Critical Reviews in Analytical Chemistry

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Analysis and Antioxidant Capacity of Anthocyanin

Pigments. Part II: Chemical Structure, Color, and
Intake of Anthocyanins

Julia Martín Bueno , Purificación Sáez-Plaza , Fernando Ramos-Escudero ,

Ana Maria Jiménez , Roseane Fett & Agustin G. Asuero

To cite this article: Julia Martín Bueno , Purificación Sáez-Plaza , Fernando Ramos-Escudero ,
Ana Maria Jiménez , Roseane Fett & Agustin G. Asuero (2012) Analysis and Antioxidant Capacity
of Anthocyanin Pigments. Part II: Chemical Structure, Color, and Intake of Anthocyanins, Critical
Reviews in Analytical Chemistry, 42:2, 126-151, DOI: 10.1080/10408347.2011.632314

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Critical Reviews in Analytical Chemistry, 42:126–151, 2012
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ISSN: 1040-8347 print / 1547-6510 online
DOI: 10.1080/10408347.2011.632314

Analysis and Antioxidant Capacity of Anthocyanin

Pigments. Part II: Chemical Structure, Color, and Intake
of Anthocyanins
Julia Martı́n Bueno,1 Purificación Sáez-Plaza,2 Fernando Ramos-Escudero,2
Ana Maria Jiménez,2 Roseane Fett,3 and Agustin G. Asuero2
1
Department of Analytical Chemistry, Higher Polytechnic School, The University of Seville, Seville, Spain
2
Department of Analytical Chemistry, Faculty of Pharmacy, The University of Seville, Seville, Spain
3
Departamento de Ciência e Tecnologı́a de Alimentos, Universidade Federal de Santa Catarina,
Florianópolis, Brazil

Anthocyanins belong to a large group of secondary plant metabolites collectively known as

flavonoids, a subclass of the polyphenol family. They are a group of very efficient bioactive
compounds that are widely distributed in plant food. Anthocyanins occur in all plant tissues,
including leaves, stems, roots, flowers, and fruits. Research on phenolic compounds through the
last century, from the chemical, biochemical, and biological points of view, has focused mainly
on the anthocyanins. Anthocyanins have structures consisting of two aromatic rings linked by
three carbons in an oxygenated heterocycle (i.e., a chromane ring bearing a second aromatic
ring in position 2). The basic chromophore of anthocyanins is the 7-hydroxyflavilyum ion. An-
thocyanin pigments consist of two or three chemical units: an aglycon base or flavylium ring
(anthocyanidin), sugars, and possibly acylating groups. Only six of the different anthocyanidins
found in nature occur frequently and are of dietary importance: cyanidin, delphinidin, petu-
nidin, peonidin, pelargonidin, and malvidin. Each aglycon may be glycosilated or acylated by
different sugars and aromatic or aliphatic acids, yielding over 600 different anthocyanins re-
ported from plants. The sugar moiety is typically attached at the 3-position on the C-ring or the
5-position on the A-ring. The chromophore of eight conjugate double bonds carrying a positive
charge on the heterocyclic oxygen ring is responsible for the intensive red-orange to blue-violet
color produced by anthocyanins under acidic conditions. Anthocyanins occur in solution as a
mixture of different secondary structures: flavylium ion, a quinoidal base, a carbinol base, and
a chalcone pseudobase. Self-association, intermolecular, and intramolecular co-pigmentation of
anthocyanins leads to the formation of tertiary structures through varying stabilization mech-
anisms. Anthocyanin composition has been used as a botanical tool for taxonomic classification
of plants. In addition, anthocyanin profiles of fruits and vegetables allow detecting adulteration
of anthocyanin-based products and are indicators of product quality. Anthocyanins are com-
mon components of the human diet, as they are present in many foods, fruits, and vegetables,
especially in berries. Moreover, anthocyanins have an antioxidant activity, depending to a large
extent upon their chemical structure. Many epidemiological studies have shown the benefits of
a diet rich in fruit and vegetables to human health, and for the prevention of various diseases
associated with oxidative stress, such as cancer and cardiovascular diseases. Anthocyanin-rich
extracts are increasingly attractive to the food industry as natural alternatives to synthetic
FD&C dyes and lakes, because of their coloring properties. Anthocyanins are also one of the
nine European Union-designated natural color classes. Various adverse effects on health have
frequently been attributed to synthetic antioxidants. For these reasons, currently, there is a
trend towards relying on antioxidants derived from natural products. Anthocyanins act as an-
tioxidants both in the foodstuffs in which they are found and in the organism after intake of
these foods. This review, like the first one of the series, intends to reflect the interdisciplinary
nature of the research that is currently carried out in this prolific area.

Keywords anthocyanins, short history, chemical structure, color, anthocyanin intake

Address correspondence to Agustin G. Asuero, Dept. of Analytical Chemistry, Faculty of Pharmacy, The University of Seville 41012, Seville,
Spain. E-mail: [email protected] 126
 ANTHOCYANIN PIGMENTS, PART II 127

INTRODUCTION R1
Anthocyanins are prominent members of the secondary plant OH
metabolite class of flavonoid compounds that belong to the B
super family of antioxidants named phenolics or polyphenolics HO O
R2
(Bueno, submitted; Quideau et al., 2011; Ferretti et al., 2010; A C
Daayf and Lattanzio, 2008). A great interest of botanists, OR3
chemists, and biochemists has been always aroused by these OH
vegetal chameleons, as they were called by the eminent botanist
Tswett (1914), discoverer of the adsorption column chromatog- FIG. 1. Structure of the major anthocyanins-3-O-glucoside
raphy (Sakodynskii, 1972). Flavonoids are perhaps the most present in fruits (Freitas and Mateus, 2006; Kähkönen and
important single group of phenolics in foods; they comprise Heinonen, 2003; Giusti et al., 1999).
a group of phytochemicals predominant in teas, honey, wines,
fruits, vegetables, nuts, olive oil, cocoa, and cereals (Bueno, native to pharmaceutical medication has been a subject of con-
submitted; Raghvendra et al., 2011; Wallace, 2011; Andersen siderable interest to researchers (Rao and Snyder, 2010; Yadav et
and Markham, 2006). Anthocyanins are the largest group of phe- al., 2009; Motohashi and Sakagami, 2008; Rajasekaran et al.,
nolic pigments and the most important group of water-soluble 2008; Wu and Prior, 2008; Shahidi and Naczk, 2006; Antal et
pigments in plants, responsible for the red, purple, and blue col- al., 2003). However, much work remains to achieve definitive
ors of many fruits, vegetables, cereal grains, and flowers, being conclusions and the need for additional basic and applied re-
odorless and nearly flavorless and contributing to taste as a mod- search in this area is evident (Wallace, 2011; Ndhlala et al.,
erately astringent sensation (He and Giusti, 2010; Gould et al., 2010; Zhang and Wang, 2010; Thomasset et al., 2009).
2009; Motohashi, 2008; Veitch and Grayer, 2008; Andersen and The anthocyanins’ health properties are due to their peculiar
Jordhein, 2006; Escribano-Bailón et al., 2004; Vidal et al., 2004; chemical structure, as they are reactive towards reactive oxygen
Williams and Grayer, 2004; Harborne and Williams, 2000; species (ROS), such as superoxide (O2· −), singlet oxygen (1O2 ),
Harborne and Grayer, 1988). Generally, the structures of the an- peroxide (RCOO·), hydrogen peroxide (H2 O2 ), and hydroxyl
thocyanins isolated from fruits and vegetative tissues are simpler radical (OH·), and reactive nitrogen species in a terminator re-
than those found in flowers (Andersen and Jordheim, 2010). action, which breaks the cycle of generation of new radicals, be-
Anthocyanins are almost universally found in higher plants cause of their electron deficiency (Magalhaes et al., 2008; Choe
(occurring in about 30 families), but in general anthocyanins and Min, 2006; Kong et al., 2003; Min and Bolf, 2002). The
seem to be absent in the liverworts, algae, and other lower antioxidant capacity of phenolic compounds is also attributed to
plants, although some of them have been identified in mosses chelate metal ions involving in the production of free radicals,
and ferns (Gould et al., 2009; Delgado-Vargas et al., 2000). thereby reducing metal-induced peroxidation (Liu, 2010).
Anthocyanin biosynthesis was one of the first branches of the Anthocyanins have long been the subject of investigation
general propanoid metabolism, for which biosynthetic enzymes by botanists and plant physiologists because of their roles as
and corresponding transcription factors were identified, given pollination attractants and phytoprotective agents (Giusti and
the ease of visualization and control of mutants and genetic Jing, 2008; Grotewold, 2006b; Delpech, 2000). It is believed
imbalances (Hichri et al., 2011; He et al., 2010; Vogt, 2010; that anthocyanins are dissolved in the cell sap found in the
Tanaka et al., 2009). vacuole of a plant’s epidermis cells and a major function of
Anthocyanins are effective donors of hydrogen. Owing to these compounds is to provide color to most flowers and fruits
anthocyanin’s positive charge (Figure 1), the number and ar- (Mlodzinska, 2009; Jordheim, 2007, Brouillard, 1983). Many
rangement of aromatic hydroxyl groups, the extent of structural other flavonoid constituents accompany anthocyanins in plant
conjugation, and the presence of electron-donating and electron- cell vacuoles. Anthocyanins are probably the most important
withdrawing substituents in the ring structure, these compounds group of visible plant pigments besides chlorophyll (Bechtold
can easily donate protons to highly reactive free radicals, thereby and Mussak, 2009). Flavonoids/anthocyanins and carotenoides
preventing further radical formation (Ignat et al., 2011; Leopol- are often present in the same organs, their combination increas-
dini et al., 2011). This protects cells from oxidative damage, ing color variety (Tanaka et al., 2008).
which leads to aging and various diseases, such as cancer, In recent years, synthetic food dyes have been banned in
neurological, and cardiovascular diseases, inflammation, dia- many countries because of their toxicity and carcinogenicity.
betes, and bacterial infections (Chahar et al., 2011; Cisowska et Anthocyanin-colored natural compounds easily obtained from
al., 2011; Siva et al., 2011; Ncube et al., 2008; Charlebois, fruits and vegetables may be considered as potential substitutes
2007; Nichenametla et al., 2006; Fernández-Pachón et al., 2005; for the banned food dyes (Ignat et al., 2011; Scotter, 2010;
Ghosh, 2005). Laboratory-based evidence was provided of the Motohashi and Sakagami, 2009). In fact, they have bright at-
potential health benefits of anthocyanins (Raghvendra et al., tractive colors, while their high solubility in water allows their
2011; Wallace, 2011). Consumption of diets rich in natural easy incorporation into aqueous food systems (Melo et al.,
bioactive components (i.e., fruits and vegetables) as an alter- 2009; Mercadante and Bobbio, 2008). Moreover, the proved
128 J. M. BUENO ET AL.

TABLE 1
Major anthocyanins-3-O-glucoside present in fruits

λmax (nm)∗
Anthocyanidin Abbrev. R1 R2 R3=H R3 = gluc Some of the produced colors

Delphinidin Dp OH OH 546 541 Purple, mauve, and blue

Petunidin Pt OH OCH3 543 540 Purple
Malvidin Mv OCH3 OCH3 542 538 Purple
Cyanidin Cy OH H 535 530 Magenta and crimson
Peonidin Pn OCH3 H 532 528 Magenta
Pelargonidin Pg H H 520 516 Orange salmon

∗
Methanol-hydrochloric acid (0.01%).
antioxidant activity of anthocyanins, related to the prevention of 2011; Biesaga and Pyrzynska, 2009; Chrzascik, 2009; Peck-
a number of degenerative diseases, as shown above, provides ad- ova et al., 2009; Pohl et al., 2009; Zima et al., 2009; Harnly et
ditional benefits to the food dyed with these natural substances al., 2007; Marszall and Kaliszan, 2007; Schoefs, 2004; Escarpa
(Bueno, submitted; Fraga, 2010; Jaganath and Crozier, 2010; and González, 2001).
Pascual-Teresa and Sánchez-Ballesta, 2008; Ghosh and Konishi,
2007). Their contribution is likely to be additive or synergistic in
combination with the many other phytochemicals present in, for ANTHOCYANINS
example, berries (Jing and Giusti, 2011; Pascual-Teresa et al., Chemically, anthocyanins are glycosilate polyhydroxy or
2010; Lila, 2009; Lila et al., 2005; Schwinn and Davies, 2004). polymethoxy derivatives of 2-phenylbenzopyrilium (Andersen
Daily intake of anthocyanins is high (estimated at ∼200 and Jordheim, 2010; He and Giusti, 2010; Castañeda-Ovando
mg/d); the consumption of other phytonutrients such as et al., 2009; Gould et al., 2009; Ghosh and Konishi, 2007),
carotenoids, vitamin E, and vitamin C are estimated at 5, 12, usually with molecular weights ranging from 400 to 1200
and 90 mg/d, respectively (Scalbert et al., 2005). The estimated (medium-size biomolecules), and containing two benzyl rings
intake (20–25 mg/d) of other dietary flavonoids such as genis- (A and B), i.e., anthocyanins are heteroxides of an aglycone unit
tein and quercetin is lower than that of anthocyanins (Wallace, (anthocyanidin), which is a derivative of flavylium ion. The ma-
2011). However, regular consumers of red wine are likely to jor anthocyanins are shown in Figure 1 and Table 1 (Kähkönen
have significantly higher intakes (Cordova and Sumpio, 2009; and Heinonen, 2003; Giusti et al., 1999), and the most com-
Mateus and De Freitas, 2009; Pascual-Teresa and Sánchez- monly accepted nomenclature numbering of their carbon atoms
Ballesta, 2008). is shown inside the structure in Figure 2. The red, violet, or blue
The analytical chemistry of polyphenols has been the subject colors of numerous fruits, flowers, and vegetables are due to the
of study in this journal from a different point of view (Escudero, presence of anthocyanins, as seen in Table 2 (Melo et al., 2009;

FIG. 2. Structure of delphinidin-3-malonylglucoside.

 ANTHOCYANIN PIGMENTS, PART II 129

TABLE 2 contrast with the very visible flavonoids present in flower petals
Major anthocyanins from selected plant sources (Harborne and Williams, 2000).
Plant sources Anthocyanins A SHORT DISCUSSION OF THE EARLY HISTORY
Strawberry, banana, red radish, potato Pelargonidin The term anthocyan derives from the Greek anthos, mean-
Apple, blackberry, elderberry, peach, pear, fig, Cyanidin ing flower, and kyanos, meaning blue, and was introduced
cherry, red onion, gooseberry, red cabbage, (Marquart, 1835; Lee and Gould, 2002) by the German phar-
rhubarb macist Ludwig Clamor Marquart (1804–1886) to designate the
Black currant, blood orange, gooseberry, red Cyanidin and blue pigments present in flowers (cornflower Centaurea cyanus
cabbage, purple carrot, seed coat of soybean delphinidin being studied originally). Nevertheless, it was shown later that
Passion fruit, eggplant, green bean, Delphinidin not only the blue color but practically all the red, blue, and pur-
pomegranate ple shades of flowers, fruits, stems, leaves, and roots are due to
Common cranberry, plum, sweet cherry, purple Cyanidin and such pigments, and this resulted in the extension of the term to
sweet potato peonidin all pigments of this group (all natural flavylium salts regardless
Bilberry, red grape Petunidin and of color). However, when it became apparent that in the plant
malvidin these pigments were combined with sugar and thus occurred as
Mango Peonidin glucosides, the ending “in” was attached to the word (Huntress,
1928a) to emphasize this chemical classification.
The word first coined by Marquardt for these substances has
been retained in the same sense to the present day. Other ri-
Delgado-Vargas and Paredes-López, 2003; Clifford, 2000; val terms now obsolete, such as crythropyll, cyanophyll, and
Timberlake, 1980; Shrikhande and Francis, 1976). In general cianin, have been also used from time to time (Onslow, 1925).
the anthocyanin concentration in most fruits and vegetables Note that we retain other more common uses of the root an-
goes from 0.1 up to 1% d.w. (Delgado-Vargas and Paredes- thos in anthology (a collection of literature, i.e., a selection of
López, 2003; Delgado-Vargas et al., 2000). Synthetic flavylium poems) and anthomania (an extravagant fondness for flowers)
salts (Bülow and Wagner, 1901), anthocyanins (Willstätter and (Huntress, 1928a). As a side anecdote, Marquardt accepted Fre-
Everest, 1913), and natural flavylium compounds (Chapman senius (who was apprenticed to an apothecary) into his private
et al., 1927; Pratt et al., 1927), discovered in this order, all laboratory (Szabadvary, 2008) and suggested that he publish
have the 2-phenyl-1-benzopyrilium chromophore in common, in a book the qualitative analysis system of cations and an-
and their history has been intertwined since the beginning (Pina ions that he had devised (Anleitung zur qualitativen chemischen
et al., 2011; Melo et al., 2009). Anthocaynins are omnipresent Analyse; Fresenius, 1841). Fresenius dedicated the first edition
in our diet, have little or no known toxicity, and are usually quite of the book to Marquardt (the second appeared with a preface
water soluble, making them particularly attractive as natural by Liebig). The book, a basic reference in analytical chemistry
substitutes for synthetic pigments and antioxidants (Zafra- history, was translated into English, French, Italian, Dutch, Rus-
Stone et al., 2007; Wrolstadt et al., 2005; Wrolstadt, 2004; sian, Spanish, Hungarian, and Chinese.
Asuero et al., 2002; Kuskoski et al., 2003, 2006; Kuskoski et Anthocyanins are easily soluble in water or alcohol but in-
al., 2002; Francis and Markakis, 1989; Jackman et al., 1987b). soluble in other organic solvents. They were first obtained in
In fruits, anthocyanins mainly exist in glycosidic forms, and a crystalline condition in 1903 and shown to be glucosides in
with the exception of blueberries, fruits usually contain antho- 1906 (Russell, 2004). Molish discovered the important fact that
cyanin derived from only one or two of the aglycone bases. anthocyanins in solution create products that crystallize well
Grapes offer a richer anthocyanin profile than many other fruits when treated with acids, and this fact was then used in the iso-
(McCallum et al., 2007). Various berries and black currants are lation and purification of these pigments. The anthocyanins are
the fruits richest in anthocyanins (Clifford, 2000). There is only usually represented as flavylium cations, which of course must
one common vegetable that contains high level of anthocyanins, also associated with anions (Timberlake, 1980), because they
the eggplant (Horbowicz et al., 2008). Although most species, were first isolated in these forms as chlorides from strongly
such as apples, plums, and pears, contain a limited number of an- acidic solutions.
thocyanin pigments, red grapes may contain a mixture of more Willstätter and Everest identified the first anthocyanin in
than 20 pigments, offering a richer anthocyanin profile than 1913, from the blue cornflower Centaurea cyanus. Arthur E.
many other fruits (Jaganath and Crozier, 2010; Clifford, 2000; Everest had taken his Ph.D. that same year at Basel under the
Mazza and Francis, 1995). Anthocyanins are found mainly in direction of Willstätter and was then a lecturer in chemistry at
the skin, except for certain types of red fruit (Pandey and Rizvi, University College, Reading. When first isolated by chemists,
2009; Manach et al., 2004), in which they also occur in the flesh many anthocyanins were named after the colorful flowers from
(cherries and strawberries). The flavonoids present in leaves are which they were extracted (Raghvendra et al., 2011), such as
completely hidden by the ubiquitous green of chlorophylls, in petunidin (petunia), rosinidin (rose), and peonidin (peonies).
130 J. M. BUENO ET AL.

The British biochemist Muriel Wheldale Onslow, a member Jeffrey B. Harborne spent two years (1953–55) as a post-
of William Bateson school of genetics (Richmond, 2001, 2007; doctoral fellow with Professor T. S. Geissman at the University
Creese, 1991; Geissman, 1949), tried in the early twentieth cen- of California, working on phenolic plant pigments (Hanson,
tury to establish the linkage between the inheritance of genetic 2002). He was a pioneer in the separation and structural iden-
factors and the production of pigments, the anthocyanins, per- tification of anthocyanins (Cooper-Driver, 2001), using both
forming plant breeding experiments and also investigating the spectral (1957, 1959) and chromatographic methods (1959),
corresponding chemistry of flower pigments of the plants (like and developing suitable solvents for separating pigments with
Erwing Bauer in Germany). J. B. Haldane used the research of varying glycosilation patterns, working also in microscale
Onslow to conclude that genes controlled the formation of large (1962–1965). At that time paper chromatography opened up
molecules, such as pigment molecules (Rayner-Canham and a whole new world, as before it had been almost impossible
Rayner-Canham, 2002). The early history of anthocyanins to the to separate mixtures of water-soluble plant phenolics (Grayer
1970s is summarized in Table 3 (Andersen and Markham, 2006; and Williams, 2007). Jeffrey Harborne formed with Bate-Smith
Sakata et al., 2006; Russell, 2004; Cooper-Driver, 2001; Whit- and Swain an eminent triumvirate in the plant polyphenol area
ing, 2001; Jackman et al., 1987a; Scott-Moncrieff, 1981; Heines, (Bueno, submitted; Quideau et al., 2011).
1972; Robinson, 1953; Mayer and Cook, 1943; Huntress, 1928a, Research into anthocyanin pigments has been responsible for
1928b; Onslow, 1925; Perkin and Everest, 1918). several important breakthroughs in molecular biology, including
R. W. Willstäter, working in the Kaiser Wilhem Institute of the isolation and identification of a plant transcription factor
Dahlem at Berlin, laid the foundations for effective progress in a gene for the first time, the isolation of one of the first cDNAs
work so masterly that it received the Nobel Prize in chemistry in for a plant cytochrome P450 enzyme, the first demonstration
1915 (Willstätter, 1920). Through the efforts of P. Karrer (Nobel of antisense RNA technology in a transgenic plant, and the
Prize in 1937) and his students at the Technische Hochschule at first description of transgene co-suppression (Mlodzinska, 2009;
Zürich, the fundamental work of Willstäter was greatly extended Davies, 2004).
and clarified (Karrer and Widmer, 1927a, 1927b; Karrer et
al., 1927). Sir Robert Robinson (Nobel Prize in 1947) made ANTHOCYANIN ANALYSIS: A BRIEF SURVEY
extensive contributions to the chemistry of anthocyanidins and Many difficulties are associated with anthocyanin analysis
anthocyanins, and he synthesized the most important examples because suitable, simple, and pure standards for anthocyanin
(Livingstone, 1987; Todd, 1987; Todd and Cornforth, 1976; compounds are not easily available and even if they were, they
Robinson and Robinson, 1931, 1935; Robertson and Robin- may not be very similar to the complex type of pigment that
son, 1926), following the publication of the classic paper might be present in the extract. The analysis of anthocanins is
by Willstäter and Everest (1913). During his five years also complex (Ignat et al., 2011) as a result of their ability to
(1923–1928) as professor of organic chemistry in Manchester undergo structural transformation and complexation reactions.
he completed the synthesis of most of the anthocyanidins and In addition, they are difficult to measure in a way independent
made headway with synthesis of anthocyanins (Cocker, 1987). of other flavonoids, as they have similar structural and reactiv-
Lady Robinson found her greatest interest in the study of plant ity characteristics and they also react with the usual reagents
pigments because of the pleasure she found in her beautiful used for phenolic analysis such as Folin-Ciocalteau and vanillin
Oxfod garden (Simonsen, 1954). The review of Blank (1947) (Biesaga and Pyrzynska, 2009; Welch et al., 2008; Rivas-
covered the two decades since the appearance of the second Gonzalo, 2003; Tura and Robards, 2002; Antolovich et al.,
edition of Onslow’s monograph (1925). 2000). The large number of chemical groups that may bind
The purification and identification of anthocyanins was to the flavylium molecule has contributed to a large variation
greatly aided by the application of paper chromatographic meth- in structure, also making the analysis of anthocyanins difficult
ods, notably by Harborne and his coworkers (Harborne, 1958). (Corradini et al., 2011; Jackman et al., 1987a).
The technique was devised by Gordon, Martin, and Synge Although spectrophotometric methods based on color are
(1943) and Consden, Gordon, and Martin (1944) and intro- possible, such as differential methods (relying on differences in
duced in the anthocyanin field by Bate-Smith (1948). Martin and color produced by pH change, requiring conversion of all chem-
Synge were awarded Nobel Prizes in 1952 for their invention ical forms to the red flavylium cation by adding normal acid),
of partition chromatography (Tiselius, 1952). Paper chromatog- the best method requires prior separation and then measuring the
raphy revolutionized and enormously simplified investigations individual compounds by chromatography (Timberlake, 1980).
in plant sciences (Thompson et al., 1959). In the United States, Isolation of anthocyanins from the sample matrix is generally
Geissman used paper chromatography (Scott-Moncrieff, 1981) a prerequisite to any comprehensive analysis scheme, although
for his study of gene action in Antirrhinium and for the study enhanced selectivity in the subsequent quantitation step may
of a large number of chalcones, aurones, and anthocyanins in reduce the need for sample manipulation (Truong et al., 2010;
order to find clues as to their biosynthesis. Research of the dis- Liu et al., 2008).
tribution of anthocyanins in plants was resumed with the work The maximum of absorption at 520 nm in the visible region
of Harborne (1967). is the most common wavelength used in the spectrophotometric
 ANTHOCYANIN PIGMENTS, PART II 131

TABLE 3
Early anthocyanin history

Year Achievement Author

1664 “Experiments and Consideration Touching Colours.” Indicator character of syrup Boyle
of violets (tincture of the flowers)
1682 Indicator character of anthocyanins in “The Anatomy of Plants” Nehemiah Grew
1835 Introduction of the term anthocynin. Examination of the color changes that take Marquart
place when extracts from flowers are heated with acids and alkalis
1836 Two types of colored chromogens in flowers: erythrogen, which turns red by the Hope
addition of acid, and xanthogen, giving rise to yellow pigments with alkalis
1837 Attempt to isolate certain of the pigments in some berries and in autumn leaves by Berzelius
precipitation of lead salts and regeneration with hydrogen sulfide; process used
successfully by Grafe (1906) and Willstäter (1916) in some cases. More than one
anthocyanin pigment existed
1849–1850 Attempt to prepare cornflower coloring matter by repeated precipitation of its Morot
aqueous solution with alcohol (the first stage in the method later developed by
Willstäter and Everest, which isolated the pure cornflower pigment)
1854 Cyanin, the 7-O-glycoside of cyanidin (present in blue cornflower), isolated in Fremy and Cloez
crude form. Belief that the red and purple flower pigments were all merely
different forms of the same blue anthocyanin, i.e., cyanin
1854 Examination of the decoloration of anthocyanins in solution; by adding acid the Filhol
color reappears, concluding that no reaction of reduction as suggested by Fremy
and Cloez occurred
1858 Attempt to separate pigments from various sources (red wine, bilberry) by the Glenard
Berzelius method or a slight modification thereof (use of ethereal hydrogen Heise and Glan (1888–1894)
chloride to decompose the lead salt)
1895 Classification of the anthocyan pigments, hereby completely dispelling the one Weigert
pigment idea that had so often been brought forward.
1899 Conclusion that a considerable number of different anthocyan pigments existed Overton
1903 Preparation for the first time of an anthocyan in a crystalline condition, from the Griffiths
scarlet pelargonium (geranium) and verbena flowers (but his record of their
composition and properties was quite erroneous)
1905 Description of crystalline anthocyanin in situ and readily followed instructions for Molish
the preparation of substances outside the plant, although on a small scale.
Beautiful, clear, and descriptive publication that spurred research in the area.
Decided in favor of their being glucosides
1906–1911 Repetition of Molish experiments on a large scale. Results of some careful analysis Grafe
of the pigment of the hollyhock (Althea rosea) in 1906, followed by further
analysis of the pigment of scarlet Pelargonium flowers in 1911 (no less than 10 g
of crystals in this case was obtained)
1913 Publication of the classic paper. Establishment of the main lines in subsequent Willstätter and Everest
investigation. Parent name anthocyanin for the glycosides and anthocyanidin for
the aglycone. First structural enquiry; it was shown to be a glucoside of a
pentahydroxy-2-phenyl-benzopyrilium salt
1914 Publication of “The Vegetable Chameleon” (because of the well-known reversible Tswett
discoloration of the pigments in acid and alkalis), with a detailed account of the
extraction and properties of colored substances in the flowers, fruits, and leaves
of plants
1914 Pelargonidine, the first anthocyanidine prepared, reported at a meeting of the Willstäter
Prussian Academy of Sciences on July 30
1920 Cyanidine, second anthocyanidin prepared, reported to the Baeyer Academy of Willstäter
Sciences and the Chemical Society at Munich
(Continued on next page)
132 J. M. BUENO ET AL.

TABLE 3
Early anthocyanin history (Continued)

Year Achievement Author

1920 Article on the anthocyanins of the young leaves of wine grape, coining the term Rosenhein
leucoanthocyanin, but failed to mention the previous work of Tswett, published
six years before
1920 New synthetic methods condensing o-hydroxybenzaldehydes with Robinson and Robinson
ω-hydroxyacetophenone were explored
1925 Publication of the second edition of the monograph The anthocyanin pigments of Wheldale Onslow
plants
1926–1939 Collaboration, with the encouragement of Haldane, with geneticists (John Innes Scott-Moncrieft
Horticultural Institute) and chemists (Sir Robert Robinson and Gerturde
Robinson Oxford team) makes major headway in illuminating the chemical
genetics of flower pigmentation
1927 Research concerning the structure of some anthocyanidins and anthocyanins Karrer et al.
1931–1932 Copigmentation, the bluing of anthocyanins by flavones and related substances is Robinson, Lawrence
suggested.
1932 Complete synthesis of several anthocyanins, including cyanin chloride, is achieved Robinson and Robinson
1936 Chromatography of anthocyanins using aqueous solutions on solid adsorbents, Karrer and Strong; Karrer and
alumina and gypsum (in 1934 the Robinsons used cellulose to study the behavior Weber
of plant pigments in water-amyl alcohol)
1939 Elegant explanation of how the resonating flavylium structure accounts for the Pauling
pigment depth and intensity in color
1947 Publication of a review on anthocyanin pigments of plants Blank
1948 Paper chromatography of anthocyanins and related substances in plant extracts Bate-Smith
1950s The availability of radioisotope tracer molecules fueled major new progress toward Melvin Calvin
eludidating the flavonoid biosynthetic patway
1958 Review on paper chromatography on anthocyanins, showing in this way later that Harborne
flavonoids and anthocyanins were present as complex mixtures of glycosides
1960 The term leucoanthocyanin was an unfortunate one, as the name was originally Swain
given by Rosenheim on an erroneous assumption that the compounds were
glucosides of a pseudo base
1967 Research on the distribution of anthocyanins in plants is resumed with the work of Harborne
Harborne
1968–1971 Series of papers dealing with quantitative methods for anthocyanins Fuleki and Francis
1970s It was established firmly that both anthocyanins and synthetic flavylium salts Brouillard and Dubois
families of compounds undergo multiple structural transformations in aqueous McClelland
solution, following the same basic mechanism

measurement of total anthocyanins, a rapid and cost-effective lent reviews of the main methods used in the characterization
determination carried out on raw materials with no sample hy- and quantification of anthocyanins by UV-visible spectroscopy.
drolysis (Wroldstad et al., 2002). This method is often adopted Anthocyanins have been analyzed by a number of separation
in the food industry to perform a rapid analysis of the total techniques including paper, thin-layer, and column chromato-
anthocyanin content in foods and beverages. Nevertheless, the graphic methods, and capillary electrophoresis (Corradini et al.,
spectrophotometric method does not provide any specifity as 2011; Ignat et al., 2011; Coté et al., 2010; Unger, 2009; Giusti
far as the molecular intensity of the anthocyanin present in and Jing, 2008; Welch et al., 2008; Mazza et al., 2004; Kong et
the material is concerned. Spectrophotometric methods provide al., 2003). Nowadays high-performance liquid chromatography
very useful qualitative and quantitative information; actually, (HPLC) is the method of choice because it is fast, sensitive, and
spectroscopy is the main technique used for the quantitation of quantitative, and almost all of the available HPLC methods for
different classes of polyphenols due to its simplicity and low the determination of anthocyanins and anthocyanidins are based
cost. Giusti and Wrolstat (2001, 2003, 2005) published excel- on reverse-phase columns. In combination with photodiode
 ANTHOCYANIN PIGMENTS, PART II 133

array and mass spectrometric techniques, structural characteri- et al., 2009; Cermak et al., 2009; Stein et al., 2002). In general,
zation of the pigments is readily achievable (Steimer and Sjöber, the level of anthocyanins in fruits is much higher than in veg-
2011; Wallace, 2010; Barnes et al., 2009; Heier et al., 2002). etables (Horbowicz et al., 2008). Each different plant and plant
Prior to about 2000, however, there were very few published part has a unique profile of anthocyanins (Hartmanova et al.,
reports on the use of liquid chromatography-mass spectrome- 2010; Nakajima et al., 2004). For this reason the anthocyanin
try (LC-MS) for analysis and characterization of anthocyanins composition has been used as a botanical tool for taxonomical
(Pascual-Teresa et al., 2002). MS techniques for anthocyanin classification (Jing and Giusti, 2011).
analysis such as fast atom bombardment (FAB), matrix-assisted Since the anthocyanin composition profile of many fruits
laser desorption ionization (MALDI), and time-of-flight (TOF) and vegetables is distinctive, it can be used to distinguish (a
have proven to be very useful for structural elucidation, but they fingerprint to classify) among species and/or varieties, to estab-
are not used routinely (Scotter, 2011b; Patel et al., 2010). The use lish the geographic origin of, for example, a wine, to identify
of nuclear magnetic resonance (NMR) is also vital for the struc- the variety of grape used in its manufacture, or in the control
tural elucidation of anthocyanins (Andersen and Fossen, 2005; of the quality and authenticity of related food (Bordonaba and
Andersen and Francis, 2004). The application of pulse technique Terry, 2008; Giusti and Jing, 2008; Giusti et al., 1999), i.e., to
and its associated Fourier transform have made NMR the most detect the adulteration of fruit juices (Tian et al., 2005; Durst
important tool for complete structure elucidation (Andersen and and Wrolstad, 2001). Considerable attention is paid to the ap-
Fossen, 2003, 2005). Recent techniques in mass spectrometry plication of anthocyanin analysis (often together with advanced
and two-dimensional nuclear magnetic resonance spectroscopy statistical methods) in classification of wine (Hartmanova et al.,
(2-D NMR) have become vital for the determination of many an- 2010). Anthocyanins, which confer the characteristic color to
thocyanin linkage positions and aliphatic acyl groups (Andersen red wine, can be used as markers to classify wines according
and Jordheim, 2010; Andersen, 2008; Jorhdeim, 2007). While to the grape variety (Vergara et al., 2010; Birse, 2007; Zimman
other more sophisticated techniques such as MS (Flamini and et al., 2004), although this requires a complex separation with
Traldi, 2010; Hayasaka et al., 2005) and NMR spectroscopy very high chromatographic efficiency, especially when dealing
are becoming more popular as research tools, their relatively with aged red wines, because of the formation of pyranoan-
high capital costs are still an impediment to their routine use in thocyanins (Rentzsch et al., 2007; Casassa and Catania, 2006).
enforcement laboratories, a fact that must be taken into consid- This family of compounds is formed through the reaction of
eration (Scotter, 2010, 2011b). anthocyanins with small molecules.
It is necessary to point out that the analysis of plant and food Pyroanthocyanins have been known for only two decades,
phenolics comprises not only rather simple compounds that are i.e., that pyroanthocyanins or Visitin A type pigments are derived
readily extracted and analyzed by HPLC, but also polymers from reactions of anthocyanins at the C-4 position with pyru-
(Quideau et al., 2011), including reaction (e.g., oxidation) prod- vic acid and other compounds to form cyclo addition products
ucts, that can prove difficult to extract, to elute from the chro- (Oliveira et al., 2009; Wrolstad et al., 2005), followed by arom-
matographic stationary phases, and/or to characterize (Fanali et atization through oxidation. The presence of this four ring in-
al., 2011; Ivanova et al., 2011; Kusznierewicz et al., 2011; An- creases stability against the nucleophilic attack water of bisulfite
dersen and Jordheim, 2010; Tarascou et al., 2010). A variety of (Jordheim, 2007; Leopoldini et al., 2010). Pyranoanthocyanins
chemical and physical parameters, such as oxygen, high temper- are thought to be responsible for the orange hues (hypsochromic
ature, and most pH values, exert an influence over anthocyanin shift) observed in red wine during maturation (Oliveira et al.,
stability (Cavalcanti et al., 2011). The various anthocyanins have 2011; Birse, 2007; Rein, 2005).
similar structures and occur often in complex mixtures, which
make them rather difficult to isolate (Andersen and Jordheim, CHEMICAL STRUCTURE OF ANTHOCYANINS
2010). Improvements in the methods and instrumentation used Anthocyanins are generally found in the form of glycosides.
for separation and structure elucidation of anthocyanins have The aglycones are rarely found in plants other than as arti-
made it easier to use smaller quantities of material for analysis, facts; the 3-deoxy forms the main exceptions appearing in,
and to achieve results at increasing levels of precision (Mullen et for example, red-skinned bananas, sorghum, and black tea.
al., 2010; Truong et al., 2010; Castañeda-Ovando et al., 2009; Glucose (the most common), galactose, rhamnose, arabinose,
Rijke, 2005; Andersen, 2008). xylose, and glucoronic acid are the sugars most commonly
Recent research on the extraction and analysis of antho- encountered, usually as 3-glycosides or 3,5-diglycosides
cyanins will be a further subject of the present review. (Pereira et al., 2009; Freitas and Mateus, 2006); 3-diglycosides
and 3-diglycoside-5-monoglycosides are less common. The
ANTHOCYANIN PROFILES AS FINGERPRINTS four main biosides encountered are rutinose, sambubiose, lath-
Total anthocyanin content among different plants or even yrose, and sophorose. In terms of glycoside distribution 3-
cultivars in the same plant varies considerably, being affected glycosides occur approximately two and half times as often
by genes, light, temperature, and agronomic factors, i.e., grow- as 3,5-diglycosides, the most ubiquitous anthocyanin being
ing conditions and maturity (Jing and Giusti, 2011; Amarowicz cyaniding-3-glucoside (Kong et al., 2003). The de-glycosilated
134 J. M. BUENO ET AL.

or aglycone forms of anthocyanins are known as anthocyani- (Quina et al., 2009). Note that chlorophylls are virtually the
dins, both parent names being introduced by Willstätter and only green pigments found in plant food. Degradation products
Everest (1913) in their classic paper on the field (Livingstone, of chlorophyll can also contribute green-brown or brown color
1987; Robinson, 1953). (Palmer, 1984).
Differences among the anthocyanins lie in the number and The most widespread anthocyanins in fruits are also gly-
position of hydroxyl and methoxyl groups, the nature and num- cosilates in the 3-OH position (3-O-monoglycosides), provid-
ber of sugars attached to the molecule, the position of this attach- ing thermal stability, and less extensively, in both 3-OH and
ment, and the degree and nature of esterification with aliphatic 5-OH (3,5-O-diglycosides) positions, and possible acylation of
or aromatic acids attached to sugars in the molecule (McGhie sugar residues with organic acids. Although not common in
and Walton, 2007; Andersen and Jordheim, 2006; Wu et al., berries, some anthocyanins may be acylated. Acylated antho-
2006; Kong et al., 2003). Structures of the main anthocyanidins cyanins have been reported in boysenberries and marionberries
are shown in Figure 1 and Table 2, with cyanidin being the (Jing and Giusti, 2011). Acylation involving hydroxycinnamic
most common. The differences in chemical structures of these acids has been reported to be an advanced character because it
six common anthocyanidins occur at the 3 and 5 positions occurs mainly in highly evolved families (Harborne, 1986). Nat-
of the B ring. Most new anthocyanins discovered in the past ural anthocyanins in which the 7 hydroxy group (a less common
15 years have been based on the six common anthocyanidins site of glycosilation) is glycosilated or replaced by a methoxy
encountered in higher plants. With regard to structural charac- group are quite rare (Scotter, 2011a; Quina et al., 2009). In rare
terization, information may be obtained (Liu et al., 2008) on: cases glycosilation has also been reported on the B ring.
(i) the structure of aglycone; (ii) the type of carbohydrates or The most frequent acylating agents are caffeic, ferulic,
other substituents present; (iii) the sequence of the glycan part; sinapic, and p-coumaric acids and 3,5-dihydroxycinnamic acids
(iv) interglycosidic linkages; and (v) attachment points of the (hydroxycinnamic acids), p-hydroxibenzoic acid, and gallic
substituents to the aglycone. Since each anthocyanidin may be acids (hydroxybenzoic acids), although aliphatic acids such as
glycosilated and acylated by different sugars and acids, at dif- acetic, malic, malonic, oxalic, succinic, and tartaric acids may
ferent positions, the number of anthocyanins is 15–20 times also occur. Common cinnamic and aliphatic acids acylated with
greater than the number of anthocyanidins (Mazza and Brouil- sugar moieties on anthocyanins are depicted in Figures 3 and
lard, 1987). All anthocyanins and anthocyanidins appearing in 4, respectively (He, 2004). Malonic acid, which is identified in
nature show an unclosed hydroxyl group in the 4 position. 25% of the anthocyanins, is the most frequently acyl moiety of
The sugar moieties of anthocyanins are normally connected anthocyanins. Up to three acylating acids may be present simul-
to the anthocyanins through O-linkages (Figure 2). Glycosi- taneously (Scotter, 2011a; Galvano, n.d.). These anthocyanins
lation position effects in many cases are tied to the effect of have some compounds designated as polyglycosides. In some
subsequent O-acylation. O-acylation, a prevalent modification, cases very complex anthocyanins may be formed, with multiple
is the addition of acid (acyl) group to the sugar residues of the glycosil and acyl groups.
anthocyanin, and anthocyanins may have single or multiple acyl The nature of the individual sugars has little general effect,
groups (Gao and Mazza, 1994). It is particularly an important but their positions in the molecule can have a profound influ-
modification because it plays a significant role in the formation ence on its reactivity (Jordheim, 2007). The possible number
of pigment-stabilizing tertiary structures. More than 60% of the of individual pigments became apparent when the possibilities
reported anthocyanins contain one or more acyl moieties, and above were considered (Andersen, 2008; Castañeda-Ovando et
the colors and functions of these pigments in plants are highly al., 2009). Other flavonoid glycosides may be attached to antho-
affected by the nature, number, and linkage positions of the acyl cyanins. These structures are formed through the bonding via
groups (Andersen and Jordheim, 2010; Andersen, 2008). ester linkages of an anthocyanin and a glycoside of a different
Anthocyanins are all amphoteric forming salts with either flavonoid type (e.g., flavones) to the carboxyl functions of an or-
acids or bases. In addition, anthocyanins occur in plants as salts ganic diacid (e.g., malonic, succinic). Such structures have been
(indicated by the positive charges on the heterocyclic ring), and found in flowers and orchids (Gould and Lister, 2006; Davies,
their color in plant cells depends mainly upon their mode of 2004).
combination. The conjugated bonds in their structures (light More than 30 monomeric anthocyanidins have been prop-
conjugated double bonds carrying a positive charge), which erly identified. Most of them are based on cyanidin (31%),
absorb light at about 500 nm, are the basis for the bright red, delphinidin (22%), or pelargonidin (18%) (Tsao, 2010; Ander-
blue, and purple color of fruits and vegetables, as well as the sen and Jordheim, 2006). The other common anthocyanidins
autumn foliage of deciduous trees (Wang and Stoner, 2008). (peonidin, malvidin, and petunidin), which contains methoxy
Every color except green has been observed (either natural or group(s) on their B-ring (Figure 1), represent together 21%
synthetic), depending on aspects such as kind of substituents of the isolated anthocyanins. One new methylated anthocyani-
present in the B-ring, the local pH, the state of aggregation din, 7-O-methylcyanidin, five new desoxyanthocyanidins, and
of the anthocyanins, complexation by organic molecules, or, a novel type of anthocyanidin called pyroanthocyanidin have
as in the case of blue color, complexation by metal cations been reported (Pereira et al., 2009; Andersen, 2008) in the past
 ANTHOCYANIN PIGMENTS, PART II 135

FIG. 3. Common cinnamic acids acylated with sugar moieties on anthocyanins (He, 2004).

decade. In spite of the structural diversity of anthocyanins, the The chemical structure of the anthocyanin influences its
three non-methylated anthocyanidins, cianydin (Cy), delphini- chemical properties and determines the stability, color, aque-
din (Dp), and pelargonidin (Pg), are the most widespread in na- ous equilibrium, copigmentation effect, antioxidant reactivity,
ture, being present in 80% of pigmented leaves, 69% of fruits, and potential biological activity (Kong et al., 2003; Prior and
and 50% of flowers (Ghosh and Konishi, 2007; Kong et al., Wu, 2006). Many factors, such as pH, temperature, light, pres-
2003). ence of other phenolic compounds, enzymes, metal ions, sugars,

FIG. 4. Common aliphatic acids acylated with sugar moieties on anthocyanins (He, 2004).
136 J. M. BUENO ET AL.

ascorbic acid, and oxygen have impact on the stability of antho- (1915a,b) on the causes of color in flowers and fruits tends to
cyanins (Cavalcanti et al., 2011; Horbowicz et al., 2008). It is be forgotten, and is seldom cited as such.” Willstätter made the
not surprising that the chemical structure of anthocyanins will striking observation that the same pigment can give rise to dif-
affect their biological properties. ferent colors (Goto and Kondo, 1991) and attributed the variety
The number of hydroxyl groups and type of sugar moieties, of flower colors to different pH values of solution. A point of
as well as the acylated groups, obviously can influence the po- note is that anthocyanins are readily distinguished from other
larity, size, and spatial conformations of individual compounds flavonoids as they undergo rearrangement in response to pH (Del
(Bjoroy et al., 2009; Guzman et al., 2009; Estevez and Mosquera, Rio et al., 2010), and they have ability for electron delocaliza-
2008; Sakata et al., 2006) and consequently have a certain im- tion and forming resonating structures following changes in pH,
pact on bioavailability. Acidity constants in general (Asuero and features that do not occur in other popular antioxidants (Zafra-
Michalowski, 2011; Jabbari and Gharib, 2011; Asuero, 1989, Stone et al., 2007; Prior and Wu, 2006). Anthocyanins, like
2007; Asuero et al. 1986a, 1986b; Herrador et al., 1986) are of flavylium salts analogs (Gomes, 2009; Gomes et al., 2009), are
vital importance in the analysis of bioactive compounds as well involved in a complex network of chemical reactions, in which
as in the interpretation of their mechanism of action; for ex- the different forms can be reversibly interconverted by changing
ample, drug transport potentials through biological membranes the pH (Figure 5). This intrinsic complexity is even increased in
(Mielczarek, 2005) or antiradical properties (Musialik et al., microheterogeneous media such as micelles, which have been
2009) are dependent on the acid-base properties shown by the shown to significantly modify the position and dynamics of the
flavonoids in question, i.e., anthocyanidins (Estevez et al., 2010; equilibria involved in anthocyanin chemistry (Lima et al., 2002).
Guzman et al., 2009; Estevez and Mosquera, 2008). However, Anthocyanins exist in solution as various structural forms in
anthocyanin structure/function relationships are not well under- equilibrium through hydration, proton transfer, and tautomeriza-
stood. In addition, impact of anthocyanin structures on their bi- tion reactions, and, depending on the particular structure, pH and
ological activities is dependent on experimental models, which temperature (Freitas and Mateus, 2006; Mazza et al., 2000); the
complicate the enigma of the structure-bioactivity relationship relative amounts of each equilibrium form vary (Freitas et al.,
(Jing and Giusti, 2011). 2011; Es-Safi et al., 2008; Asenstorfer et al., 2006). The color
of an anthocyanin solution is determined by the proportions of
the different anthocyanin forms, namely, red flavylium cation
ACID-BASE EQUILIBRIA: A PRIMER (AH+), violet quinoidal base (A), pseudobase or carbinol (col-
As indicated by Melo et al. (2007): “It is worth noting that orless water adduct) (B), and yellow chalcones (C) (Ibrahim
the crucial and fundamental paper by Willstätter and Mallinson et al., 2011; Dougall and Baker, 2008). Interconversion between

FIG. 5. Structural transformations of cyanidin-3-O-glucoside in strongly acid to alkaline solutions.

 ANTHOCYANIN PIGMENTS, PART II 137

these four structures takes place according to: isms, i.e., they can behave as optical memories and logic gate
systems: a write-read-erase molecular switch (Pina et al., 2011;
AH + ↔ A + H + acid − base equilibria (Ka ) Gomes et al., 2010; Pina et al., 2003). By using natural dyes as
AH + H2 O ↔ B + H + hydration equilibria (Kh )
+
wide band-gap semiconductors in dye-sensitized solar cells, a
B ↔ Cc ring − chain tautomeric equilibria (KT ) successful conversion of visible light into electricity is possible.
Cc ↔ Ct isomerization (Ki ) Anthocyanin plant extracts may be used as TiO2 dye sensitizers
(Polo et al., 2006).
Previous work carried out by Brouillard (1983) is the basis of
our current understanding of the pH-induced changes of an- COPIGMENTATION
thocyanins, in particular, a crucial point of the kinetic process: Willstätter and Zollinger (1916) observed long ago the in-
at moderately acidic pH values B is formed from hydration tensification of color on the addition of tannin to acidic oenin
of AH+ and not from A. At pH 1–3 the flavylium cation is (malvidin-3-glucoside) solutions, the major pigment that they
red colored, at pH 5 the resultant (nucleophilic attack of wa- had isolated from Alicante grapes, resulting in a much bluer
ter) carbinol pseudobase (B) is colorless, and at pH 7–8 the color. They also observed that the effect was not found in
blue purple quinoidal base (A) is formed. The ring-opening of cyanin (cyanidin 3,5-diglucoside) solutions. Similar observa-
the hemiketal B ring leads to cis-chalcone Cc. Finally, the trans- tions on the resultant additive complexes were made by others
chalcone Ct is formed from its cis isomer. In an alkaline medium (i.e., Lawrence, 1932; Shibata et al., 1919), but the physico-
the anionic quinoidal base (A−) is formed. Vacuolar pH is gen- chemical feature of such phenomenon was initially described
erally regulated by vacuolar ATPase and pyrophosphate. Nature and investigated in detail in the pioneering works of Robinson
has developed strategies for stabilizing the red color of antho- and Robinson and was designated copigmentation (Robinson
cyanins at pH values around 5, i.e., the pH of plant cell vacuoles and Robinson, 1931; Robinson, 1933; Geissman, 1941), i.e.,
in which anthocyanins are located in vivo (Mazza et al., 2000). intermolecular stacking (formation of complexes and stabiliza-
Various forms—including the colorless hydroxyl-adducts tion) with other flavonoids or by self-association, ranking almost
known as carbinol bases, pseudobases, or hemiketals—have 30 different cofactors by their ability to provide a blue shade to
been examined using pH-jump methods, UV-visible and fluores- acidic oenin solutions (Boulton, 2001). By suitable combination
cence spectroscopy, and NMR spectroscopy. Using traditional of anthocyanins and copigments at appropriate concentrations
methods obviously makes it very difficult to estimate simultane- and pH levels, Asen et al. (1972) were able to reproduce the
ously the number, concentration, and pure spectra profile of so spectra of flower petals.
many species present in such a complex mixture system with- Copigmentation is almost always a variation towards blue-
out spectral resolution (Marco el al., 2011). Multivariate curve ness and is induced by the presence of substances that are them-
resolution (de Joan and Tauler, 2006) allows to investigation of selves colorless or only slightly colored (Lambert et al., 2011;
the kinetic (photo) degradation of anthocyanins at different pH Yawadio and Morita, 2007). Intramolecular copigmentation is
values, with and without radiation exposure (up to nine different due to the acylation of molecules, being more effective than
species were detected and resolved by this method). intermolecular copigmentation. It has been suggested that in
While the structural transformations of anthocyanins are well acylated anthocyanins the acyl groups interact with the basic
known, few results have been reported on the physicochemical anthocyanin structure, thus avoiding the formation of the hy-
properties of anthocyanin-derived pigments (Quijada-Morin et drated species. The basic role of copigments is to protect the
al., 2010). The intrinsic pH-dependent set of chemical reactions colored flavylium cation from the nucleophilic attack of the
involving synthetic flavylium compounds has been confirmed to water molecule (Quina et al., 2009). The copigmentation com-
be basically identical to those of natural anthocyanins in acidic plexes are easily disrupted by dilution returning anthocyanins to
and neutral mediums. Substituent effects on the position of hy- the dependent pH equilibria between their structural forms. This
dration, tautomerization, and chalcone isomerization equilibria is the basis for characterizing the color due to copigmentation
of flavylium salts can be correlated by linear free energy rela- (González-Manzano et al., 2008). Copigmentation of flavonoids
tionships (LRERs) employing Hammett parameters (Freitas et other than anthocyanins is also possible, but it is either a rare or
al., 2011). Based on these relationships, it is possible to pre- an understudied phenomenon (Davies, 2004).
dict values of the apparent pKa of flavylium ions that were not Cyanidin and delphinidin with this 3 -4 -dihydroxy group
included in the original data set, as well as those of several readily chelates metal ions with bathochromic shifts in the visi-
naturally occurring anthocyanins. ble spectra (Smyk et al., 2008). As a result, anthocyanins with the
Systematic investigations of photochemical and thermal re- ortho-dihydroxyl groups have the potential to scavenge hydroxyl
actions carried out with flavylium compounds reveal that these radicals through the inhibition of ·OH generation by chelating
examples of multistate/multifunctional chemical systems may iron, and to prevent iron-induced lipid peroxidation. The
be used for information processing at the molecular level (Pina ortho-dihydroxyl group also helps to form anthocyanin-metal-
and Branco, 2011; Pina et al., 2011) according to principles copigment complexes at physiological pH ranges with various
similar to those that govern information transfer in living organ- organic compounds such as ascorbic and partially through this
138 J. M. BUENO ET AL.

mechanism to spare Vitamin C (He, 2004). The methoxy group the color of young red wines (Oberholster, 2008; Birse, 2007;
ortho in relation to the position 4 on the B ring in malvidin Zimman et al., 2004; Lapidot et al., 1999; Mazza and Francis,
lowers the ability to form complexes with metal ions. Metal 1995). Food content of anthocyanins is generally proportional
ions such as Al3+ and Fe3+ play a critical role in the generation to color intensity and reaches values up to 2–4 g/kg fresh weight
of blue flowers in hydrangeas and tulips, respectively (Tanaka et in blackberries. These values increase as the fruit ripens (Pandey
al., 2010; Schreiber et al., 2010; Tanaka et al., 2008). The most and Rizvi, 2009).
interesting molecular complexes of anthocyanins are the metal- Color differences between anthocyanins are largely deter-
loanthocyanins (Yoshida et al., 2009; Goto and Kondo, 1991). mined by the structure, pH, temperature, type of solvent, and the
In certain products, such as matured red wine and ports, presence of copigments. Their color greatly depends on the num-
chemical and enzymatic transformations occur, and an increas- ber of hydroxyl groups on the B ring; the larger the number of
ing number of anthocyanin-derived polyphenols are being found groups, the bluer the color (Tanaka et al., 2008). Aromatic acy-
(Crozier et al., 2009; Birse, 2007; Zimman et al., 2004). Two lation causes a blue shift and stabilizes anthocyanins. Aliphatic
groups of flavonoids, the anthocyanins and flavonols, are par- acylation does not change the color but increases the stability
ticularly important to the quality of red wines (Cheynier et al., and solubility (Bakowska-Barczak, 2005; Giusti and Wrolstad,
2006). Flavonols exist as catechin monomers and as oligomers 2003). Details regarding the acyl groups of anthocyanins includ-
and polymers, also called condensed tannins or proanthocyani- ing some chemataxonomic considerations are treated by Honda
dins (Bueno, submitted). and Saito (2002).
The most important red wine pigments are those derived Substitution of the third ring of aglycone with –OH and
from the parent anthocyanins, malvidin-3-glucoside being the –OMe groups at the 3 - or 5 - positions produces a range of
most abundant in Vitis vinifera. These antocyanins-like wine changes in the electron distribution over the molecules that re-
pigments include a variety of pyranoanthocyanins pigments, sult in a shift (Curtright et al., 1994) from the orange color of
visitin A and B, vinyl-like pigments, and a large but transient pelargonidin (strawberries) to the deep blue color of malvidin
group of ethyl-linked anthocyanin-flavanol pigments. All of (blueberries) (Table 1). In 1939 Linus Pauling gave an elegant
these pigments share similar characteristics of the parent explanation of how the resonating flavylium structure accounts
anthocyanin in that they participate in pH-dependent acid-base for the pigments’ depth and intensity in color (MacKinney, 1940;
(protonation-deprotonation) equilibria and are subject to attack Pauling, 1939). Pigments of pelargonidin and cyanidin deriva-
(to different extents) by water to form colorless hydrated tives usually produce red and purple, respectively, whereas those
species (Oliveira et al., 2011). of delphinidin show purple or blue color. Blue flower color is
restricted to the more highly evolved angiosperm family (Har-
COLOR OF ANTHOCYANINS borne and Williams, 2000). Blue flower color development is
Flavonoids and their colored class, anthocyanins, contribute one of the most interesting topics in anthocyanin research and
to a wide range of colors: pale yellow, orange, red, magenta, has been the subject of a recent detailed review (Yoshida et al.,
violet, and blue (Andersen and Jordheim, 2009). Carothenoids 2009). Classically bred roses, carnations, chrysanthemums, and
are ubiquitously distributed in plants as essential components lilies lack delphinidin-based anthocyanins, and this is the pri-
of photosynthesis and confer a yellow or red color on flowers mary reason there are no blue/violet varieties in these species
when they are present (Tanaka and Ohmiya, 2008; Mlodzinska, (Yoshida et al., 2009). However, most delphinidin glycosides
2009). Betalains also result in yellow and red color, but only the are mauve in color, and the shift to blue region usually requires
families of Carophyllales (except for Carophyllaceae and Mol- the presence of a flavone copigment and occasionally one or
luginaceae) produce betalains. To date, no plants producing both more cations (Shiono et al., 2005).
anthocyanins and betalains have been discovered (Tanaka et al., Anthocyanin copigmentation is a way to stabilize natural
2010; Stintzing and Carle, 2004). Anthocyanins, carotenoids, colors (Cavalcanti et al., 2011; Gallan et al., 2007; Mazza
and other pigments contribute to the UV patterns that are vis- and Brouillard, 1987; Markakis and Jurd, 1974). Good early
ible to insects and serve to signal flowers that are attractive to reviews of anthocyanins as food colorants are the ones by
pollinators (Ellestad, 2006; Mortensen, 2006). Bridle and Timberlake (1997), Francis and Markakis (1989),
Edible berries and selected fruits are rich sources of both an- Timberlake and Henri (1988), Jackman et al. (1987b), and
thocyanins and flavonol glycosides, which are responsible for Mazza and Brouillard (1987). A number of good reviews
the red, violet, purple, and blue color of the fruits (Ellestad, have recently published on this topic (Aberoumand, 2011;
2006). Berries are popularly consumed in North America in Scotter, 2011a, 2011b; Melo et al., 2009; Motohasi and
both fresh or frozen forms and in a variety of processed foods Sakagami, 2009; Garzon, 2008; Mercadante and Bobbio, 2008;
as yogurts, beverages, jams, canned fruits, and jellies (Seeram, Bakowska-Barczak, 2005; Wrolstad et al., 2005; Wrolstad,
2008). Berry extracts are being commercialized as nutraceuti- 2004) covering the various aspects of the field.
cals and as dietary supplements to meet consumer demands (Jing In summary, the major factors that cause the wide range of
and Giusti, 2011). Anthocyanins extracted from grape skins dur- plant color due to the presence of anthocyanin pigments include:
ing wine making are also the main compounds responsible for (i) the nature and concentration of the anthocyanidin; (ii) type of
 ANTHOCYANIN PIGMENTS, PART II 139

anthocyanin glycosidation and acylation; (iii) metal complexes; The European Union permits natural food color additives
(iv) anthocyanidin secondary structures (equilibrium forms); in foods and beverages, and anthocyanins (E 163) are one of
and (v) the nature of copigmentation (Andersen and Jordheim, the nine designated natural color classes. Commission Direc-
2010). Very small differences in chemical structures of the an- tive 2008/128/EC defines anthocyanins as colorants obtained
thocyanins were found to have a very marked effect upon a by extraction with sulfited water, acidified water, carbon diox-
flower color (Scott-Moncrieff, 1981). Anthocyanins with dif- ide, methanol, or ethanol from natural strains of vegetables and
ferent aglycones and sugar moieties may have quite different edible fruits. Anthocyanins contain common components of the
responses in terms of their bioavailability and potential health source material, namely, anthocyanin, organic acids, tannins,
effects (Wu et al., 2006). Several in vivo studies suggest that sugars, minerals, etc., but not necessarily in the same propor-
the food matrix has a significant effect on the absorption and tions as found in the source material (European Union, 2008).
metabolism of anthocyanins (Wallace, 2011; Yang et al., 2011). Nevertheless, in spite of it being possible to extract color from
The desire to understand and identify the source of natural any of the above raw materials (Table 2), the most common
colors has fascinated practicing chemists and their predecessors source, for economic reasons, is grape skins, a by-product of
since before the Renaissance (Andersen, 2008). In spite of this, it the wine industry. However, EU legislation permits the use of
is only in the past two decades that there has been an increasing sources that include the concentrated juice of black currants,
interest in anthocyanins, mainly due to their potential health- strawberries, cranberries, elderberries, cherries, and red cab-
promoting properties and their use as natural food colorants on bage. Other less familiar sources include radishes and black
one hand and to their appearance in cultivars and plant mutants carrots.
with new colors and shapes on the other hand. As a result, the With the current trend away from synthetic colors it is to be
demand for natural plant colorants is increasing as they provide expected that the production and use of anthocyanins as natural
an alternative to synthetic dyes such as FD&C red no. 40 (Allura colorants will increase in the future. Recently, interest in using
red) and the banned FD&C red no. 2 (amaranth). Note that in some natural colorants in foods as “multifunctional food addi-
the United States, FD&C numbers are given by the Food and tives” is rising due to their additional nutritional and therapeutic
Drug Administration (FDA) for synthetic food dyes that do not effects. The anthocyanin-based natural colorants, with a variety
exist in nature that are approved for use in foods, drugs, and of colors, ranging from orange to blue, are water soluble and
cosmetics (Code of Federal Regulations, 2010), while in the nontoxic. They are also well known as multifunctional food ad-
European Union, E numbers are used for all additives, both ditives with a quenching effect against ROS, which contributes
synthetic and natural, that are approved in food applications. to the prevention of coronary diseases (Wada et al., 2007).
Color appearance of food products is one of the major con- Anthocyanins are gradually being incorporated into food and
cerns of the food industry because color is an important factor beverage products as a food colorant and as a functional food
for consumers when choosing a food product (Estupiñan et al., or dietary supplement. Increased development of anthocyanins
2011; Downham and Collins, 2000). In a certain way, color with enhanced stability and prolonged shelf life will increase
may act as a “fingerprint” of a food product, being related to its food applications and overall consumption and thereby increase
flavor and at the same time an estimate of its overall quality (Ma- its positive role in human health (Shipp and Abdel-Aal, 2010).
teus and Freitas, 2009; Chattopadhyany et al., 2008; Grifitths, Anthocyanins in blue and purple corn have been used for the
2005). Color is also the first attribute to be perceived in foods and production of naturally colored blue tortillas (Sugiyama Chem-
beverages and is usually positively correlated with standards of ical Institute, 1977) for two decades. Raddish and potato ex-
quality by the consumer. Note that there is not legal definition of tracts have color characteristics very similar to those of Allura
a natural color; hence, several disparities in categorization arise red (a red dye used in food applications) and therefore have
(Scotter, 2010, 2011b). Natural colorants can be extracted from the potential to be incorporated as food colorants (Giusti and
plants, animals, fungi, and microorganisms and classified by Wrolstad, 2003). Anthocyanins are widely used as natural food
their origins or chemical structures (Aberoumand, 2011). Food colorants, for example, the cyanidin acylglucosides from red
colorants may be organized in three different types, depending cabbage (Brassica oleracea var. capitata) and Perilla frutescens
on their source (Mateus and Freitas, 2009; Henry, 1996): (Tanaka et al., 2008). The anthocyanin colorant obtained from
purple corn is approved in Japan and listed in the “Existent Food
Additive List” as purple corn color; it is used for coloring bev-
(a) Natural colors: organic colorants derived from natural edi- erages, jellies, candies, and so on in that country (Aoki et al.,
ble sources such as anthocyanins. 2002). Extracts of several anthocyanin-containing fruits, espe-
(b) Nature-identical colors: manufactured by chemical synthe- cially grapes, have long been used as food colorants for food
sis so as to be identical to colorants found in nature (e.g., or beverages having a pH of 4 or below; above this pH value
β-carotene and riboflavin). anthocyanins are rapidly decolored (Mazza and Francis, 1995);
(c) Synthetic colors: colors that do not occur in nature, pro- blue-grey color changes occur at higher pH values. Acidic foods
duced by chemical synthesis (e.g., tartrazine and carmoi- include soft drinks, fruit preserves (usually fresh/frozen rather
sine). than sulfited/canned), sugar confectioneries (particularly high
140 J. M. BUENO ET AL.

boiling/pectin jellies), dairy products (acidic products such as (Clifford, 2000). These values are thought to be higher for
yogurt), frozen products (water ice at about pH 3), dry mixes regular red wine consumers. Indeed, despite not being the best
(acidic dessert mixes and drink powders), and alcoholic drinks source of anthocyanins compared to fruit berries, anthocyanins
(Scotter, 2011b). from red wine may complex with other wine components;
Generally, natural colorants have higher coloring capacities thereby their consumption levels should be significantly higher
than synthetic ones, as seen from their relative absorptivities. than initially expected (Mateus and de Freitas, 2009).
Natural anthocyanins have powerful coloring properties, as only However, Wu et al. (2006) estimated the mean daily intake
small doses of anthocyanins are required to display the color de- of anthocyanins to be 12.5 mg/person in the United States;
sired in several food matrixes (e.g., 30–40 ppm for soft drinks such a huge difference of total anthocyanin daily intake esti-
and 20–60 ppm for fruit preserves). Anthocyanins are almost mation must result from different food intake data. The influ-
exclusively required to provide a red color for foodstuffs, but in ence of methodological differences in the assessment, as well
some cases betalains are preferred. In recent years, several ap- as nutritional, social, and cultural differences of the investigated
proaches have been made aiming to apply different anthocyanin populations, may also explain the wide range of anthocyanin
extracts to food matrixes (Sidani and Makris, 2011; Yang et al., consumption estimated by different authors. Intake estimations
2011; Fang and Bhandar, 2010; Mateus and Freitas, 2009). have almost exclusively been conducted in adult populations.
To date no plants producing both anthocyanins and betalains However, anthocyanin intake and its trends in German infants
have been discovered (Tanaka et al., 2010), with one exception, and toddlers have been studied (Drossard et al., 2011). In a sur-
Mammillaria vivapara var. vivapara (Chapman et al., 2006). A vey with Italian subjects, anthocyanin daily intake was in the
molecular explanation for the observed mutual exclusion of the range 25 to 215 mg/person, depending on gender and age (and
anthocyanins and betalains is an unresolved issue (Grotewold, this intake is large enough to induce pharmacological effects)
2006a). Anthocyanins and betalains that are mutually exclusive (Delgado-Vargas et al., 2000). The daily estimated intake of
in nature were also mixed to investigate their potential for food anthocyanins is high compared with the intake of other dietary
coloring purposes (Stintzing et al., 2006). Anthocyanins have flavonoids such as genistein and quercetin (estimated as 20–25
extensively investigated by horticulturists and food scientists mg/d) (Wallace, 2011).
(Wrolstad, 2004) due to their importance to the color quality In processed foods to which foods containing anthocyanins
of fresh and processed fruit and vegetables. However, there is were added as raw materials, such as canned food, bread, ce-
still much to be learned about the effects of processing and food reals, and baby foods, anthocyanins generally could not be de-
matrix on anthocyan stability (Cavalcanti et al., 2011; Sidani tected. Anthocianins were barely detected in baby foods pre-
and Makris, 2011; Yang et al., 2011). pared from fruits high in anthocyanins such as blueberries.
However, Chun et al. (2007) estimated the mean daily total
ANTHOCYANIN INTAKE flavonoid intake to be 189.7 mg/person, of which about 1.6%
Anthocyanins were incorporated into the human diet many was anthocyanidins. It has been suggested that a combination
centuries ago (Kuskoski et al., 2002). They were components of of different redox-active components (i.e., antioxidants or re-
traditional herbal medicines used by North American Indians, ductants) may be needed for proper protection against oxidative
Europeans, and the Chinese and were habitually derived from damage, cooperating in an integrated manner as in plant cells
dried leaves, fruits, berries, storage roots, and seeds (Krenn et (Halvorsen et al., 2006). A glass of red wine provides around
al., 2007; Konczak and Zhang, 2004). Anthocyanins are widely 115 mg of polyphenols, contributing towards a total intake of
ingested by humans, mainly due to consumption of red fruits phenolic compounds of 1171 mg/person/day (Saura-Calixto and
(like berries and red grapes), vegetables such as red cabbage, red Goñi, 2006; Scalbert et al., 2005). The scientific aspects of the
wines, cereals, and purple corn (Galvano et al., 2004). Accurate Mediterranean diet have been the subject of study (Martı́nez-
estimation of anthocyanin content in foods and daily intake is González et al., 2009; Giugliano and Esposito, 2008; Serra-
critical in food science, nutrition, and other related research Majem et al., 2006; Covas et al., 2002).
fields. Anthocyanins are found in many foods because they are re-
It has been calculated that consumption of anthocyanins sponsible for the cyan and red colors of several fruits such as
could exceed 200 mg/day (Robards et al., 1999). Depending red berries (grapes, elderberries, cranberries, raspberries, black
on nutritional habits, the daily intake of anthocyanins for currants, mulberries, etc.), cherries, pomegranates, and plums,
individuals has been estimated to range from several milligrams and also of eggplants, beetroots, and pink potatoes (Valls et al.,
to hundreds of milligrams per person. Anthocyanins in foods 2009; Giusti and Jing, 2008; Andersen and Markham, 2006). All
are typically ingested as components of complex mixtures of these fruits are regularly consumed in diets, and consequently
flavonoid components (Raghvendra et al., 2011), their intake they have also been used for a long time by the food industry to
being increased by consuming flavonoid supplements, i.e., produce juices, soft drinks, alcoholic beverages, and pies, among
grape seed extract, ginkgo biloba, or pycnogenol. In the United other products (Krenn et al., 2007). This traditional use has ex-
States, average daily intake of anthocyanins has been estimated ploited the organoleptic characteristics of anthocyanins to create
at 215 mg during the summer and 180 mg during the winter colorful and attractive products (Valls et al., 2009; Bechtold and
 ANTHOCYANIN PIGMENTS, PART II 141

TABLE 4
Some areas of study in the field of anthocyanins with some selected examples of applications

Area of study Reference

Development of analytical techniques for Scotter, 2010, 2011b; Valls et al., 2009; Welch et al., 2008; Krenn et al., 2007;
their purification and separation Mazza et al., 2004
Applications in foods Yang et al., 2011; Day et al., 2009, Meulenberg, 2009; Motohashi and Sakagami,
2009
Identification and distribution in plants Corradini et al., 2011; Truong et al., 2010; Horbowicz et al., 2008; Tian et al., 2005
Tracking color and pigment changes Aberoumand, 2011; Cavalcanti et al., 2011; Raghvendra et al., 2011; He and Giusti,
2010
Biosynthesis Hichri et al., 2011; He et al., 2010; Tanaka et al., 2008, 2010; Vogt, 2010
Quantitative analysis using Corradini et al., 2011; Fanali et al., 2011; Coté el al., 2010; Hartmanova et al., 2010;
chromatographic and electrophoretic Ignat et al., 2011; Ivanova el al., 2011; Kusznierewicz et al., 2011; Wallace, 2010;
techniques Barnes et al., 2009; Liu et al., 2008
Effects in the plant stress Mlodzinska, 2009
Chemotaxonomic investigations and wine Bordonaba and Terry, 2008; von Baer et al., 2008; Honda and Saito, 2002
adulteration
Health beneficial properties Bueno et al., 2011; Cisowska et al., 2011; Jing and Giusti, 2011; Siva et al., 2011;
McCune et al., 2011; Visioli et al., 2011; Wallace, 2011; Yang et al., 2011;
Pascual-Teresa et al., 2010; Pascual-Teresa and Sánchez Ballesta, 2009;
Thomasset et al., 2009; Motohashi and Sakagami, 2008; Seeram, 2008; Wang and
Stoner, 2008; Wu and Prior, 2008; Ghosh and Konishi, 2007; McGhie and Walton,
2007; Zafra-Stone et al., 2007

Mussak, 2009). Intake of anthocyanin is increasing because ex- stability and water solubility to the parent aglycone. Intensity
tracts and juices with high anthocyanin contents from fruits and and type of the color of anthocyanins are affected by the number
vegetables are now much more commercially available than of hydroxyl and methoxyl groups: if there are more hydroxyl
earlier (He, 2004). Due to antioxidant and other potential bene- groups, the color goes toward a more bluish shade; if there are
ficial properties, grapes, various berries, red cabbage, and other more methoxyl groups, then redness is increased (Giusti and
anthocyanin-rich foods are becoming more popular (McCune et Jing, 2008; Heredia et al., 1998). A free hydroxyl group at one
al., 2011; Rao and Snyder, 2010). Berry extracts are also being of the 5, 7, or 4 positions is essential for generating the in situ
commercialized as nutraceuticals and dietary supplements to colors responsible for plant pigmentation, which arises mainly
meet consumer demands (Jing and Giusti, 2011). from the loss of an acidic hydroxyl hydrogen from the flavylium
structure (Scotter, 2011b). Position 3 is always occupied by a
sugar (whether acylated or not), and glucose can occur addition-
CONCLUDING REMARKS ally in position 5, 7, 3 , and possibly 4 . The possible number
Anthocyanins are one of the flavonoid groups that have been of individual pigments becomes apparent when the possibilities
most widely studied in recent decades (Valls et al., 2009). As a indicated above are considered (Andersen, 2008; Castañeda-
matter of fact, the heaviest concentration of chemical, biochem- Ovando et al., 2009).
ical, and biological research on phenolic compounds over the The glycosilation, acylation, and methylation of anthocyani-
past century has focused on the anthocyanins (Whiting, 2001). A dins are the primary sources of structural diversity (Tanaka et
number of topics, i.e., anthocyanidin equilibrium forms, biosyn- al., 2010). The nature and accumulation of anthocyanins are
thesis and molecular biology, and anthocyanin localization in principally regulated by genetic factors, but also by external
plant cells, have seen important progress recently (Andersen, factors, such as light and temperature. In spite of the increas-
2008). The main areas of study are compiled in Table 4. The ingly large number of structures derived from only about 30
most prolific area of flavonoid and anthocyanin research is con- different anthocyanidins, almost 94% of the new anthocyanins
nected with their possible applications to human health benefits. discovered are in fact based on only the 6 common anthocyani-
Anthocyanins usually contain a single glucoside unit but dins: pelargonidin, cyanidin, delphinidin, peonidin, petunidin,
many anthocyanins contain two, three, or more sugars at- and malvidin. Anthocyanins show a λmax between 465 and 550
tached at multiple positions, or occurring as oligosaccharide nm, as well as a significant absorption in the UV range between
side chains. Anthocyanidins are rarely found in nature because 270 and 280 nm (Wallace, 2011; He and Giusti, 2010; Stintzing
of their poor stability. Glycosilation confers increased structural and Carle, 2004).
142 J. M. BUENO ET AL.

Depending on the pH, anthocyanins in aqueous solutions However, in spite of the advantages of synthetic pigments with
exist in the form of different species in equilibria, hydration, respect to heat, light, pH, stability, and purity in comparison
proton-transfer, tautomerization, and isomerization equilibria with natural colorants, i.e., carotenoids, anthocyanins, and beta-
being involved in their rich chemistry. Color variation and sta- lains, they are increasingly being rejected by consumers owing
bilization of anthocyanins in aqueous solution may have causes to health concerns.
such as self-association, copigmentation, and intermolecular or Although widespread use of anthocyanins as food dyes has
intramolecular hydrophobic interactions (sandwich-type stack- long been envisaged, problems of chemical and color stability
ing); with, for example, flavones and aromatic acids, hydrogen in processing, formulation, and storage conditions still repre-
bonds and charge transfer interactions may also being involved. sent a significant barrier to this application at a commercial
Copigmentation protects the anthocyaidin molecule from the scale. There is still much to be learned about the effects of pro-
hydration reaction and also controls somewhat its color in re- cessing and food matrix on anthocyanin stability (Cavalcanti et
sponse to its lightness (Gallan et al., 2007; Sakata et al., 2006). al., 2011), as shown above. Anthocyanin chemical stabilization
Copigmentation occurs with all six common anthocyanidins, (Sidani and Makris, 2011; Yang et al., 2011; Fang and Bhandar,
in contrast to metal chelation, which occurs only with antho- 2010; Falcao et al., 2009) is the main focus of recent studies due
cyanins that have an ortho-dihydroxyl system linked to the B to their abundant potential applications, their beneficial effects,
ring (Asen et al., 1972); therefore, cyanidin and delpinidin can and their use as alternatives to artificial colorants. Since antho-
form complexes while malvidin cannot. cyanins are highly reactive compounds that readily degrade or
The innumerable shades of blue, purple, violet, mauve, and react with other constituents in mixtures to form colorless or
magenta, and nearly all the reds that appear in flowers, fruits, brown compounds, it is necessary to increase their stability in
leaves, and stems are due to anthocyanin pigments. Plants adopt order to retain their color and functionality (Shipp and Abdel-
many sophisticated mechanisms to exhibit and stabilize the Aal, 2010).
flower color they need, especially blue colors (Yosida et al.,
2009). Apart from their physiological roles in plants, antho- ACKNOWLEDGMENTS
cyanins are regarded as important components in human nu- This work was supported by the Junta de Andalucı́a (Spain)
trition (Ross and Kasum, 2002). Anthocyanins present in vari- through grant excellence research project P06-FQM-02029, for
ous fruits and vegetables, especially in edible berries, provide which the authors are grateful.
natural pigmentation and exhibit a wide range of antioxidant
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