascular diseases and type 2 diabetes.1 Several medical authorities outside the US, including in the UK, Denmark, Canada, and Europe, have proposed that nonfasting metabolic parameters are an effective screening tool for dysmetabolism in primary care.2 While not officially standardized, an “oral fat tolerance test” (OFTT) has been used to research dyslipidemia for more than 30 years and may reflect metabolic efficiency more accurately.3
The study under review here used OFTT in the form of an English muffin, sausage, egg, and cheese with added palm oil (62% of total calories). As predicted, LPS, a marker of endotoxemia, rose with the high-fat meal (HFM) and set off a cascade of dysmetabolic effects known to raise cardiometabolic risk.4,5 The lessening of dysmetabolism by phytochemicals is 1 of the ways that plant-based diets are assumed to increase longevity.6 Of the many classes of phytochemicals that may be responsible for this effect, perhaps none has been better studied than polyphenols.
Chemistry aside, the simplest means of helping patients find anthocyanins is to have them look for red, blue, and purple plant foods.
As a class of compounds, polyphenols include thousands of different phytochemicals, broken down into flavonoids (flavones, flavonols, flavan-3-ols, flavanones, isoflavones, and anthocyanins) and nonflavonoids (phenolic acids, stilbenes, lignans, and “others”).7 There is a synergistic effect when consuming various polyphenols at once in whole foods, such that the effective dose from a mixture is likely lower than any single type of polyphenol being tested in a research setting.8 Adding yet another layer of complexity to research on phytochemicals, the gut microbiota produces active secondary metabolites, which are influenced by the type of microbes inhabiting the gut.9
That said, the medical literature leans heavily toward flavonoids being the polyphenol type that renders many of the benefits of plant foods. Among the subtypes of flavonoids, anthocyanidins have long been studied for their ability to reduce risk of chronic degenerative diseases. A 2021 review of 44 randomized controlled trials and 15 cohort studies found that anthocyanidin intake correlated with lower coronary artery disease, cardiovascular disease (CVD) incidence, and CVD deaths.10
Anthocyanins are a subgroup of flavonoids that contains over 500 different water-soluble compounds.11 The name comes from the Greek word for flower, anthos, and the word kyanous, which means dark blue.12 Traditionally used as pigments or dyes, the color range spans the spectrum from red-orange (pH<3) to blue-green (pH>11), with red-purple-blue naturally occurring (pH 6-8).13 The term anthocyanidin, which is often used interchangeably with anthocyanin, actually denotes the chemical structure when it is lacking a sugar moiety. In chemical terms, anthocyanins are the glycosylated forms of anthocyanidins (aglycones).
Chemistry aside, the simplest means of helping patients find anthocyanins is to have them look for red, blue, and purple plant foods. Often, the color is their name (blueberries, red raspberries, purple cabbage, purple potatoes). When the name has “black” in it, that actually indicates a deep purple (blackberries, black currants, black rice). Another patient-friendly tool to help gauge the anthocyanin content in each plant food is this: If it will stain your clothing red, blue, or purple, then it is high in anthocyanins. Berries, fresh or frozen, are 1 of the most practical sources.
The study under review here used 320.4 mg of anthocyanidins per test meal. An equivalent amount of anthocyanidins can be easily added with side dishes, berry-focused desserts, salad fixings, and toppings or garnishes. Some examples include purple corn (68–1,642 mg AC/100 grams), blackberries (83–326 mg/100 grams), red cabbage (250–322 mg/100 grams), and pomegranate juice (9–765 mg/L). A combination of several different red/blue/purple plant foods can add up to 320 mg quickly. In comparison, a study in 2006 found the typical US person consumed merely 12.0 mg/day of anthocyanins.14
At first, this study may not seem like a game changer for practicing integrative-medicine clinicians. One of the most consistent findings in epidemiology is that communities with greater longevity eat a diet rich in colorful vegetables and fruits. But with the meager anthocyanin intake, maybe adding them to the meal, no matter what else the meal contains, will provide some benefit?
As clinicians, even in the absence of conclusive evidence, we must give patients our opinion. “Eat the rainbow” is common dietary shorthand for eating a varied and colorful plant-based diet. This study implies that we should consider recommending specifically high-anthocyanin foods, particularly if the overall meal is nutrient-poor and high in fat. This is relevant for all clinicians since high-fat/low-nutrient meals are typical Western-diet choices: a burger and fries, steak and potatoes, pancakes and bacon, eggs and home fries, and nearly any fast-food meal. While none of these may be an ideal meal, meeting people where they are is part of every clinician’s responsibility in serving their patients.
Lastly, one of the toughest things to do is to change old habits, including dietary habits. Mealtime comes with all sorts of social and cultural expectations. Food is comfort. Food is customary. Food can be ceremony. Food can convey love. In short, a shared meal is far more than the nutrients it contains (or doesn’t). There are times when the ceremony of a meal takes precedence over its nutritional value. During those times, when the meal may be perfect in every way but nutrition, bringing high-anthocyanin food(s) to the table may be a clever means of having your cake and eating it too.