anthocyanins are members of the flavonoid group of 
 phytochemicals , a group predominant in teas , honey , wines , 
 fruits , vegetables , nuts , olive oil , cocoa , and cereals . the flavonoids , 
 perhaps the most important single group of phenolics in foods , 
 comprise a group of over 4000 c15 aromatic plant compounds 
 with multiple substitution patterns ( www.nal.usda.gov/fnic/foodcomp/index.html ) . the primary players in this group include the anthocyanins ( eg , cyanidin , 
 pelargonidin , petunidin ) , the flavonols ( quercetin , kaempferol ) , 
 flavones ( luteolin , apigenin ) , flavanones ( myricetin , naringin , 
 hesperetin , naringenin ) , flavan-3-ols ( catechin , epicatechin , 
 gallocatechin ) , and , although sometimes classified separately , the 
 isoflavones ( genistein , daidzein ) . phytochemicals in this class 
 are frequently referred to as bioflavonoids due to their 
 multifaceted roles in human health maintenance , and anthocyanins in food are typically ingested as 
 components of complex mixtures of flavonoid components . daily 
 intake is estimated from 500  mg to 1  g , but can be 
 several g / d if an individual is consuming flavonoid supplements 
 ( grape seed extract , ginkgo biloba , or pycnogenol ; see , eg , 
 ) . the colorful anthocyanins are the most recognized , 
 visible members of the bioflavonoid phytochemicals . the 
 free - radical scavenging and antioxidant capacities of anthocyanin 
 pigments are the most highly publicized of the modus operandi 
 used by these pigments to intervene with human therapeutic 
 targets , but , in fact , research clearly suggests that other 
 mechanisms of action are also responsible for observed health 
 benefits [ 2 , 
 3 , 4 , 
 5 ] . anthocyanin isolates and 
 anthocyanin - rich mixtures of bioflavonoids may provide protection 
 from dna cleavage , estrogenic activity ( altering development of 
 hormone - dependent disease symptoms ) , enzyme inhibition , boosting 
 production of cytokines ( thus regulating immune responses ) , 
 anti - inflammatory activity , lipid peroxidation , decreasing 
 capillary permeability and fragility , and membrane strengthening 
 [ 6 , 
 7 , 8 , 
 9 , 10 ] . the chemical structure ( position , number , 
 and types of substitutions ) of the individual anthocyanin molecule also has a 
 bearing on the degree to which anthocyanins exert their bioactive 
 properties [ 11 , 12 ] and the structure / function relationships also influence the intracellular localization of the pigments 
 . the anthocyanin literature includes some controversy 
 over the relative contributions of glycosylated anthocyanins 
 versus aglycones in terms of bioavailability and bioactive 
 potential [ 7 , 
 13 , 14 , 
 15 , 16 ] . originally , it was assumed 
 that only aglycones could enter the circulation circuit , however , 
 absorption and metabolism of anthocyanin glycosides has now been 
 demonstrated . the nature of the sugar conjugate and the aglycone 
 are important determinants of anthocyanin absorption and 
 excretion in both humans and rats . the roles of anthocyanin pigments as medicinal agents have been 
 well - accepted dogma in folk medicine throughout the world , and , in 
 fact , these pigments are linked to an amazingly broad - based range 
 of health benefits . for example , anthocyanins from 
 hibiscus sp have historically been used in remedies for liver 
 disfunction and hypertension ; and bilberry ( vaccinium ) anthocyanins have an 
 anecdotal history of use for vision disorders , microbial infections , 
 diarrhea , and diverse other health disorders [ 17 , 
 18 , 19 ] . but 
 while the use of anthocyanins for therapeutic purposes has long been 
 supported by both anecdotal and epidemiological evidence , it is only 
 in recent years that some of the specific , measurable pharmacological 
 properties of isolated anthocyanin pigments have   been conclusively verified 
 by rigorously controlled in vitro , in vivo , or clinical research 
 trials . in many other cases , the exact roles of the 
 anthocyanins in human health maintenance versus other 
 phytochemicals in a complex mixture from a fruit extract or whole 
 food have not been completely sorted out . in fact , some reports 
 suggest that anthocyanin activity is potentiated when delivered in 
 mixtures [ 20 , 21 , 
 22 ] . for example , visual acuity can be markedly improved through 
 administration of anthocyanin pigments to animal and human subjects , 
 and the role of these pigments in enhancing night vision or overall 
 vision has been particularly well documented . oral intake 
 of anthocyanosides from black currants resulted in significantly 
 improved night vision adaptation in human subjects , and 
 similar benefits were gained after administration of anthocyanins 
 from bilberries . three anthocyanins from black currant 
 stimulated regeneration of rhodopsin ( a g - protein - coupled receptor 
 localized in the retina of the eye ) , and formation of a regeneration 
 intermediate was accelerated by cyanidin 3-rutinoside . these studies strongly suggest that enhancement of rhodopsin 
 regeneration is at least one mechanism by which anthocyanins enhance visual acuity . in both in vitro and in vivo research trials , anthocyanins have 
 demonstrated marked ability to reduce cancer cell proliferation 
 and to inhibit tumor formation [ 27 , 
 28 , 29 , 
 30 ] . the capacity 
 of anthocyanin pigments to interfere with the process of 
 carcinogenesis seems to be linked to multiple potential mechanisms of 
 action including inhibition of cyclooxygenase enzymes and potent 
 antioxidant potential . hou et al   revealed that anthocyanins 
 inhibit tumorigenesis by blocking activation of a mitogen - activated 
 protein kinase pathway . this report provided the first indication of 
 a molecular basis for why anthocyanins demonstrate anticarcinogenic 
 properties . in other research , fruit extracts with significant 
 anthocyanin concentrations proved to be effective against various 
 stages of carcinogenesis [ 18 , 
 28 , 31 , 
 32 ] , but the individual 
 role of anthocyanins versus other components was not determined , in 
 part because the anthocyanins were too easily degraded during 
 bioassays if separated from stabilizing cofactors such as other 
 phenolic constituents . the role of anthocyanins in cardiovascular disease protection is 
 strongly linked to oxidative stress protection . since endothelial 
 dysfunction is involved in initiation and development of vascular 
 disease , four anthocyanins isolated from elderberries were 
 incorporated into the plasma lemma and cytosol of endothelial 
 cells to directly examine the protective roles . these 
 tests   demonstrated not only that anthocyanins could be directly 
 incorporated into endothelial cells , but that significant 
 oxidative stress protection was the result . delphinidin , but not 
 malvidin or cyanidin , provided endothelium - dependent 
 vasorelaxation in the rat aorta , providing a pharmacological 
 benefit comparable to red wine polyphenolics . in a rat 
 model , little influence of feeding purified anthocyanins 
 ( cyanidin 3-o - glucoside ) or anthocyanin - rich extracts 
 from elderberry or blackcurrant could be detected on 
 cholesterol levels or fatty acid patterns in liver , but the pigments were 
 capable of sparing vitamin e . crude anthocyanin 
 extracts from bilberry have been administered both orally and via 
 injection to reduce capillary permeability . in other 
 research related to cardiovascular impairment , the roles of 
 anthocyanin pigments versus other flavonoids delivered in the 
 phytochemical extract have not been completely sorted out . 
 protection from heart attacks through administration of grape 
 juice or wine was strongly tied to the ability of the 
 anthocyanin - rich products to reduce inflammation and enhance 
 capillary strength and permeability , and to inhibit platelet 
 formation and enhance nitric oxide ( no ) release 
 . similarly , delivery of a black currant concentrate 
 with intense anthocyanin content caused endothelium - dependent 
 vasorelaxation in rat aorta rings in vitro . the 
 mechanism of vasorelaxation was attributed to increased levels of 
 no production , but the active compounds in the concentrate 
 were not isolated . when rats were pretreated to create increased 
 susceptibility to oxidative damage , then fed anthocyanin - rich 
 extracts , significant reduction in indices of lipid peroxidation 
 and dna damage resulted . ingestion of these extracts , 
 which contained mixtures of delphinidin , cyanidin , petunidin , 
 peonidin , and malvidin in the 3-glucopyranoside forms , also 
 increased plasma antioxidant capacity . tsuda et al   recently provided evidence that anthocyanins 
 extracted from purple corn , when provided to mice in tandem with 
 a high - fat diet , effectively inhibited both body weight and adipose 
 tissue increases . typical symptoms of hyperglycemia , hyperinsulinemia , 
 and hyperleptinemia provoked by a high - fat diet did not occur when 
 mice also ingested isolated anthocyanins . the experiments suggest 
 that anthocyanins , as a functional food component , can aid in the 
 prevention of obesity and diabetes . anthocyanins have been credited with capacity to modulate 
 cognitive and motor function , to enhance memory , and to have a 
 role in preventing age - related declines in neural function . cho 
 et al   reported that administration of isolated 
 semipurified anthocyanins from purple sweet potato enhanced 
 cognitive performance as assessed by passive avoidance tests in 
 ethanol - treated mice , and also effectively inhibited lipid 
 peroxidation in rat brain tissues . by administering blueberry 
 extracts with significant anthocyanin content ( but not purified 
 pigments ) , it was noted that the blueberry - supplemented diets led 
 to effective reversal of age - related deficits in various neural 
 and behavioral parameters ( memory and motor functions ) . further investigations by this laboratory team demonstrated that 
 anthocyanins ( in particular , cyanidin-3-sambubioside-5-glucoside 
 and cyanidin-3 , 5-diglucoside ) were highly bioavailable in 
 endothelial cells , which was linked to their roles in prevention 
 of atherosclerosis and neurodegenerative disorders [ 34 , 
 41 ] . anthocyanins exerted multiple protective effects against pleurisy 
 in a rat model and were capable of attenuating inflammation . the antimicrobial 
 activity of anthocyanins in general has been well established , 
 including significant inhibition of aflatoxin biosynthesis . the experimental evidence demonstrating anthocyanin benefits for 
 diabetes and pancreatic disorders has also accumulated in recent 
 years , and again the efficacy is attributed to the multiple , 
 simultaneous biological effects these pigments cause in the body , 
 including prevention of generation of free radicals , decreased 
 lipid peroxidation , reduced pancreatic swelling , and decreased 
 blood sugar concentrations in urine and blood serum [ 43 , 
 44 ] . an enigma is defined as anything that perplexes because it is 
 inexplicable , hidden , or obscure ; something that serves as a 
 puzzle to be solved . the whole realm of anthocyanin consumption 
 and human health fits into this definition , because several 
 aspects of anthocyanin 's pharmacological roles have remained 
 elusive to the scientist . in most of the interventions of 
 anthocyanins in human health , details on the mechanisms of action 
 for bioactivity , uptake , absorption , bioavailability , whole body 
 distribution , and tissue localization are still not fully elucidated . there are at least four primary obstacles that have impeded the 
 formulation , by medical professionals , of robust dietary or 
 prescriptive guidelines on consumption of anthocyanins . probably the most complicated piece of the puzzle is that , in 
 terms of biological activity in the human body , an anthocyanin 
 pigment is ( almost ) never acting independently . typically , 
 anthocyanins and other flavonoid components , or anthocyanins and 
 other nonflavonoid phytochemicals , are interacting together in 
 order to provide full potency . interactions between phytochemicals 
 within a plant are a key evolutionary strategy for the host plant . there are over 4000  flavonoids described , with multiple 
 substitution patterns and often large complex structures in the 
 mixtures . bioflavonoids like anthocyanins occur in mixtures within 
 edible foods and are ingested in mixtures . the 
 anthocyanins and related flavonoids are secondary products typically 
 produced by plants as defensive , protective , or attractive agents , and 
 it makes good evolutionary sense for the plant to use a variety of 
 strategies and multiple fronts of attack to accomplish these 
 functions , rather than single compounds to which a pathogen or 
 predator could become resistant . this same multiplicity in bioactive 
 phytochemical synthesis is also a bonus for animals and humans who 
 ingest the plant material donors , and benefit from the interaction of 
 the flavonoids with therapeutic targets . when the interactions between 
 co - occurring phytochemicals are positive ( eg , additive effects or 
 synergies ) , they are called potentiating interactions . in other 
 cases , components in the donor plant can actually inhibit the 
 bioactivity of the flavonoid compound ( eg , pectin interference with 
 antioxidant capacity in in vitro assays ) , and in other cases , 
 concomitant compounds which are not themselves bioactive may work 
 together with a bioflavonoid to enhance bioavailability or absorption . synergy among flavonoids including anthocyanins has been cited as 
 responsible for antiplatelet activity of red wine and grape juice , 
 with strong interactions between components of grape skin and grape 
 seed required to potentiate antiplatelet activity in human and animal 
 systems . co - occurring flavonoids working synergistically to 
 antagonize hydrogen peroxide formation are most effective in 
 depressing platelet function . traditional bioprospecting 
 approaches , which search for single purified plant - derived compounds 
 as a means of drug discovery , will not capture the full potency of a 
 plant extract when multiple potentiating interactions are responsible for bioactivity . another common well - recognized handicap to scientists exploring 
 the bioactive properties of the flavonoids , and the second part 
 of the anthocyanin puzzle , is the fact that these phytochemicals 
 can be of an evanescent nature . the susceptibility of 
 anthocyanins to oxidation and degradation is one of the concerns 
 of food processors who wish to maximize the shelf life of products 
 enhanced with natural pigment colors . in particular , many of the 
 classic phytochemical methods ( including column chromatography ) , used 
 to extract from plant tissues and fractionate components out of a 
 crude extract , can degrade anthocyanins and/or inactivate them during 
 purification steps . as a result , research that aims to identify 
 bioactive entities and gauge potencies of extracts can easily fail to 
 assess the actual sources of biological activity in situ . strict attention to the ephemeral nature of some flavonoid 
 constituents in berries ( especially during extraction / fractionation 
 sequences ) led to the adaptation of a vacuum chromatography technique 
 in our laboratory , which was designed to ( as much as feasible ) 
 preserve the integrity of the compounds and keep natural mixtures 
 intact until final separation for purposes of identification 
 [ 32 , 
 47 ] . using whole individually - quick - frozen berries as a 
 starting point , fruits are extracted in a waring blender with 70% 
 aqueous acetone ( 2l solvent kg fruit ) then filtered 
 through cheesecloth . acetone is removed from the filtrate under 
 vacuum in a 40c water bath , and water is then removed 
 by lyophilization , resulting in a dark purple powder . a portion of 
 the crude dry extract is then redissolved in water and poured over 
 a toyopearl resin polymer column for vacuum chromatography . vacuum 
 chromatography on   toyopearl with a series of solvents ( water , 50% 
 meoh , 100% meoh , 100% acetone , and 50% acetone ) 
 elutes 5  tp fractions which are then concentrated under vacuum , 
 and water is removed by lyophilization . sugars are very quickly and 
 efficiently removed in the first fraction , which greatly simplifies 
 the handling and analysis of remaining fractions . once bioactive fractions are identified , a second , third , and 
 subsequent rounds of separation are accomplished on silica gel , 
 also by vacuum , sometimes open column gravity chromatography . at 
 each step of the procedure , isolated mixtures are compared using 
 silica gel thin layer chromatography and 2 spray reagents 
 ( vanillin - hcl and dichromate reagent ) in order to gauge the 
 composition and number of components in each fraction . in general , 
 this fractionation strategy has permitted rapid separation of 
 relatively large volumes of extract , with less exposure to damaging 
 and expensive solvents , less exposure to column support materials and 
 air , minimal losses , and reliable separation of flavonoids . in tandem 
 with all of the fractionations is a consistent sequence of bioassays 
 ( for multiple stages of carcinogenesis ) because the fractionation 
 scheme is bioactivity - guided . as fractions become more highly 
 purified , analysis with hplc , hplc - ms , and nmr can be used to 
 conclusively determine the origins of the bioactivity . a third piece of the puzzle is the inducible nature of many of 
 the bioactive flavonoids including anthocyanins . as is true of a 
 plethora of secondary plant products , the initial production and 
 accumulation of phytochemicals is triggered by physical or chemical 
 microenvironmental triggers , usually a stress factor . a 
 researcher intent on maximizing production of anthocyanin pigments 
 must recognize the induction factors and deliberately elicit 
 production of bioactive flavonoids by providing these stimuli to the 
 plant material of interest . elicitation mimics stresses that provoke 
 secondary product formation in nature , and activates otherwise 
 dormant biochemical pathways . this triggering of productivity can , of 
 course , be very difficult to accomplish in a field setting , but can be 
 accomplished reliably in controlled growth facilities . the final puzzle piece in the  anthocyanin enigma  is the 
 inability of the scientist or medicinal practitioner to track 
 metabolic progress of anthocyanins after ingestion , due to the 
 plethora of metabolic breakdown products that are rapidly produced 
 in situ . there is substantial current interest in the quest to follow 
 the transport of bioflavonoids through the body , to determine 
 absorption and bioavailability , and to see where breakdown products 
 accumulate and for how long . however , since these phytochemicals are 
 highly metabolized after consumption of anthocyanin - rich foods or 
 supplements , metabolic tracking has not been possible . despite active 
 research and increasing   interest in the realm of natural products and 
 health maintenance , there is a paucity of information on the 
 absorption , biodistribution , and metabolism of anthocyanins and 
 interacting flavonoids . various plant secondary products have been 
 implicated in the promotion of good health or the prevention of 
 disease in humans , but little is known about the way they are 
 absorbed in the gut , or in which tissues they are deposited throughout 
 the body . while these issues could be studied if the phytochemicals 
 were isotopically labeled , generating labeled molecules often is 
 problematic because many compounds of interest can be synthesized 
 only in planta at present . the development and optimization of plant cell culture systems 
 which reliably and predictably synthesize anthocyanins in a controlled 
 environment has provided a unique and useful model for in - depth 
 research on anthocyanins , and has helped at least in part to 
 circumvent the obstacles presented in all four cases of the 
  anthocyanin enigma  as described above . callus and cell suspension 
 cultures from a wide and diverse range of plant genera have been 
 cultivated to produce anthocyanin pigments in vitro 
 [ 48 , 49 , 
 50 , 51 , 
 52 , 53 , 
 54 , 55 , 
 56 ] . in most of these past 
 reports , the overall goal of the plant cell culture production system 
 was to explore an alternative resource for natural plant pigments , 
 for possible use as food colorants . more recently , some 
 anthocyanin - producing plant species have been intensively cultured 
 in vitro in order to harvest the bioactive pigments and related 
 phytochemicals as medicinally - active compounds [ 47 , 
 57 , 58 , 
 59 ] . 
 by controlling both the physical and the chemical microenvironment of the 
 plant cell cultures , anthocyanin production could in many cases be 
 boosted to higher concentrations than available in the parent plant 
 in vivo . some of the most intensively - researched cell culture 
 production systems used selections from the genus vitis ( grape ) , where 
 scaled - up bioreactor - based systems approached semicommercial 
 productivity [ 60 , 61 ] . the cell culture systems can be quite 
 stable , and many have been selected for high anthocyanin yield and 
 lack of dependence on irradiance . anthocyanin profiles from cell 
 cultures do not necessarily mirror the profiles from the parent plant , 
 and isolation of pigments from the simplified cell culture tissues is 
 substantially more streamlined than from complex fruit or vegetative 
 tissues [ 47 , 53 , 
 58 ] . this simplicity can be a particular 
 advantage for investigation of the health properties of bioflavonoids including anthocyanins . in most cases , the systems begin with vegetative plant materials 
 ( leaves , petioles , stems ) and not fruit tissues . explants from 
 in vivo plants are surface - disinfested and introduced into cell 
 culture to produce rapidly proliferating callus , then cell suspension 
 cultures , which are eventually induced to produce flavonoids ( usually 
 with a trigger such as light , elevated carbohydrate , changed nitrogen 
 profile , or elicitation with a fungal extract or other chemical elicitor ) . because cell culture anthocyanin production systems are comprised 
 of simple tissues which can be engineered to reliably and 
 predictably accumulate pigment , these systems circumvent many of 
 the obstacles in the anthocyanin enigma . interactions between 
 potentiating phytochemicals are still in force in cell culture 
 systems , but because the tissues are much simpler to extract , the 
 nature of phytochemical interactions is much easier to sort out 
 and to quantitatively test . cell cultures permit rapid and 
 efficient isolation without many of the interfering compounds 
 ( pectins , excess polysaccharides , enzymes ) that can complicate 
 extraction or bioassay from fruits . aqueous extracts of 
 an anthocyanin - producing sweet potato line   exhibited 
 higher potency ( antiproliferative and antimutagenic potential ) than extract from field - grown crops 
 . similarly , when antioxidant capacity of cell cultures 
 and various fruit extracts were compared side by side in a 
 galvinoxyl free radical assay , the potency of the cell culture 
 extract was substantially higher than that of 
 all fruit extracts , and only grape seed proanthocyanidins 
 exhibited higher activity . because these other 
 substances are not present , the flavonoids are much easier to 
 isolate without the degradation that can occur rapidly when 
 isolating slowly   from complex , recalcitrant fruit or vegetative tissues . while the flavonoid content of a fruit may comprise only 1% or less 
 of the total substance , a cell culture can be crafted to accumulate 
 much higher concentrations of flavonoids , in the range of 20%30% 
 by volume . many flavonoids , in particular , high - molecular weight 
 proanthocyanidin oligomers or complex anthocyanin isomers , are either 
 not available commercially or   prohibitively expensive . by 
 producing these phytochemicals in volume in cell cultures , cell cultures are a superb model system for testing the effects 
 of elicitation on the inducible bioflavonoid genes , which is a 
 means of resolving yet another aspect of the anthocyanin enigma . in fact , elicitation of cell cultures ( using chemical or 
 environmental triggers to production ) is a recognized and 
 efficient means of maximizing anthocyanin pigment towards 
 commercialization of product recovery [ 55 , 
 56 , 62 , 
 63 ] , and 
 since the in vitro   production environment is rigorously controlled , 
 investigators have the opportunity to test multiple elicitation 
 triggers without interference from uncontrolled environmental conditions in field settings . perhaps the most significant advantage to investigation using in 
 vitro anthocyanin - accumulating cell cultures is that the cultures 
 can serve as a vehicle for delivery of isotopic labels ( 13c or 14c ) 
 to the metabolizing cells while the pigments are being biosynthesized 
 [ 59 , 64 , 
 65 ] . by using a radioisotope - labeled source of 
 compounds , an administered phytochemical can be included   in a defined 
 diet and can be discerned from preexisting , endogenous sources of 
 the same phytochemical or breakdown product . these large molecules 
 must be synthesized in planta . in this emerging research area , radiolabel or isotopic label has been introduced to metabolizing 
 cell cultures using a carbohydrate source ( sucrose or glucose ) or a 
 precursor which is much further along the metabolic pathway to 
 anthocyanin synthesis , such as phenylalanine . levels of incorporation 
 range between 15% and 30% , and levels achieved now allow organ and 
 neuronal localization of the c - labeled compounds and 
 monitoring using autoradiography and scintillation counting . accelerator mass spectrometry ( ams ) technology will even permit 
 monitoring of small levels in human systems . with these innovations , it is clear that the effective use of cell - culture - produced 
 anthocyanins can now elucidate previously hidden roles of 
 anthocyanin pigments in human health and metabolism .
anthocyanin pigments and associated flavonoids have 
 demonstrated ability to protect against a myriad of human 
 diseases , yet they have been notoriously difficult to study with 
 regard to human health . 
 anthocyanins frequently interact with 
 other phytochemicals to potentiate biological effects , 
 thus contributions from individual components are difficult to 
 decipher . 
 the complex , multicomponent structure of   compounds in 
 a bioactive mixture and the degradation of flavonoids during 
 harsh extraction procedures obscure the precise assignment of 
 bioactivity to individual pigments . 
 extensive metabolic breakdown 
 after ingestion complicates tracking of anthocyanins to assess 
 absorption , bioavailability , and accumulation in various organs . 
 anthocyanin pigments and other flavonoids that are uniformly , 
 predictably produced in rigorously controlled plant cell culture 
 systems can be a great advantage for health and nutrition 
 research because they are quickly , easily isolated , lack 
 interferences found in whole fruits , can be elicited to provoke 
 rapid and prolific accumulation , and are amenable to biolabeling 
 so that metabolic fate can be investigated after ingestion .