Content and main anthocyanins in foodstuffs.
The objectives of this chapter are to summarize and discuss (i) the anthocyanins structure and content in foodstuffs and their dietary intake (ii) the anthocyanins bioavailability and human metabolic pathways and (iii) the in vitro and in vivo potent anti-neuroinflammatory effects of anthocyanins and their metabolites. Indeed, anthocyanins are polyphenolic compounds belonging to the group of flavonoids, and are one of the most commonly consumed polyphenols in a normal diet. They are responsible of red, blue and purple color of several fruits and vegetables and their intake has been related with several human health benefits. The anthocyanins structures diversities as well as their content in various fruits, vegetables and cereals is addressed. Moreover, despite the growing evidence for the protective effects of anthocyanins, it is important to highlight that the in vivo bioavailability of these compounds is relatively low in comparison to their more stable metabolites. Indeed, after consumption, these bioactives are subjected to substantial transformations in human body. Phase I and II metabolites generated by intestinal and hepatic enzymatic reactions, and phenolic acids produced by gut microbiota and their metabolized forms, are the most important metabolic anthocyanins forms. For this reason, the study of the biological properties of these circulating metabolites represents a more in vivo realistic situation. Although the anthocyanin bioavailability researches in humans are limited, they will be discussed together with a global metabolic pathway for the main anthocyanins. Moreover, several works have demonstrated that anthocyanins can cross the blood brain barrier, and accumulate in brain endothelial cells, brain parenchymal tissue, striatum, hippocampus, cerebellum and cortex. Consequently, the study of anthocyanins as potent therapeutic agents in neurodegenerative diseases has gained relevance and the principal and the most recent studies are also discussed in the book chapter.
- phenolic acids
Anthocyanins (deriving from the Greek
Apart of being responsible for the color of many foods and beverages, anthocyanins also have numerous health benefits resulting of their antioxidant and anti-inflammatory activities, among others. Although the dietary intake of anthocyanins depends on the nutritional habits , they have received less attention than other flavonoids compounds. This may be due to the fact that anthocyanins are poorly absorbed, highly metabolized, and rapidly excreted in the urine . In addition, their bioavailability and the metabolites formed by intestinal, hepatic enzymatic reactions, and gut microbiota depend on the chemical structure of anthocyanin.
This book chapter will summarize and discuss (i) the anthocyanins structure and content found in fruits, vegetables and cereals as well as the global dietary intake (ii) the anthocyanin bioavailability and human metabolic pathways and (iii) the
2. Anthocyanins: chemistry, intake and dietary sources
From a structural point of view, anthocyanins are glycosylated, polyhydroxy or polymethoxy derivatives of 2-phenylbenzopyrylium (or flavylium cation) containing two benzoyl rings (A and B) separated by a heterocyclic (C). The number of hydroxyl groups and their degree of methylation, the nature and number of the sugar and the position of the attachment, as well as the nature and number of aliphatic or aromatic acids attached to the sugars, determine their different structural variations [3, 7].
Regarding sugars, they can be attached at different positions: 3-monoglycosides, 3-diglycosides, or 3-triglycosides, 3,5-diglycosides and to a lesser extent 3,7-diglycosides. Glucose is the most common sugar moiety but other monosaccharides as rutinoside, rhamnose, galactose, arabinose, xylose are found. Furthermore, the disaccharides as sambubioside or sophoroside and as well as trisaccharides like as xylosilrutinoside or glucosylrutinoside can also be present [8, 9]. The linking of acyl substituents to sugars make possible a further degree of complexity of anthocyanins. Among them, aliphatic (acetic, malonic, succinic, malic) and cinnamic acids (
Anthocyanins are sensible to different factors such as temperature, light, oxygen or enzymes but pH represent one of the most important factors affecting them. Four different equilibrium species can co-exist, the flavylium cation (red; pH 1), the quinonoidal base (bleu, pH 4), the carbinol pseudobase (colorless or pale yellow, pH 5) and the chalcone (
The determination of the dietary intake of flavonoids, and among them, the mean consumption of anthocyanins has been the subject of several studies over the last two decades. In United States the daily consumption of these compounds in adults has been estimated in 12.5 mg/day, representing cyanidin anthocyanins the 44.7% of the total intake followed by delphinidin, malvidin, petunidin, peonidin and pelargonidin anthocyanidins . Another study in adults (17900 individuals) showed a lower anthocyanidin intake, 9.20 ± 0.79 mg/day. In addition, they stated differences among anthocyanin consumption according to gender (women’s consume higher anthocyanins than men’s) and sociodemographic and lifestyle factors such as education, alcohol consumption and activity levels . Concerning European data, the European Prospective Investigation into Cancer and Nutrition (EPIC) study estimated a mean of anthocyanin intake of 31 mg/day. At the same time, they also observed that these values vary according to the country, age, sex, body mass index (BMI), level of education, smoking status and physical activity level . Between European countries, significant differences were reported. Indeed, Italy, France and Germany displays the greater mean values, from 35.1 to 42.3 mg/day, whereas Netherlands and Sweeden are the countries with a lower anthocyanin consumption (22.6 and 20.9 mg/day, respectively). More recently, after the study of the dietary habits of 30000 subjects in 14 European countries the mean intake of anthocyanins was estimated to be 19 mg/day . In other continents and countries such as Australia (12153 subjects) or China (1393 subjects) the estimated anthocyanin mean intake was calculated at 24.2 mg/day and 27.6 mg/day, respectively, which are very closed values to European levels [17, 18].
Apart of being present in many colored fruits and vegetables they appeared also in beverages as red wine or juices and in processed foods as jams. Both, the type and the concentrations of anthocyanins are influenced by genetics (cultivar, species), cultivation, climate, soil, and processing . However, one of the best sources of anthocyanins are berries. Among them, bilberries, blueberries and blackcurrants can be reach values greater than 1000 mg/100 g of fresh weight (FW) (Table 1). Among vegetables and cereals, red cabbage, cauliflower and colored corn and rice represent good sources of anthocyanins. The most common anthocyanins are cyanidin glucosides, but some fruits contain other predominant anthocyanin (Table 1). For example, pelargonidin-3-
|Apples (Red)||0.1–315 mg/Kg peel||Cy-3-gal|||
|Apricot||1.9–230.4 mg/100 g FW||Cy-3-rut|||
|Bilberry||933–1017 mg/100 g FW||Delp-3-gluc/ Delp-3-ara/ Delp-3-gal||[22, 23]|
|Blackberry||84–201 mg/100 g FW||Cy-3-gluc/ Cy-3-rut|||
|Blueberry||232–438 mg/100 g FW||Mv-3-gluc/ Mv-3-gal/ Delp-3-gal/Delp-3-ara|||
|Cherry||6.3–60 mg/100 g FW||Cy-3-gluc/ Cy-3-rut||[25, 26]|
|Cranberry||12.4–207.3 mg/100 g FW||Cy-3-gal / Cy-3-ara/ Peo-3-gal / Peo-3-ara|||
|Blackcurrant||146.15–403.66 mg/100 g FW||Cy-3-rut/Cy-3-gluc/Delp-3-rut/ Delp-3-gluc|||
|Red grapes||11.5–29.8 g/Kg DM||Mv-3-gluc/ Mv-3-acetylgluc (|||
|Mv-3,5-digluc (other than |
|Pomegranate juice||8.9–346.6 mg/L||Cy-3,5-digluc/ Cy-3-gluc/ Delp-3,5-digluc/Delp-3-gluc/ Pel-3-gluc|||
|Strawberry||8.5–66 mg/100 g FW||Pel-3-gluc/ Cy-3-gluc/ Pel-3-rut|||
|Black beans||32 mg/g DW||Delp-3-gluc/ Pet-3-gluc/ Mv-3-gluc|||
|Red cabbage||2.32 mg/g DW||Cy-3-digluc-5-gluc/ Cy-3-coumaroyldigluc-5-gluc/Cy-3-sinapoyldigluc-5-gluc|||
|Purple carrot||168.7 mg/100 g FW||Cy-3-xylosyl-coumaroylglucosyl-gal/ Cy-3-xylosyl-feruloylglucosyl-gal/Cy-3-xylosyl-gal|||
|Purple cauliflower||7.18–201 mg/100 g FW||Cy-3-coumarylsoph-5-gluc/Cy-3-coumarylsoph-5-sinapylgluc||[35, 36]|
|Eggplant (skin)||12.1 mg/ 100 g DW||Delp-3-rut|||
|Colored potatoes||14.42–25.79 mg/g DW||Pel-3-coumaroylrut-5-gluc/ Pel-3-feruloylrut-5-gluc (red)|||
|Pet-Pe and Mv-3-coumaroylrut-5-gluc (blue–purple)|
|Red onions||48.5 mg/ 100 g FW||Cy-3-gluc/ Cy-3-laminaribioside/Cy-3-malonylgluc/Cy-3-malonyllaminaribioside|||
|Radish||32 mg/100 g FW||Pel-3-coumaroylsoph-5-gluc/Pel-3-feruloylsoph-5-gluc/Pel-3-feruloylsoph-5-malonylgluc/Pel-3-coumaroylsoph-5-malonylgluc|||
|Colored Barley||8–679 mg/Kg DW||Cy-3-gluc/ Peo-3-gluc (purple and blue)||[40, 41]|
|Dep-3-gluc/Peo-3-gluc/ Mv-3-gluc (purple)|
|Purple, blue, Red, black corn||27–1439 mg/Kg DW||Cy-3-gluc/Cy-3-malonylgluc/Cy-3-dimalonylgluc||[41, 42]|
|Purple, red, black rice||68–5101 mg/Kg||Cy-3-gluc/Peo-3-gluc (black)/Mv (red)|||
|Black and red sorghum||32–680 μg/g DW||3-deoxyanthocyanins (Luteolinidin and apigeninidin)|||
|Purple, blue, black wheat||10–212 mg/Kg DW||Cy-3-gluc/Peo-3-gluc/ Cy-malonylgluc/Cy-succinylgluc|||
3. Anthocyanins bioavailability and human metabolic pathways
To validate the prominent health-promoting effects revealed in many
3.1 Anthocyanins absorption
Despite having different molecular sizes and types of sugars or acetylated groups attached, anthocyanins can be absorbed intact [48, 49]. Moreover, anthocyanins were found in the blood stream within minutes of consumption in humans  suggesting that they can be quickly absorbed from the stomach. This fact is supported by the fact that anthocyanin urine concentrations were fivefold higher when introduced through nasal tubes into the stomach as opposed to the jejunum in patients with colorectal liver metastases after administration of a bilberry extract . In fact, thanks to the low stomach pH (1.5–4) the anthocyanin stability increase permitting their absorption under their glycoside forms. Because anthocyanins are hydrophilic molecules, an organic anion membrane carrier named bilitranslocase, which is expressed in the gastric mucosa has been proposed to mediate anthocyanin transport . Another hypothesis is the involvement of glucose transporter 1 in the transport of anthocyanin glucosides . However, the main site of anthocyanin absorption is the small intestine. They undergo deglycosylation mediated by β-glucosidase in the intestinal lumen and lactasephloridzin hydrolase in the brush border of the intestinal epithelial cells. Alternatively, anthocyanins can enter the enterocyte without deglycosylation via the sodium-coupled glucose transporter after which deglycosylation can occur by cytosol β-glucosidase . These proposed mechanisms are based, in contrast, on
3.2 Anthocyanins metabolism
Anthocyanin aglycones that enter the intestinal epithelial are metabolized before reaching portal circulation. This metabolism includes oxidation, reduction, and hydrolysis reactions (phase I metabolism) and conjugation reactions (phase II metabolism). In the intestine, anthocyanins can undergo methylation, sulfation, and glucuronidation by catechol-
Anthocyanin aglycones can alternatively undergo degradation rendering different phenolic compounds within the intestinal lumen or epithelial cells. Anthocyanin fragmentation can also be a result of the colonic microbiota activity. The microbiota gut can release many deglycosylation enzymes giving rise to aglycones that further undergo ring-opening to produce different benzoic acids or aldehydes such as gallic, vanillic, protocatechuic and syringic acids or aldehydes [46, 54]. Consequently, the phenolics acids portion increases whereas ingested anthocyanin forms portion decreases along the gastrointestinal tract. These products of anthocyanin degradation may be absorbed from the intestine and be transported and further metabolized in the liver and kidneys . The specific anthocyanins metabolism will be described below.
3.3 Anthocyanin’s distribution
The protective effects of flavonoids have been associated with diseases occurring in various tissues, but such claims are mainly based on
Anthocyanin distribution in tissues has been evaluated in rodents and pig models but never in humans [56, 57, 58, 59]. In a study in which Wistar rats were fed during 15 days with blackberry extract (370 nmol anthocyanin/day), total averaged anthocyanins concentrations were found in jejunum (605 nmol/g), in stomach (68.6 nmol/g), in kidney (3.27 nmol/L), in liver (0.38 nmol/g) and in brain (0.25 nmol/g) . In pigs, anthocyanins were identified in the liver (1.30 pmol/g), in eyes (1.58 pmol/g), in cortex (0.878 pmol/g) and in cerebellum (0.664 pmol/g) after being supplemented with 0, 1, 2, or 4% w/w blueberries for 4 weeks . In anesthetized rats received cyanidin-3-
3.4 Anthocyanin excretion
Anthocyanins can be excreted in urine, bile and even though in air. Around 5% of 13C-label was recovered from urine after the [13C]-cyanidin-3-
Finally, volatile metabolites produced from [13C]-cyanidin-3-
3.5 Anthocyanin’s behavior
Researching the xenobiotic methylation and hydroxylation of anthocyanins is challenging based on MS/MS because anthocyanidins are themselves differentiated by hydroxyl and methyl groups on the B-ring. For example, 3’-
3.5.1 Cyanidin metabolism
Cyanidin is the best-studied anthocynidin as it is the most widely distributed. Isotopically-labeled cyanidin-3-
Recently, a human study has been carried on to investigate the metabolic pathways and human bioavailability of anthocyanins of red-fleshed apple in which 22% of phenolic compounds are anthocyanins and the main is cyanidin-3-
Protocatechuic acid (PCA) and dihydroxyphenylpropionic acid (dihydrocaffeic acid) were respectively detected in these studies [55, 69]. PCA has been observed at maximum concentrations of 147 nM, thus suggesting that it is not a major metabolite of anthocyanins. The A-ring-derived degradation product, phloroglucinolaldehyde, was present at concentrations greater than either cyanidin-3-
Hippuric acid has been identified as the major metabolite of anthocyanins, reaching a maximum concentration of 1962 nM in serum . The detection of 13C2-labeled hippuric acid in this study indicates that PCA and its conjugates are likely further metabolized to form benzoic acid, which is conjugated with glycine to form hippuric acid, or alternatively, formed from the α-oxidation and dihydroxylation of hydroxyphenylacetic acids . PCA might have been formed by β-oxidation of dihydroxyphenylpropionic acid. Then, this phenolic acid could either be further degraded by the action of the gut microbiota to catechol metabolites (α-oxidation), pyrogallol metabolites (hydroxylation) and hydroxybenzoic acid (dehydroxylation), or methylated to vanillic acid [55, 69].
Colonic metabolism has long been speculated to be a major contributor to the overall metabolism of anthocyanins . It has been proposed that phenylpropenoic acids arise from cyanidin-3-
On the basis of the findings of these studies, the metabolic pathway of cyanidin-3-
3.5.2 Pelargonidin metabolism
As it was shown before, demethylation and dihydroxylation of highly substituted anthocyanins gives rise to pelargonidin, that helps to explain the high apparent recovery of pelargonidin-based metabolites . Indeed, pelargonidin glucuronide has been detected in urine after the ingestion of boysenberry (rich in four cyanidin glycosides and without pelargonidin) in humans . Furthermore, strawberry pelargonidin was found to be metabolized to 4-hydroxybenzoic acid in humans when 13 healthy volunteers consumed 300 g of fresh or stored strawberries . In which 4-hydroxybenzoic acid plasma recovery was 23 and 17 mmol, corresponding to the percentages of 54 and 56% of pelargonidin-3-
3.5.3 Delphinidin, petunidin and malvidin metabolism
After administration of Concord grape juice in humans, delphinidin-3-
4. Anti-neuroinflammatory effects on anthocyanins and their metabolites
As it was discussed above, several works have demonstrated that anthocyanins can cross the blood brain barrier, and accumulate in brain endothelial cells, brain parenchymal tissue, striatum, hippocampus, cerebellum and cortex [74, 75, 76]. Consequently, the study of anthocyanins as therapeutic agents in neurodegenerative diseases has gained relevance.
Neuroinflammation is a common physiopathological hallmark in neurodegenerative diseases as Alzheimer, Parkinson or amyotrophic lateral sclerosis, among others. This process is mediated by microglial cells, the immune cells of central nervous system. Their functions are related with the host defense by destroying pathogens, promoting tissue repair and facilitating tissue homoeostasis . Nowadays it is well establish that these cells can adopt different phenotypes depending on the brain environment to shift into pro-inflammatory/neurotoxic or anti-inflammatory/neuroprotective phenotypes. The stimulation agent will be the responsible of trigger one or another phenotype. Thus, when microglial cells are stimulated with lipopolysaccharide (LPS) and interferon gamma (IFN-γ), microglia develop a classically phenotype or M1, while when it is activated with IL-4 microglia show an alternative activated phenotype or M2 . On the one hand, M1 microglia type is characterized by the production of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) [79, 80] and by the expression of inflammatory chemokines and cytokines, such as interleukin (IL)-6, IL-12, IL-1β, IL-23, and tumor necrosis factor (TNF)-α. All this culminates in the influx of new immune system cells to combat the infection. When neuroinflammation becomes chronic, it can ultimately lead to neuronal cell death. On the other hand, M2 microglia is characterized by a suppression of IL-12 secretion and an induction of the release of IL-10, transforming growth factor beta (TGB-β), IL-1R . Furthermore, the expression of arginase-1 instead of iNOS, switching arginine metabolism from production of NO to ornithine, and also the increase of polyamines production for extracellular matrix and collagen synthesis, promotes the neuroregeneration and tissue repair .
Not only the reduction of IL-1β and TNF-α but also the reduction of IL-10 induced by LPS was observed after the treatment with 100 mg/Kg of anthocyanin obtained from
Other rich anthocyanins fruits as bilberry has exhibited promising results. In fact, the administration in food or in water of an bilberry extract (20 mg/Kg day) on APP/PSEN1 mice and their littermates downregulates the expression of several inflammatory factors (TNF-α, NF-κβ, IL-1β, IL-6, COX-2, iNOS and cluster of differentiation 33 (CD33), the chemokine receptor CX3CR1, but also and for the first time, the microglia homeostatic factors (TREM2 and TYROBP) and the Toll-like receptors (TLR2 and TLR4) .
As was explained above, circulating concentrations of phenolic acid metabolites derived from anthocyanin degradation such as protocatechuic, gallic, syringic and ferulic acids have been observed at up to eight times to that of the parent anthocyanins . Two papers have been very recently published showing that a mixture of anthocyanin metabolites can have anti-neuroainflammatory activities. Indeed, an
However, any anti-neuroinflammatory activity has been reported for glucuronidated, sulfated and
Anthocyanins represents one the most consumed polyphenols in human diet. However, their type, complexity and quantities depends on the foodstuff. For example, anthocyanins in vegetables and cereals are chemically more complex in comparison with fruits, but berries are the major source of these compounds. Anthocyanin bioavailability has been reported to be very low, with recovery of less than 1% of the ingested anthocyanin dose. However, nowadays much greater bioavailability values have reported taking into account not only the phase I and phase II metabolites but also the microbiota catabolites. One of the peculiarities of anthocyanin metabolism is their capacity of interconversion between them. For example, dehydroxylation reaction can arise pelargonidin from cyanidin and methylation reactions can convert delphinidin into petunidin and malvidin. For this reason, metabolism data after anthocyanin ingestion is more straight forward to interpret. Regarding metabolism, cyanidin is the most studied anthocyanin due to ubiquitous character in the nature. However, more studies are necessary to better understand the similarities and differences with the other less studied anthocyanins. Even though several papers have reported the potential anti-neuroinflammatory effect of rich anthocyanin extracts, anthocyanins or their metabolites, the number of papers are very scarce. The most important limitation to study the activity of anthocyanin metabolites is the lack of commercial phase II and microbiota catabolites compounds. Thus, the chemical synthesis is the most employed technique to obtain standards although more developments are requires in order to obtain greater quantities. Moreover, little is known about the molecular mechanisms implicated in the observed effects. Furthermore, the majority of works are based on the study of the microglia M1 phenotype, so more studies are necessary to know if anthocyanins and their metabolites are able to induce an anti-inflammatory phenotype. To sum up, more research is necessary to stablish if anthocyanins and their metabolites are efficacious in slowing the progression of brain aging or of neurodegenerative diseases with an inflammatory component.