1School of Public Health (Shenzhen), Sun Yat-sen University, Guangzhou, China

2Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Sun Yat-sen University, Guangzhou, China

3Guangdong Engineering Technology Center of Nutrition Transformation, Sun Yat-sen University, Guangzhou, China

4China-DRIs Expert Committee on Other Food Substances, Sun Yat-sen University, Guangzhou, China

1School of Public Health (Shenzhen), Sun Yat-sen University, Guangzhou, China

2Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Sun Yat-sen University, Guangzhou, China

3Guangdong Engineering Technology Center of Nutrition Transformation, Sun Yat-sen University, Guangzhou, China

4China-DRIs Expert Committee on Other Food Substances, Sun Yat-sen University, Guangzhou, China

1School of Public Health (Shenzhen), Sun Yat-sen University, Guangzhou, China

2Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Sun Yat-sen University, Guangzhou, China

3Guangdong Engineering Technology Center of Nutrition Transformation, Sun Yat-sen University, Guangzhou, China

4China-DRIs Expert Committee on Other Food Substances, Sun Yat-sen University, Guangzhou, China

1School of Public Health (Shenzhen), Sun Yat-sen University, Guangzhou, China

2Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Sun Yat-sen University, Guangzhou, China

3Guangdong Engineering Technology Center of Nutrition Transformation, Sun Yat-sen University, Guangzhou, China

4China-DRIs Expert Committee on Other Food Substances, Sun Yat-sen University, Guangzhou, China

1School of Public Health (Shenzhen), Sun Yat-sen University, Guangzhou, China

2Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Sun Yat-sen University, Guangzhou, China

3Guangdong Engineering Technology Center of Nutrition Transformation, Sun Yat-sen University, Guangzhou, China

4China-DRIs Expert Committee on Other Food Substances, Sun Yat-sen University, Guangzhou, China

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

Objective: The associations between intake of anthocyanins and anthocyanin-rich berries and cardiovascular risks remained to be established. We aimed to quantitatively summarize the effects of purified anthocyanins and anthocyanin-rich berries on major surrogate markers of cardiovascular diseases (CVDs) and the longitudinal associations between dietary anthocyanins and CVD events.

Methods: Meta-analysis of randomized controlled trials (RCTs) and prospective cohort studies.

Results: We included 44 eligible RCTs and 15 prospective cohort studies in this study. Pooled analysis of RCTs showed that purified anthocyanin supplementation could significantly reduce blood LDL cholesterol (weighted mean difference (WMD): −5.43 mg/dL, 95% CI: −8.96, −1.90 mg/dL; p = 0.003) and triglyceride (WMD: −6.18 mg/dL, 95% CI: −11.67, −0.69 mg/dL; p = 0.027) while increase HDL cholesterol (WMD: 11.49 mg/dL, 95% CI: 7.43, 15.55 mg/dL; p < 0.001) concentrations. Purified anthocyanins also markedly decreased circulating tumor necrosis factor alpha (WMD: −1.62 pg/mL, 95% CI: −2.76, −0.48 pg/mL; p = 0.005) and C-reactive protein (WMD: −0.028 mg/dL, 95% CI: −0.050, −0.005 mg/dL; p = 0.014). Besides, administration of anthocyanin-rich berries could significantly lower blood total cholesterol (WMD: −4.48 mg/dL, 95% CI: −8.94, −0.02 mg/dL; p = 0.049) and C-reactive protein (WMD: −0.046 mg/dL, 95% CI: −0.070, −0.022 mg/dL; p < 0.001). Neither purified anthocyanins nor anthocyanin-rich berries could cause any substantial improvements in BMI, blood pressure, or flow-mediated dilation. In addition, meta-analysis of prospective cohort studies suggested that high dietary anthocyanins were related to lower risk of coronary heart disease (CHD) (relative risk (RR): 0.83, 95% CI: 0.72, 0.95; p = 0.009), total CVD incidence (RR: 0.73, 95% CI: 0.55, 0.97; p = 0.030), and total CVD deaths (RR: 0.91, 95% CI: 0.87, 0.96; p < 0.001).

Conclusion: Habitual intake of anthocyanins and anthocyanin-rich berries could protect against CVDs possibly via improving blood lipid profiles and decreasing circulating proinflammatory cytokines.

Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO, identifier: CRD42020208782.

Cardiovascular diseases (CVDs) remain the leading cause of premature death globally, which have exerted persistent and tremendous burdens on healthcare systems in the recent decades (1). Predominant risk factors of CVDs include but not restrict to overweight, hypertension, and elevated blood atherogenic lipoproteins (2). It is estimated that successful management of blood lipids could lead to about 30% less CVD events among Chinese hypertensive adults (3). Besides, CVDs could result in over 60% deaths in patients with diabetes, and they suffered from a worse prognosis for survival than patients with CVD without diabetes (4). Circulating biomarkers of chronic low-grade inflammation, such as C-reactive protein (CRP) and tumor necrosis factor alpha (TNF-α), could also serve as independent predictors of future CVD events (5).

Diet modification is the pivotal strategy for CVD prevention (6, 7). Firm epidemiological evidence has established strong inverse associations between CVD risks and dietary intake of plant foods and plant-based bioactive constitutes (8–10). Anthocyanins are polyphenolic pigments, which are rich in dark-colored plant foods including berries, grapes, onions, and black rice (11, 12). Increasing research interest has focused on the health benefits of anthocyanins and anthocyanin-rich foods (13–15). Owing to rich hydroxyl groups in their chemical structures, anthocyanins also represent one of the largest families of phenolic pigments with antioxidant and anti-inflammatory properties (16). Habitual consumption of anthocyanins and anthocyanin-rich foods was suggested to reduce the risks of various chronic diseases including CVD, neuroinflammatory process, and liver steatosis (17). A previous meta-analysis of prospective studies found that frequent intake of anthocyanin-rich foods was related to 9% lower risk of coronary heart disease (CHD) (18). Both clinical and preclinical investigations have demonstrated strong lipid-lowering effects of anthocyanins (19, 20). In addition, anthocyanin intake could substantially improve endothelial function and alleviate arterial stiffness among subjects with high cardiovascular risks (21). However, the effects of anthocyanins on adiposity, blood pressure, and chronic low-grade inflammation were still conflicting (20, 22, 23). Our recent study unraveled that anthocyanins could dose-dependently reduce blood ceramides, newly identified predictors of CVDs, in the dyslipidemia subjects (24).

Berries comprised of about 10% of total fruit consumption in the United States (25) and served as the main dietary sources of anthocyanins regardless of commercialized anthocyanin supplements (11). Although the anthocyanin contents vary dramatically across berry species and are profoundly influenced by cultivation, preservation, and processing (11), blueberry, cranberry, bilberry, and blackcurrant basically rank the most plentiful in anthocyanins among berry fruits, which could contain 100 to 200 mg anthocyanins per 100 g edible portions (Supplementary Table 1). In turn, anthocyanins composed the largest proportions of bioactive polyphenols in ripe berries and were suggested to make the greatest impacts on the physiological improvements from berry intake (11, 26). Regular consumption of anthocyanins and anthocyanin-rich berries has been widely recommended due to their potential cardioprotective benefits (26, 27), even though the associations and causal effects were still elusive (28, 29). Therefore, we aimed to quantitatively summarize current eligible randomized controlled trials (RCTs) and prospective cohort studies to investigate the associations of anthocyanins and major anthocyanin-rich berries with cardiovascular health in this study.

The present meta-analysis was reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement (30). This study has been registered at the International Prospective Register of Systematic Reviews (PROSPERO, registration ID: CRD42020208782).

Two investigators (LX and HC) searched the PubMed, Embase, and Cochrane Library for eligible studies up to December 31, 2020. Literature search for RCTs and prospective cohort studies were conducted independently. Because we did not identify any RCTs reporting CVD events in the preliminary search, we alternatively focused on the major surrogate markers of CVD in the meta-analysis of RCTs. For RCTs, the search terms were anthocyanins or anthocyanin-rich berries combined with major CVD risk factors including adiposity, blood pressure, blood lipids, and inflammation (see Online Supplementary Materials for details). For prospective cohort studies, the search terms were anthocyanins combined with fatal or non-fatal CVD events including CHD, stroke, total CVD incidence, and total CVD mortality (see Online Supplementary Materials for details). Because the anthocyanin intakes in observational studies were generally derived from various food items in diet records or food frequency questionnaire (FFQ) in our preliminary literature search, we only analyzed the relationship between anthocyanin intake and CVD events regardless of their dietary sources. We also searched reviews and meta-analysis articles concerning the effects of anthocyanins and anthocyanin-rich berries on cardiovascular health. Literature search was restricted to those published in English. We screened the titles and abstracts of all retrieved publications and then determined the eligibility via checking the full text.

Two investigators (LX and HC) independently performed study inclusion and exclusion. Any discrepancies were resolved by discussion with other research team members until a consensus was reached. For RCTs, studies were included if they meet the following criteria: (1) were either parallel- or crossover-designed; (2) conducted in adults; (3) with a intervention duration longer than 2 weeks; (4) used purified anthocyanins or anthocyanin-rich berries including blueberry, cranberry, bilberry, and blackcurrant as the intervention approach; (5) adopted placebo or other adequate controls as the comparators; and (6) provided sufficient data for calculating changes in any of the following CVD biomarkers before and after intervention: BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), flow-mediated dilation (FMD), total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), triglyceride (TG), CRP, and TNF-α. Studies were excluded if they (1) were acute feeding trials; (2) conducted in pregnant or lactating women, or critically ill patients (e.g., subjects with advanced cancer, end-stage cardiac insufficiency, or end-stage nephropathy); (3) had a multifactorial design; and (4) used crude plant or herb extractives as the intervention approach making it difficult to isolate the effects of anthocyanins or anthocyanin-rich berries.

For prospective cohort studies, studies were included if they (1) were prospective cohort studies; (2) conducted in adults; (3) reported baseline dietary anthocyanin intake as the exposure; (4) reported fatal or non-fatal CVD events as the outcome, including CHD incidence and mortality, stroke incidence and mortality, total CVD incidence, and total CVD mortality; and (5) provided relative risk (RR) or hazard ratio (HR) with corresponding 95% confidence intervals (CIs) or sufficient data to calculate them. Studies were excluded if they were case-control or retrospective studies.

Quality assessments of eligible RCTs and prospective cohort studies were performed according to the National Heart, Lung, and Blood Institute (NHLBI) Quality Assessment of Controlled Intervention Studies and the NHLBI Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, respectively. A study was considered as high quality if it met at least 11 of the 14 criteria (about 80%), otherwise it was regarded as low to moderate quality.

We estimated heterogeneity among studies using the Cochrane's Q test, and a p-value < 0.1 or a I2 statistic >50% indicated substantial between-study heterogeneity. Pooled estimates were calculated using the DerSimonian–Laird random-effects model to address potential between-study heterogeneity. Statistical significance set at a p-value < 0.05. For RCTs, crossover studies were treated as parallel studies in a way that each intervention phase was treated as an independent arm of a parallel study. For prospective cohort studies, HRs were treated as RRs. To explore the sources of potential between-study heterogeneity, we performed pre-specified subgroup analysis stratified by study characteristics. We evaluated the robustness of pooled estimates via leave-one-out sensitivity analysis. We assessed the publication bias using funnel plots and also the Begg's tests. The trim and fill methods were used to correct theoretically missing studies, if any. All statistical analyses were performed in Stata/SE version 16.0 (College Station, Texas, US).

We identified a total of 44 eligible RCTs consisting of 52 comparison groups and 2,353 subjects in the present meta-analysis (Supplementary Figure 1). Detailed characteristics of included studies can be found in Supplementary Table 2. Briefly, 15 of the included studies investigated the effects of purified anthocyanins, all of which were produced from berries. For the remaining anthocyanin-rich berry studies, interventions were blueberry in 13 studies, cranberry in 12 studies, bilberry in three studies, and blackcurrant in one study. Seven of the 44 studies were crossover trials with the rest parallel-designed. Most studies were conducted in Asia (n = 12), Europa (n = 14), and the United States (n = 15). The intervention durations ranged from 2 weeks to 24 months with a median of 8 weeks. Thirty-one of the included studies recruited subjects that were at high risks of CVDs such as patients with obesity, dyslipidemia, diabetes, and history of CVDs. Nearly half of the included studies (n = 21) clearly claimed that they received research grants from berry industry or industry associations.

We included 15 eligible prospective cohort studies including 16 independent cohorts and 5,54,638 subjects in the present meta-analysis (Supplementary Figure 2). Briefly, seven of the included cohort studies were conducted in the United States with another three in Australia and four in Europa. The follow-up periods ranged from 4.3 to 24 years with a median of 12 years. Most of the included cohort studies used FFQ to assess dietary anthocyanin intake and only three of them used dietary records (31–33) (see Supplementary Table 3 for detailed study characteristics).

Allocation concealment was adequate in 35 of the 44 included RCTs (Supplementary Tables 4, 5). Group assignment was sufficiently blind to both participants and clinical investigators in 33 studies. The overall dropout rates at end point were Open in a separate windowFigure 1

Forest plot for the pooled effects of purified anthocyanins on circulating low-density lipoprotein cholesterol stratified by anthocyanin doses. Between-study heterogeneity was examined using the Cochrane's Q test. The diamonds represented the pooled effect sizes which were calculated using the DerSimonian–Laird random-effects model. WMD, weighted mean difference.

Pooled effects of purified anthocyanins and anthocyanin-rich berries on circulating LDL cholesterol.