Cardioprotective effects of anthocyanins

By Roberto Mattioli, Antonio Francioso, Luciana Mosca and Paula Silva

See the full paper at: https://doi.org/10.3390/molecules25173809

Increased lifespan and a better quality of life have dramatically improved life expectancy of the world population. However, wide availability of hypercaloric foods and the increase in consumption of processed foodstuff, particularly in the western countries, has led to the epidemic of chronic non communicable diseases such as cardiovascular, metabolic, and neurodegenerative diseases. In this perspective, the consumption of fresh foods containing non-nutrient bioactive compounds should be promoted, as they confer health protection at many different levels. Indeed, fresh foods, particularly plant food, contain a plethora of bioactive compounds like the polyphenolic compounds, able to modulate different pathways and processes in our body, as they display antioxidant, anti-inflammatory, anticancer, glucose regulating and neuroprotective activities.

Among polyphenols, an interesting class of compounds is represented by anthocyanins. These compounds are water soluble vacuolar pigments present mostly in fruits and flowers, but also in vegetative organs. They have a strong impact on food sensory properties, as they confer the characteristic red to blue colour to fruits and vegetables. In plants they play a key role in pollination and by absorbing light, protect plants from UV rays-induced damage and from cold stress. The colour of some organs, such as the petals of the flowers, can change during development both through the synthesis of a greater or lesser amount of anthocyanins and through a different acidification of the vacuole. While the variation of anthocyanin concentration alters the colour intensity, the different vacuolar acidification changes the hue. The main function of the anthocyanins, contained in flowers or in fruit epidermis, is to attract animals and pollinating insects to easily disseminate the seeds or to facilitate the spread of pollen. However, evidence that the synthesis of anthocyanins is induced during the establishment of adverse conditions suggests their involvement also in both biotic and abiotic stresses.

Besides the use as food colorants, these compounds are potentially useful as nutraceutical ingredients, as they confer numerous beneficial health effects. Many in vitro, animal and human studies have evaluated the biological and pharmacological potential of these molecules and demonstrated that they possess the capacity to counteract oxidative stress, to act as antimicrobial substances, and to counteract the onset and progression of numerous non-communicable diseases such as neurodegenerative, cardiovascular, metabolic diseases and cancer. They are also well known because they protect visual function along with vitamin A and carotenes. The activities of anthocyanins have been attributed to their free-radical scavenging capacity and to their action on an array of enzymes like cyclooxygenase and mitogen-activated protein kinase, and on inflammatory cytokines signalling. No negative effect of anthocyanin derivatives has been reported, even after ingestion of very high doses, hence their use in the prevention or treatment of numerous diseases is an appealing possibility. Here we make an attempt to review the most recent literature on the chemistry and biochemistry of these very interesting and potentially helpful pigments.

Natural sources of anthocyanins

Anthocyanins are widespread in red/blue fruits and vegetables and their content in plants varies markedly among different species, depending on cultivar or variety, growing area, climate, farming methods, harvest time, ripening, seasonal variability, processing and storage, temperature and light exposure. Berries such as strawberries, blueberries, blackberries, blackcurrant, redcurrant and raspberries are a rich source of anthocyanins, with levels ranging from about 100 to about 700 mg/100 g of fresh product, but the highest content is found in elderberries and chokeberries, which can contain up to 1,4-1,8 g of anthocyanins per 100g of product. Other good sources of anthocyanins include purple corn, cherries, plums, pomegranate, eggplant, wine, grapes, and red/purple vegetables such as black carrots or red cabbage and purple cauliflower which may contain from a few milligrams up to 200-300 mg/100 g of product.

Cyanidin, having two hydroxyl groups on the B-ring, is the most widely distributed pigment among plants. The most represented anthocyanin in edible plants is cyanidin-3-glucoside, followed by delphinidin, pelargonidin and peonidin glucosides. In general, hydroxylation confers a blue hue and reduces stability, whereas methylation induces red hue and improves stability. The hydroxyl groups may be modified by glycosylation or acylation. Both modifications affect the physical and chemical properties of anthocyanins and modify the chemical reactivity and polarity of the molecules.

Fruits are the most common dietary source of anthocyanins, providing up to 70% of daily intake, predominantly from apples, pears, berries, stone fruits and grapes. Wine may contribute up to 25% of intake across Europe. In the US and Northern Europe, the main dietary sources are berries. Anthocyanins are not considered as essential nutrients hence no recommended daily intake has been established, however China has recently suggested a daily intake of 50 mg. Though the evaluation of anthocyanin daily intake is cumbersome and inaccurate, mainly due to the incomplete data on the anthocyanin quantities in food, it has been estimated that the daily intake is about 12.5 mg/day in the US, while in Europe mean intake ranges from 19 to 65 mg/day for men, and from 18 to 44 mg/day for women. An Australian study reports that the mean anthocyanin intake is about 24 mg/day, whereas in Finland the daily intake has been estimated to be up to 150 mg/day, with the primary source being the consumption of berries.

Given the health protecting effects of anthocyanins, promoting the intake of fresh fruits and vegetables may be desirable to guarantee an adequate level of antioxidant and protecting substances at the plasma level. Indeed, a regular intake of fruits and vegetables is an important factor of a healthy lifestyle and can confer protection against chronic and degenerative diseases. For instance, the adherence to Mediterranean diet which is rich in food containing anthocyanins (fruits, berries, vegetables, beans and cereals) has been associated to a reduction of inflammation markers and to a lower risk of various diseases, including obesity, diabetes, cancer, and cardiovascular disease. Conversely, low intake of fruits and vegetables account for an estimated 1.7 million deaths globally, including but not limited to those caused by gastrointestinal cancer (14%), ischemic heart disease (11%), and stroke (9%).

Anthocyanins as natural food and beverages colorants

Food industry uses many chemical substances as food colorants. However this use poses a number of problems, mainly due to health risks. Indeed, synthetic dyes have been suspected to cause adverse behavioural and neurological effects. Being anthocyanins safe and potentially health protective, they represent an attractive alternative to synthetic substances. Indeed, the use of anthocyanins as food colorants in foods and beverages is widely permitted within Europe (E163), Japan, the United States, and many other countries.

Products which could benefit from anthocyanin addition include soft drinks, syrups, jams, jellies, sweets, bakery or dairy products, and powders. Besides conferring colour to the food, anthocyanins can provide an additional double advantage. They can act as antioxidants protecting the food to which they are added, but they can also confer a distinctive quality to food as they can increase the nutritional potential, exerting health promoting effects for the consumers. However, the use of anthocyanins as natural food colorants poses several problems. In the first place, their stability is not optimal, as they tend to quickly degrade mainly depending on light, oxygen, enzymes, metals, presence of other oxidants, pH and temperature. Secondly, they are quite expensive compared to synthetic molecules. In the third place, they could confer off-flavours to food products, such as anthocyanins extracted from red radish.

Various methods and techniques have been applied to improve anthocyanins’ stability, including microencapsulation, oxygen exclusion, co-pigment addition with colourless molecules in solution and chemical derivatization such as acylation. Microencapsulation or complexation with biopolymers seem to confer an efficient protection, particularly when maltodextrins and beta-glucans are used. Microcarriers may be produced by spraydrying or freezedrying. An alternative approach to microencapsulation is represented by nanoformulations such as nanoliposomes or nanoemulsions.

Sources of anthocyanins as potential food colorants are: grape skin, radishes, red potatoes, red cabbage, black carrots, and purple sweet potatoes, black beans, chokeberry, Thymus moroderi, prunes, Hibiscus sabdariffa and many others. Generally, acylated anthocyanins are preferred as food colorants as they are more stable than nonacylated anthocyanins, though some fruits such as elderberry and chokeberry can be used to extract high amounts of nonacylated anthocyanins at low cost, thus they also have potential use in the food industry .

Cardiovascular diseases

Cardiovascular diseases (CVDs) are the principal cause of morbidity and mortality worldwide and deaths caused by neurodegenerative diseases more than doubled in six years, moving from the 14th to the 5th position in global causes of deaths list between 2016 and 2020.

Several randomized controlled trials have been carried out to find cause−effect relationships between anthocyanins and CVDs prevention and treatment. Regarding atherosclerosis, and like in the animal studies, clinical trials also demonstrate that anthocyanins effects occur in different atherosclerotic stage. In areas of atherogenic lesions, the activation of endothelial cells can occur due to chronic infection, free radicals, hypertension, diabetes and cigarette smoking. This activation prompts the expression of genes such as MCP-1 mRNA, involved in induction of transcription factors responsible for shear stress-mediated effects. When overweight/obese individuals, with impaired glucose intolerance or diabetes type 2, ingest a diet supplemented with a standardized (36 % (w/w) anthocyanins) concentrated bilberry extract, no changes in plasma levels of MCP-1 is observed. However, the ingestion of the bilberry extract reduces postprandial glycaemic response. Anthocyanins isolated from berries were given to hypercholesterolemic individuals and an improvement of endothelium-dependent vasodilation through the activation of the nitric oxide–cyclic guanosine-3′,5′-monophosphate signalling pathway was observed.

A randomized, double-blind trial carried out on 150 subjects with hypercholesterolemia, consuming purified anthocyanin mixture (320 mg/day) or a placebo twice a day for 24 weeks, showed that anthocyanin consumption reduces serum levels of high sensitivity C-reactive protein (hsCRP) and plasma IL-1β compared to the placebo. This could mean that anthocyanins reduce the inflammatory response activated by the interaction(s) between endothelial and white blood cells. The reduction in the inflammatory response reduces monocyte activation and, subsequently decreases the affinity of monocyte ligands to adhesion molecules, as demonstrated by the decrease in plasma level of soluble VCAM-1. Purified anthocyanins supplementation for 24 weeks reduces serum levels of LDL-cholesterol and increases the HDL-cholesterol levels in subjects with moderate hypercholesterolemia. Additionally, the change of LDL-cholesterol positively correlates with the hsCRP change after 24-week anthocyanin intervention. The improvement of the lipid profile sems to be correlated with the decrease observed in the serum levels of CRP, VCAM-1 and IL-1β observed in hypercholesterolemic subjects supplemented with an anthocyanin mixture to for 24 weeks. Moreover, in hypercholesterolemic individuals, the decrease in LDL-C, hsCRP and IL-1β levels are correlated with the decrease in plasma levels of the platelet chemokines. These pro-inflammatory molecules released by activated platelets mediate the pro-atherogenic effects that promote recruitment, activation or differentiation of other cell types including endothelial cells and leukocytes. Anthocyanins supplementation (MEDOX^® capsules, 320 mg/day) for four-weeks also decreases blood levels of the proinflammatory cytokines’ tumour necrosis factor α (TNF-α), IL-6 and CCL2 in lean, overweight and obese population. Proinflammatory markers decrease with a bilberry rich diet in individuals with features of metabolic syndrome. Bilberry rich diet also promoted a differential regulation of the genes related to TLR signalling, cytoplasmic ribosomal proteins, and to B‐cell receptor signalling pathway as well as the differential expression of MMD (monocyte to macrophage differentiation associated) and CCR2 (CCL2 receptor) transcripts representing monocyte and macrophage function associated genes.

Regarding anthocyanins effects on lipid profile there are different results among clinical trials. Some report that consumption of anthocyanins does not produce favourable effects on lipoprotein concentrations of healthy subjects, as observed after 1 month-daily ingestion of anthocyanins derived from blood orange juice that did not reduce LDL-cholesterol nor any biomarkers associated with vascular function and CVD risk. Also, short-term (2 weeks) supplementation with cranberry juice, in a group of young, healthy volunteers, did not influence several biomarkers of blood lipid profile (TC, HDL and LDL). On other hand, there are many reports that anthocyanins decrease both LDL-cholesterol and TG and increase HDL-cholesterol. Furthermore, when hypercholesterolemic subjects receive 160 mg of anthocyanins twice daily or placebo (n = 61 of each group) for 24 weeks in a double-blind, randomized, placebo-controlled trial, anthocyanin supplementation also increased the activity of HDL-PON1 and cholesterol efflux capacity (20.0% increase) compared with placebo group. Inhibition of HDL-PON1 activity strongly prevents HDL antioxidant effect and cholesterol efflux capacity attenuation [211]. Supplementation with purified anthocyanin at 0–320 mg/day, over a 12-week period, has beneficial effects on the lipid profile and cholesterol efflux capacity in a dose–response manner. Supplementation with 80 and 320 mg/day of anthocyanin can produce moderate and strong improvements, respectively. In conclusion, results suggest that anthocyanin has a beneficial effect on the lipoprotein profile, which includes a decrease in LDL-cholesterol and TG, and an increase in HDL-cholesterol concentrations.

Daily intake of boysenberry juice is beneficial for reducing systolic blood pressure in subjects with higher systolic blood pressure and so decreasing the cardiovascular risk. Anthocyanins present in roselle maybe also responsible for the antihypertensive effect of this herb as observed in a double-blind, lisinopril-controlled clinical trial involving 171 hypertensive patients for 4 weeks and when administered to patients with metabolic syndrome (125 mg/kg/day for 4 wk).

In summary, animal and clinical studies suggest that anthocyanins could reduce oxidative stress and ameliorate inflammation, being a potential, safe candidate for prevention and therapy of CVDs.