Jackman 1996

Machine Translated by Google
8 Anthocyanins and betalains

RL JACKMAN and JL SMITH
8.1 Summary
Anthocyanins occur widely in plants, being responsible for their blue,

purple, violet, magenta, red and orange coloration; while betalains,
consisting of red-violet betacyanins and yellow-orange betaxanthins,
occur exclusively in families of the order Caryophyllales. The occurrence
of these two classes of pigments is mutually exclusive. Their stability is
markedly influenced by environmental and processing factors such as
pH, temperature, O2, enzymes and condensation reactions. Due to their
inherent instability these pigments, and betalains in particular, have not
been widely used as food colorants. However, structural variants of the
anthocyanins, ie polyacylated and copigmented species, have
demonstrated remarkable stability and have great potential for use as stable natural dy
Anthocyanin and betalain preparations can be used to color a variety of
foods and pharmaceuticals with compatible physicochemical properties,
yielding highly colored and high quality products. Their application could
be enhanced, however, with new sources and stable structural variants,
modification of current processes and foods, and technological advances
(eg industry-scale extractions/purifications, microbial purifications, bio-
technology) that would make purer and more stable preparations avail-
able. In this chapter the chemistry and biochemistry of anthocyanins and
betalains-their structure and distribution, functions, biosynthesis, factors
influencing their stability, methods of extraction and analysis, and current
and potential sources and uses-are reviewed.
8.2 Introduction
Anthocyanins are among the best known of the natural pigments, being
responsible for the blue, purple, violet, magenta, red and orange species
color of a majority of plant and their products [1, 2]. Like the most
widespread anthocyanins, the betalains, a class of water-soluble pigments
consisting of the red-violet betacyanins and the yellow-orange betaxan-
thins, occur abundantly and uniquely in flowers, fruits and leaves of plants
that contain them [3, 4]. Betalains are also responsible for the coloration of
GAF Hendry et al. (eds.), Natural Food Colorants

© Springer Science+Business Media Dordrecht 1996
ANTHOCYANINS AND BETALAINS 245
some fungal species, eg toadstools [5,6], often accumulate in the stalk of

the plants, and in the red beet they are found in high concentration in the
root. Interestingly, these biogenetically and structurally distinct pigments,
the anthocyanins and betalains, have not been detected together in the
same species or even in different species of the same family: their
occurrence is mutually exclusive [7].
The striking colors associated with anthocyanins and betalains often
serve as the basis for identification and consumer acceptance of foods
containing these pigments. However, despite their obvious aesthetic appeal
and familiarity, the use of anthocyanins and betalains as colorants in the
food industry has been somewhat limited. They possess inherent instability
as a function of pH, temperature and light, low tinctorial strength, low yields,
in some cases they are associated with other properties such as flavour,
and they are generally difficult and/or expensive to extract and purify from
their natural sources. However, concern over the toxicological safety of
synthetic colorants and the banning of several red dyes [8, 9] has
encouraged the development and application of anthocyanins, betalains and
other natural colorants as food ingredients.
Anthocyanin powder obtained by physical means from fruit or vegetable
materials, eg mainly from the skins of grapes and other by-products of wine
manufacture and from red cabbage, and beetroot powder are permitted
food color additives in most countries of the world [10, 11 ].
Food colorants extracted from 'natural' sources are exempt from the rigorous
and costly toxicological testing that synthetic dyes must undergo prior to
their clearance as safe food ingredients; Natural colorants are generally
considered safe due to their presence in edible plant material.
Reports that have provided toxicological data on anthocyanins and betalains
[12-15] support the long-held view that these pigments pose no threat to
human health. Furthermore, these pigments have demonstrated therapeutic
or medicinal properties, including antioxidant or antilipoper-oxidant activity
[16--19], antiinflammatory activity [20], anticonvulsant activity [21], the ability
to reduce serum cholesterol [22, 23] and serum lipid levels [24], and to act
as chemoprotectors in certain cancer therapies [25]. Further investigation of
these properties, with respect to structural dictates and mechanisms of
action, is required.
A knowledge of the properties of natural colorants and the factors that
influence them, including their interaction with other food components, is
germane to their successful application in a wide variety of food products.
This chapter will review the chemistry and biochemistry of anthocyanins and
betalains: their structure and distribution, in vivo functions, biosyn-thesis,
factors that influence their stability, methods of extraction and analysis, and
current and potential sources and uses. In addition to books edited by
Mazza and Miniati [2] and Markakis [26], several comprehensive reviews
have been recently published concerning the anthocyanins
246 NATURAL FOOD COLORANTS
[9, 27-32]. Betalains and related topics have also recently been reviewed [3, 4,
33, 34].
8.3 Functions
The mutually exclusive occurrence of anthocyanins and betalains, and similarity in

developmental and tissue distributions, suggests that their functions in plants that
produce them are essentially identical [35]. Both pigments play a definitive role as
pollination factors, floral and fruit coloration serving to attract insect, bird and
animal vectors, thereby aiding in plant reproduction. However, the function of
anthocyanins and beta-lains in vegetative tissues (eg leaves, stems, roots) is not
well defined.
Their accumulation at wound or injury sites in plants that synthesize them normally
suggests that they may function as phytoalexins, whereby they aid in defense
against viral and/or microbial infection. These particular functions are of
technological interest, with respect to the use of these pigments in health
beverages and/or as therapeutic agents. Both anthocyanins and betalains have
been shown to participate in biological oxidations, enzyme inhibition, viral
resistance and inhibition of microbial growth and respiration [36-40]. Their
accumulation as a result of various environmental stresses such as chilling,
drought, high salinity or nutritional deficiency may be a type of wound response.
The suggestions by Stafford [35] that anthocyanins and betalains may function
also as photoreceptors and/or as filters needs to be more closely examined. When
associated with proteins, these pigments could function as green light
photoreceptors: only small amounts of pigment would be required to fulfill this
function. Also, since their major absorption peaks occur in the 465-560 nm range,
when present in large concentrations these natural pigments might act as green
light filters and thereby counteract the repression of plant growth that green light
has been reported to induce. Since these pigments also strongly absorb UV light,
they are suggested to play a protective role against the adverse effects of UV
irradiation [41a,b]. In plants that produce them, betalains may also serve as
nitrogen reservoirs [42]; the presence of endogenous betalain-specific decolorizing
enzymes [43, 44] is consistent with this tenet.
8.4 Anthocyanins
8.4.1 Structure
Anthocyanins possess the characteristic C6C3C6 carbon skeleton of other natural

flavonoids, and the same biosynthetic origin [45, 46]. Yet, they
differ from other natural ftavonoids by strongly absorbing visible light. The
range of colors associated with the anthocyanins results from their ability
to form resonance structures, from distinct and varied substitution of the
parent C6C3C6 nucleus, and various environmental factors.
The anthocyanins are glycosides of eighteen different naturally occurring
anthocyanidins, these being polyhydroxy and polymethoxy derivatives of 2-
phenylbenzopyrylium (ftavylium) salts (Table 8.1). An additional antho-
cyanidin, ricciniodin A, and its dimer linked at the 3' and 5' position and
referred to as ricciniodin B, have recently been isolated from the cell walls
of Ricciocarpus natans [47]. The red monomeric form is hydroxylated at
positions 6, 7.3' and 4' and possesses an ether linkage between the 3 and
2' positions (Figure 8.1). Ricciniodin A constitutes the first reported naturally
Table 8.1 Structure of naturally occurring anthocyanidins
Anion"
Rs
Anthocyanidin Substitution pattern (R) Color
3 Yes 6 7 3' S'
Pelargonidin (Pg) OH OH H OH HH Orange

Cyanidin (Cy) OH OH H OH OH H Orange-red
Delphinidin (Dp) OH OH H OH OH OH Blue-red
Peonidil'l (Pn) OH OH H OH OMe H Orange-red
Petunidin (Pt) OH OH H OH OMe OH Blue-red
Malvidin (Mv) OH OH H OH OMe OMe Blue-red
Apigeninidin (Ap) H OH H OH H h Orange
Luteolinidin (Lt) H OH H OH OH H Orange
Tricetinidin (Tr) H OH H OH OH OH Red
Aurantinidin (Au) OH OH OH OH HH Orange
6-Hydroxy-Cy (60HCy) OH OH OH OH OH H Grid
6-Hydroxy-Dp (60HDp) OH OH OH OH OH OH Blue-red
Rosinidin (Rs) OH OH H OMe OMe H Grid
Hirsutidine (Hs) OH OH H OMe OMe OMe Blue-red
S-Methyl-Cy (SMCy) OH OMe H OH OH H Orange-red
Pu1chellidin (PI) OH OMe H OH OH OH Blue-red
Europinidin (Eu) OH OMe H OH OMe OH Blue-red
Capensinidin (Cp) OH OMe H OH OMe OMe Blue-red
occurring anthocyanidin in which the Band C rings are connected; the

effect of the ether linkage on pigment stability has yet to be determined.
The occurrence of ricciniodins A and B in other liverworts [47] suggests
that these pigments may be more widespread than is currently realized.
With the exception of the 3-deoxy forms, which are relatively stable
[48], and ricciniodin A, anthocyanidins are rarely found in their free form
in plant tissues [45]. A free hydroxyl group in positions C-6, -8, -3' and,
especially, -3 destabilize flavylium chromophores, while hydroxylation of
positions C-5, -7, -2' (rare in natural anthocyanins) and -4' have a
stabilizing effect [49, 50]. Hydroxyl substitution at the C-7 position
generally has a greater stabilizing effect than at C-5. Without exception,
the 3-hydroxyl group is glycosylated, this conferring stability and aqueous
solubility to the anthocyanin molecule [51]. Loss of the 3-g1ycosyl moiety
is accompanied by rapid decomposition of the aglycone (ie anthocyanidin)
with irreversible loss of color [52]. If a second site is glycosylated it is
most often at the C-5 hydroxyl when present. Glycosylation of 7-, 2'-, 3'-, 4'-
and/or 5'-hydroxyl groups may also occur, although glycosylation at both
the 3'-OH and 4'-OH is sterically hindered [53], and thereby contribute
further to pigment stabilization by blocking the reactive hydroxyl groups,
reducing the electrophilic character of the chromophore and, as a con-
sequence, reducing its susceptibility to nucleophilic attack, eg by water.
Anthocyanins have been classified into eighteen groups based on the
position of attachment and number of sugar residues [54]; the 3-
monosides, 3-biosides, 3,5-diglycosides and 3,7-diglycosides occur most
often [1, 2, 45]. The most common glycosyl group is glucose, although a
number of different monosaccharides (eg rhamnose, galactose, xylose,
arabinose or fructose), disaccharides (mainly rutinose, sambubiose or
sophorose; lathyrose, gentiobiose or laminariobiose occur less often) or
trisaccharides (homo- or mixed glycans) may also serve as a glycosyl
moiety. The nature of the sugar residue(s) appears to have a greater
influence on anthocyanin stability than the nature of the aglycone,
OH
H.O.
H.O.
Figure 8.1 Structure of the anthocyanidin riccionidin A.

Although further investigation in this regard is required: anthocyanin

stability decreases for glycosyl moieties in the order glucose > galactose >
arabinose [55, 56].
The sugar residues attached to anthocyanins are often acylated with
aromatic acids such as p-coumaric, caffeic, ferulic, sinapic, gallic or p-
hydroxybenzoic acids, and/or with aliphatic acids such as malonic, acetic,
malic, succinic or oxalic acids. The latter acyl groups are particularly labile
in dilute hydrochloric acid, the traditional solvent used for extraction of
anthocyanins, and are more difficult to identify from spectra than the
aromatic acids [9]. In recent years, the use of weaker acids such as
perchloric, formic or acetic acids for anthocyanin recovery, and of more
selective and sensitive methods for structural elucidation, has revealed
that the aliphatic acids serve as acyl groups more often than was once thought.
Acyl substituents are usually bound to the C-3 sugar [57], esterified to the
6-0H or less frequently to the 4-0H group of the sugar [58, 59].
Anthocyanins containing two or more acyl groups have been reported,
including several with rather complex acylation patterns, eg cyanodelphin
[60], rubrocinerarin [61, 62], the ternatins [63-67] and lobelinins [68], and
the major anthocyanin of Tradescantia pallida [69]. Acylation has a
stabilizing influence on anthocyanins via intramolecular copigmentation, ie
through sandwich-type stacking of the acyl group(s) with the pyrylium ring
of the anthocyanin molecule (Figure 8.2) [27, 31, 53, 70, 71]. The effect is
more apparent for aromatic acyl groups. Deacylated pigments fade
immediately after their dissolution in neutral or slightly acidic media, similar
to the behavior of non-acylated anthocyanins [72]. Monoacylated
anthocyanins do not generally display the color stability of di-, tri- or
polyacylated anthocyanins, indicating that at least two constituent acyl
groups are required for good color stability/retention in neutral or slightly
acidic media. Based on limited data, the nature of the acyl moiety does
seem to have some influence on anthocyanin stability, eg anthocyanins
acylated with p-coumaric acid are less stable than those acylated with
caffeic acid [73, 74].
Methyoxylation of anthocyanidins and their glycosides also occurs, most
frequently at the C-3' and -5' positions but also at position C-7 and -5. It is
no coincidence that natural anthocyanins have not been reported in which
glycosylation or methoxylation occurs at all of the C-3, -5, -7 and -4'
positions. A free hydroxyl group at any of the C-5, -7 or -4' positions is
essential for formation of a quinonoidal (anhydro) base structure [53].
Anthocyanins normally exist in this colored form in plant tissues, where
they are localized in cell vacuoles in weakly acidic or neutral aqueous
solution (eg pH 2.5-7.5) [31, 70, 75-77].
Under acidic conditions the color of non- and monoacylated antho-
cyanins is determined largely by substitution in the B-ring of the aglycone
(Table 8.1). Increased hydroxyl substitution yields a bluer colour, while
Intramolecular stacking
(Sandwich-type)
Acylation
Intermolecular stacking
(Chiral-type)
Copigmentation Self-association
[
anthocyanidin copigment acyl group sugar
eg flavone
Figure 8.2 Schematic presentation of mechanisms of anthocyanin stabilization.
Methoxylation causes the chromophores to become more red [2]. Thus,

aqueous extracts containing primarily pelargonidin and/or cyanidin glyco-
sides appear orange-red, those with peonidin glycosides are deep red, and
those containing glycosides of delphinidin, petunidin and/or malvidin exhibit
bluish-red coloration. The nature of the glycosyl group has no apparent
influence in anthocyanin coloration, although increased glycosyl substitution
and/or substitution specifically at the C-7 hydroxyl does generally lead to
increased color intensity [9]. Glycosylation of B-ring
Hydroxyl groups also generally increase perceived redness. Acylation of

glycosyl groups may have a slight blueing effect.
8.4.2 Distribution
A thorough survey of the distribution of anthocyanins in nature has

revealed that they are particularly characteristic of the angiosperms or
flowering plants, and are more prominent in fruits and berries than in other
plant parts [2, 32, 78, 79]. The pigmentation of plants and plant parts is
rarely due to a single anthocyanin. This is evident in Table 8.2, which lists
the individual anthocyanins in a number of food plants.
The major food sources of anthocyanins belong to the families Vitaceae
(grape) and Rosaceae (cherry, plum, raspberry, strawberry, blackberry,
apple, peach, etc.). Other families containing anthocyanin pigmented food
plants include the Solanaceae (tamarillo, aubergine), Saxifragaceae (red
and black currant), Ericaceae (blueberry, cranberry) and Cruciferae (red
cabbage). Most anthocyanin-containing food plants exhibit colors that are
characteristic of their constituent aglycone types; However, it should be
emphasized that many of the food plants listed in Table 8.2 also contain
pigments other than anthocyanins (eg chalcones, aurones, carotenoids,
chlorophylls), which influence the actual perceived color (hue, tint,
intensity) [146]. The most commonly occurring anthocyanins are based on
cyanidin, followed by pelargonidin, peonidin and delphinidin, then by
petunidin and malvidin [2, 32, 78, 79]. 3-Glycosides occur approximately
two and a half times as often as 3,5-diglycosides, the most ubiquitous
anthocyanin being cyanidin-3-g1ycoside.
8.4.3 Biosynthesis
The biosynthesis of anthocyanins follows a pathway common to other

flavonoids in plants, a pathway that is now well defined at both the
biochemical and genetic levels through the use of genetic mutants, cell
and tissue cultures and substrate feeding studies [40, 46,147-152] .
Biosynthesis is controlled by a number of regulatory and structural genes
[153, 154] that appear to be functionally conserved among plant species
[155]. Mutants at most steps of the pathway have been identified and, at
least in maize, snapdragon and petunia, many of the regulatory and
structural genes have been cloned [154]. The structural genes, those
encoding specific enzymes of the biosynthetic pathway, appear to be
expressed coordinately and in a tissue-specific fashion. Control of
expression of the structural genes appears to occur at the level of transcription factors [1
Table 8.2 Anthocyanins in selected food plants
Botanical name Common name Anthocyanins present References

(organ)
Alium cepa Red onions Cy 3-glucoside and 3- 80-82

laminariobioside, nonacylated and
acylated with malonoyl ester;
Cy 3-galactoside and 3-
diglucoside; Pn 3-glucoside
Brassica oleracea Red cabbage (leaf) Cy 3-sophoroside-5-glucoside 83-86
acylated with malonoyl and
mono- and di-p-coumaroyl,
-feruloyl and -sinapoyl esters
Citrus sinensis blood orange Cy and Dp 3-glucosides 87
Cyphomandra betacea Tamarillo Cy, Pg and Dp 3-glucosides and 3- 88
rutinosides
Dioscorea alata Purple yam (tuber) Cy 3-glucoside, 3-diglucoside, 89-92 3-rhamnoside,
3-gentiobioside acylated with
sinapoyl ester, and 3-glucoside
acylated with feruloyl ester; Pn 3-
gentiobioside acylated with

sinapoyl ester
Ficus spp. Fig (skin) Cy 3-glucoside, 3-rutinoside and 93.94
3,5-diglucoside; Pg 3-
rutinoside
Fragaria spp. Strawberries Pg and Cy 3-glucosides 95-97
Glycine maxima Soybean (seedcoat) Cy and Dp 3-glucosides 98

Ipomoea batatas Purple sweet potato Cy and Pn 3-sophoroside-5- 99,100
glucosides acylated with
feruloyl and caffeoyl esters
Litchi chinensis Lychee fruit (skin) Cy 3-rutinoside and 3- glucoside; 101
Mv 3-acetylglucoside
Malus pumila Apple (skin) Cy 3-galactoside, 3-glucoside, 3- 102-104
xyloside, and 3- and 7-
arabinosides; free and acylated
Mangifera indica Mango Pn 3-galactoside 105
Passiflora edulis Passion fruit Pg 3-diglucoside; Dp 3-
glucoside 106
Phaseolus vulgaris Kidney bean Pg, Cy and Dp 3-glucosides and 107 3,5-
(seedcoat) diglucosides; Pt and Mv 3-
glucosides
Prunus avium sweet cherry Cy and Pn 3-glucosides and 3- 108-110
rutinosides
Prunus cerasus cherry tart Cy 3-glucoside, 3-rutinoside, 3- 109-113
sophoroside, 3-
glucosylrutinoside and 3-
xylosylrutinoside; Pn 3-
glucoside, 3-rutinoside and 3-
galactoside
Prunus domestica Plum Cy and Pn 3-glucosides and 3- 97,110, rutinosides
114
Punica granatum Pomegranate Cy, Dp and Pg 3-glucosides and 115 3,5-

diglucosides
Table 8.2 continued
Botanical name Common name Anthocyanins present References

(organ)
Raphanus sativus Red radish (root) Pg and Cy 3-sophoroside-5- 57, 116,

glucosides acylated with p- 117
coumaroyl, feruloyl and
caffeoyl esters
Rheum rhaponticum Rhubarb (stem) Cy 3-glucoside and 3-rutinoside 117-119
Ribes nigrum blackcurrant Cy and Dp 3-glucosides, 3- 120, 121
diglucosides and 3-rutinosides
Ribes rub rum redcurrant Cy 3-glucoside, 3-rutinoside, 3- 110, 122,
sambubioside, 3-sophoroside, 3- 123
glucosylrutinoside and 3-
xylosylrutinoside
Rubus fruticosus blackberry Cy 3-glucoside and 3- 110, 124,
rutinoside; free and acylated 125
Mv 3-biosides
Rubus ideaus red raspberry Cy and Pg 3-glucosides, 3- 109, 126--
diglucosides, 3-rutinosides, 128
glucosylrutinosides, 3-
sophorosides, 3-sambubiosides, 3,5-
diglucosides and 3-
rutinoside-5-glucosides
Solanum melongena Aubergine Dp 3-galactoside, 3-rutinoside, 129, 130 3,5-
diglucoside, 3-rutinoside-5-
glucoside and 3-
glucosylrhamnoside-5-glucoside
acylated with p-coumaroyl ester
Solanum nigrum Huckleberry Pt and Mv 3-rutinoside-5- 131,132
glucoside; free and acylated with
mono- and di-p-coumaroyl esters
Synsepalum dulcificum Miracle fruit (skin) Cy and Dp 3-galactosides, 3- glucosides and 3- 133
arabinosides
Vaccinium Lowbush blueberry Cy, Dp, Pn, Pt and Mv 3- glucosides, 134
angustifolium 3-galactosides and 3-arabinosides
Vaccinium Highbush blueberry Cy, Dp, Pn, Pt and Mv 3- glucosides 135, 136
corymbosum and 3-galactosides;
Dp, Pn, Pt and Mv 3-
arabinosides
Vaccinium Common cranberry Cy and Pn 3-galactosides, 3- arabinos 137-140
macrocarpon ides and 3-glucosides
Vaccinium oxycoccus Small cranberry Pn and Cy 3-glucosides, 3- 141
galactosides and 3-arabinosides;
Dp, Pt and Mv 3-glucosides
Vitis spp. Grape Cy, Pn, Dp, Pt and Mv mono- 142 and
diglucosides; free and acylated
Zea mays purple corn Cy, Pg and Pn 3-glucosides, and 143-145

Cy 3-galactoside; free and acylated
a Abbreviations: Cy = cyanidin; Pg = pelargonidin; Pn = peonidin; Dp = delphinidin; Pt = petunidin; Mv =

malvidin.
patterns of expression have been observed in different plants, suggesting that the
control of biosynthesis by phytohormones such as gibberellic and abscisic acids
may vary among plant species [158-161]. Anthocyanin accumulation is also
influenced by environmental factors such as light, temperature, plant nutrition,
mechanical damage and pathogenic attack, light being the most important of these.
In general, anthocyanin accumu-lation arising from prolonged exposure to red and
far-red light is mediated by phytochrome, while responses to blue and UV light are
mediated by cryptochrome (ie UV-Alblue light-photoreceptor) and/or a UV-B
-photoreceptor [162]. The identity of the UV photoreceptors is still uncertain, but
there is some evidence to suggest that it may be or involve riboflavin [163, 164].
The signal transduction of the phytochrome system is apparently different from and
independent of that of cryptochrome [165] and the UV-B-photoreceptor [166]. UV-
light is often required in addition to active phytochrome for enzyme induction.
Anthocyanin biosynthesis proceeds according to the scheme in Figure 8.3. Initial

steps of the pathway lead to formation of a CIs-chalcone intermediate synthesized
in the plant from condensation of a molecule of activated cinnamic acid (ie coenzyme
A-ester of mainly p-coumaric acid) with three molecules of malonyl-CoA, catalysed
by chalcone synthase (CHS). The free activated cinnamic acid is derived from L-
phenylalanine by general phenylpropanoid metabolism [147, 148, 150]; malonyl-
CoA is formed from the acetyl-CoA carboxylase reaction [167]. Cyclization of the
yellow colored chalcone by chalcone isomerase (CHI) leads to formation of colorful
isomeric flavanones which themselves are converted to dihydroflavonols (flavan
3-01s) by flavanone 3-hydroxylase (F3H) [46,147].
F3H is a 2-oxoglutarate-dependent dioxygenase requiring Fe2+, oxygen and

ascorbate as cofactors [168]. Dihydroflavonols serve as the immediate precursors
to anthocyanidins and, specifically dihydrokaempferol, act as direct substrates for
specific B-ring hydroxylases (eg flavonoid 3'-
hydroxylase, flavonoid 3' ,5'-hydroxylase) [150, 169] and methyltransfer-ases [148,
152] that lead to variation in B-ring substitution. Substitution in the B-ring may also
occur at the cinnamic acid level; CHS from several plant sources has exhibited in
vitro activity towards caffeoyl-CoA and feruloyl-CoA esters [150]. However, the
coenzyme A-ester of p-coumaric acid appears to be the main physiological substrate
for the enzyme. CHS and the B-ring hydroxy lases are cytochrome P-450-like
monooxygenases that may be associated with microsomal membranes, and require
NADPH and O2 as cofactors [152, 170]. Intermediates in the conversion of
dihydroflavonols to anthocyanidins are leucoanthocyanidins (flavan-3,4-
diols), formed via oxidative reduction of the 4-keto group and catalysed by a
dihydroflavonol reductase (DFR) with NADPH serving as a cofactor [148, 171]. A
similar enzyme, flavanone 4-reductase, catalyses the reduction of flavanones to
flavan-4-0Is, precursors of the unusual 3-
ANTHOCY ANINS AND BETALAINS 255
I POOtooyn-1
co, + H,O
......... ~
CHO
Yo
HCOH
co, to® Yo
3AcelylCoA
HCOH
"
CH,
tH,O®
3CO'-i
~/
Yo Yo
ShIdmI< Acid Pathway 3 MaIonyf-CoA
COOH Co-SCoA
6;~6~
-C4H 0 .4CL - 0 OH OH
L -Phenytolanlne CInnamiC AcId
p-Coumar1cAcid p-Coumar1cAcid
OH
H.O.
HOWO :
Yo
OH CHI CHS
~
Yo
--
OH 0
Chalcone
Flavanone
(yellowW)
(colo!1ess)
F3H 1 OH
H.O.
H.O. OFR
-+H,
OH 0 OH OH
FlaVan 3-d leucocyanldln
(colo!1ess) (colorless)
FOH lH,O
r OH
Anthocyanin
(coloUred)
~J
FGT +--
r OH
Anlhocyanldln
(coloUred)
Figure 8.3 The anthocyanin biosynthesis pathway. Abbreviations: PAL, phenyl ammonia
lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl-coenzyme A ligase; CHS, chalcone
synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol4-
reducease; FDH, flavan-3,4-diol dehydroxylase; FGT, flavonoid glycosyltransferase.
Gly = glycosyl group.
deoxyanthocyanidins (Table 8.1) [172, 173]. Subsequent dehydration of

leucoanthocyanidin into the corresponding anthocyanidin involves a puta-
tive dehydratase [46, 147, 171]. Since the naturally occurring anthocyan-
idins are unstable and somewhat insoluble in aqueous media [174], under
physiological conditions they are thought to be immediately glycosylated.
Glycosylation proceeds in a stepwise manner to higher glycosylated
forms of the anthocyanins, beginning with attachment of sugars to the 3-
hydroxyl residue catalysed by UDP-glycosyl:flavonoid 3-0-
glycosyltransferase. Uri-dine diphosphate (UDP)-sugars serve as glycosyl
donors for all of the glycosyltransferases. Glycosylation is presumed to
be one of the latter steps in anthocyanin biosynthesis. Acylation generally
occurs subsequent to glycosylation, catalysed by acyltransferases with
aliphatic- or aromatic- CoA esters serving as acyl donor substrates [148,
152, 175-177]. Evidence for the enzymatic formation of hydroxycinnamic
acid (RCA)-acylated anthocyanins with RCA-glucose esters serving as
acyl donors has also been provided [178].
Anthocyanins do not accumulate in all cells of plants that synthesize
them. Rather, they are typically localized in flower and fruit tissues, and
in the epidermal and hypodermal cell layers of leaves and stems [46].
Anthocyanin biosynthesis is mediated via a membrane-associated enzyme
complex [179-181]. The first enzyme, phenylalanine ammonia lyase
(PAL), is located on the lumen face of the endoplasmic reticulum (ER)
where it has access to a pool of phenylalanine. Transmembrane
cinnamate 4-hydroxylase channels its product to the cytoplasmic face of
the ER, likely in close proximity to the vacuolar membrane or tonoplast,
where further enzymes of the flavonoid pathway are located. Their
products, dihydro-flavonoids, are further transformed to anthocyanidins
by membrane-associated hydroxylases and cytosolic methyltransferases
[182], and membrane-associated oxido-reductases. Subsequent
glycosylation and acylation of anthocyanidins by glycosyltransferases and
acyltransferases, respectively, may be a requisite for translocation across
the tonoplast into the central or cell vacuole [183-185] where they
accumulate in highly pigmented spherical structures called
anthocyanoplasts. These structures are apparently not restricted to the
cell vacuole, having also been observed free in the cytoplasm [186]. They
occur widely in both dicotyledons and monocotyledons [46] as high
density, strongly osmiophilic globules that lack internal structure [187].
The self-association of anthocyanins in the formation of anthocyanoplasts
may serve to protect individual pigment molecules from degradative reactions to which
The enhanced production of anthocyanins and of vacuolar anthocyan-
oplasts in plants or cell/tissue cultures as a result of low nitrate and high
sugar concentrations [188, 189] suggests that a nitrate-sensitive tonoplast
ATPase may be involved in the translocation and accumulation of
anthocyanins in cell vacuoles [190].
8.4.4 Factors influencing anthocyanin color and stability
As with most natural colorants, anthocyanins suffer from inherent instability;

they are most stable under acidic conditions. They may degrade by any of a
number of possible mechanisms to soluble colored and/or brown colored and
insoluble products. Degradation may occur during extraction/purification and
normal food processing and storage. A know-ledge of the factors governing
anthocyanin stability, and of putative degradation mechanisms, is vital to the
efficient extraction/purification of anthocyanins and to their use as food colorants.
Such knowledge could also lead to more judicious selection of pigment sources
and development of more highly colored food products. The major factors
influencing anthocyanin stability are pH, temperature and the presence of oxygen
and light, but enzymatic degradation and interactions with food components (eg
ascorbic acid, metal ions, sugars, copigments) are no less important. The
interdependence of these various factors precludes their evaluation in strict
isolation.
8.4.4.1 pH. The presence of the oxonium ion adjacent to the C-2 position in
anthocyanins is responsible for their characteristic amphoteric nature. Thus, non-
and monoacylated anthocyanins behave somewhat like pH indicators, existing
as either an acid or a base depending on pH. The structural transformations of
anthocyanins as a function of pH are fundamental to their color and stability.
Anthocyanin-containing solutions generally display their most intense red
coloration at acid pH (eg < pH 3). With increasing pH, anthocyanin-containing
extracts normally fade to the point where they may appear colorful before finally
changing to purple or blue at pH > 6. At any given pH, an equilibrium exists
between four main anthocyanin/aglycone structures (Figure 8.4): the blue quinon-
oidal (anhydro) bases (collectively denoted by A), the red flavylium cation (AH+)
and the colorful carbinol (pseudo)bases or hemiacetals (collectively denoted by
B; the equilibrium concentration of B4 is usually considered to be negligible)
and chalcones (CE and Cz , collectively denoted by C). At neutral or slightly
acidic pH the anthocyanins exist predominantly in their non-coloredforms. The
color of anthocyanin-containing solutions and the relative concentrations of each
of the colored (AH+, A) and colorful (B, C) species at equilibrium (Figure 8.5)
are dependent on the values of the equilibrium constants controlling the acid-
base or proton transfer (Ka '), hydration (K{,) and ring-chain tautomeric (KT )
reactions, where:
K; = ([A]/[AH+])aH+ (8.1)
K': = «[B] + [C])/[AH+])aH+ (8.2)
KT = [C]/[B] (8.3)
either
ccB° w90 I
~ ~ 0GIy
0GIy
"
TO·
7
Yo
~
4 •
. ~ ....:....:
~I ....:
0GIy
~.
0GIy
*00 * 4
~~
OGIy
TO,
0GIy ~I
0GIy
...-:::
TO,.
0GIy
~~
TO
t OGIy 1
AH'
+H,O/-H"Y ~+H'OI-H"
OH
~
OH
H.O.
0GIy
B,
b.
OIL
-eleven
OH
very slow
4
GIyO OH
•
0GIy
OH C,
C,
Figure 8.4 Structural transformations of anthocyanins (ie cyanidin 3,5 diglycoside): flavy-
lium cation (AH+); carbinol (pseudo)bases or hemiacetals (B4, B2); chalcones (CE , Cz);
neutral quinonoidal bases (A4" -A7); ionized quinonoidal bases (A4" -A7). Gly = glycosyl
moiety.
100
c: b
0 ;:;
IV
~
-c:
Q)
(.)
c:
00 50
Q) > ;:;
IV
Cii
0:::
0~
c
TO
0
4 5 6
pH
Yo
Mixture of I Mixture
Flavylium ion of : neutral and
flavyllum cation Neutral qulnonoidal
predominates I ionized
and quinonoidal predominate bases
bases quinonoidal
I bases
Figure 8.5 pH-distribution of anthocyanin species and predominant chromophores at equilibrium

for cyanidin 3,5-diglycoside at 25°C: flavylium cation (AH+); carbinol bases (B); chalcones (C);
quinonoidal base(s) (A). pKn' = 2.23; pKh ' = 3.38; p(Kh ' KT ) = 3.03 (redrawn from Brouillard
[53] and Mazza and Brouillard [30] with permission).
and aH + is the hydronium ion or proton activity (pH = -log aH +) [53, 70, 191, 192].
The expression for Kh ' is often simplified, where the overall conversion of AH+ into
the colorless forms (hemiacetal and cha\cones) are considered together in the
notation B. In highly acidic media (ie < pH 2) the red flavylium cation (AH+ ) is
essentially the only anthocyanin species present (Figure 8.5). With an increase in
pH the flavylium cation yields the quinonoidal base (A) through rapid loss of a
proton. For the naturally occurring anthocyanins so far investigated, pKa' values
(pKa' = -log Ka') generally range from 3.36 to 4.85 [53, 70, 191]. The hydroxyl
groups at C-4', -7 and -5 (if present) have similar ionization constants; therefore,
the quinonoidal species normally exists as a mixture (eg A4"
A7; Figure 8.4). Anthocyanins substituted with more than one free hydroxyl group
become further deprotonated at pH 6.0 to 8.0 to yield a mixture of resonance-
stabilized quinonoidal anions (eg A4,-, A7-).
Unlike f1avylium salts that are unsubstituted at positions C-3 and -5 (ie synthetic
salts), for which hydration reactions occur more readily with quinonoidal base
than cationic forms [193-195], equilibrium between the quinonoidal bases and
hemiacetal of naturally occurring anthocyan ins occurs exclusively via the
f1avylium cation [192, 196]. The concentration of the quinonoidal bases (A), which
become apparent above pH 4, generally remains low due to their thermodynamic
instability relative to the hemi-acetal (B) [197]. On standing, at pH values of about
3.0 to 6.0, nucleophilic addition of water to the C-2 position of the f1avylium cation
slowly yields the colorful hemiacetal (B). Equilibration of this species with the
open cis-chalcone (CE ) is extremely rapid, ie in the range of 1 s or less.
However, the cis-trans equilibrium of CE with the corresponding (Z)-form occurs

very slowly [192]. The hydration-<iehydration equilibrium is usually characterized
by pKh ' values (pKh ' = -log Kh ') of 2-3 [53]. Any process that reduces the
efficiency of the hydration reaction, that is, induces an apparent decrease of Kh ',
should enhance the stability of the colored anthocyanin species. According to
Brouillard [70], a 102-103-fold reduction in the value of Kh ' is sufficient for such
stabilization, whereby the neutral and/or ionized quinonoidal bases are both the
kinetic and thermo-dynamic products of equilibrium reactions. In vivo,
copigmentation (see section 8.4.4.8) strongly favors the anthocyanin chromophores
by reducing Kh ' values, and thereby confers color to plant products within a pH
range in which the anthocyanins generally display poor color properties (eg pH
3.0 to 7.0 ). Anthocyanins may have greater application as food colorants if
polyacylated forms or copigmentation were exploited by food manufacturers. Both
means of anthocyanin stabilization have received considerable attention in recent
years [31, 198].
8.4.4.2 Temperature. As with most chemical reactions the stability of anthocyanins

and the rate of their degradation is markedly influenced by temperature. In
general, structural features that lead to increased pH- stability (ie methoxylation,
glycosylation, acylation) also lead to increased thermal stability [199-201].
Anthocyanin degradation is virtually pH-independent under anaerobic conditions
[202-204]. However, in the presence of oxygen increased methoxyl, glycosyl and/
or acyl substitution generally leads to an increase in the pH at which maximum
thermal stability occurs [127, 199, 205-207]. Deprotonation of the f1avylium cation
by solvent (AH+ ~ A ~ A -) is exothermic, whereas cation hydration (AH+ ~ B) and
pyrylium ring opening (B ~ CE) are both endothermic and associated with positive
entropy changes [208]. Thus, unless structural features or other factors are
successful in reversing the equilibrium towards formation of the f1avylium cation
or quinonoidal base, formation of chalcone is favored by increasing temperature
(viz. during storage and processing) at the expense of quinonoidal, f1avylium and
hemiacetal species .
Coumarin glycoside has been identified as a common product of thermal

degradation of anthocyanidin 3,5-diglycosides; however, anthocyanidin 3-
Glycosides do not form coumarin derivatives [209]. While deglycosylation
may not be required for anthocyanins to undergo thermally-induced
degradation [210], it occurs readily at temperatures approaching 100°C and
higher [202, 203, 211] and is facilitated by the activity of certain enzymes
( see section 8.4 .4.5). The kinetics of deglycosylation vary with the number
and nature of glycosyl constituents, thereby influencing the rate and,
possibly, the mechanism of anthocyanin thermal degradation. Once
deglycosylation has occurred, all anthocyanidins undergo a similar thermal
degradation pattern with chalcone serving as an intermediate product [212,
213]. Subsequent breakdown of the chalcone leads to carboxylic acids (ie
substituted benzoic acids) originating from the B-ring and carboxy-
aldehydes (ie 2,4,6-trihydroxybenzaldehyde) from the A-ring of the parent
anthocyanin [212-214]. a-Diketone, protochuic acid, quercetin and
phloroglucinol have also been identified as thermal degradation products
of anthocyanidin 3-glycosides [202, 212-215]. All of these products may
take part in further reaction to form melanoidins, rather ill-defined brown-
colored complexes that may be evident as a precipitate in fruit juices and
other anthocyanin-containing beverages [204, 210, 215-218].
With few exceptions anthocyanin degradation follows first-order kin-etics.
The mechanism of anthocyanin degradation appears to be temperature-
dependent. At storage temperatures (eg < 40°C) activation energy (Ea)
and z values of around 70 kJ mol-1 and 25°C, respectively, have been
reported; while at processing temperatures (eg > 70°C) Ea values of 95 to
113 kJ mol-1 and z values of around 28°C have been reported [216, 219-
222]. (The z value is the temperature change required to change the
thermal destruction time by a factor of ten; it reflects the temperature-
dependence of thermal degradation.) It would appear, then, that antho-
cyanins have relatively greater stability at higher temperatures, despite that
such conditions favor the formation of the colorful anthocyanin species,
particularly the chalcones. The protective effect of various constituents in a
given system, and of condensation reactions, cannot be overlooked. The
concentration of polymeric pigments has been shown to increase with
temperature and storage time, and to contribute to the coloration of juices
and red wines [223-226]. Several authors have recommended high-
temperature short-time processing to achieve maxi-mum pigment retention
in anthocyanin-containing food products [210, 221, 224, 227]. The short
times involved may be sufficient to prevent significant degradation of
anthocyanins and/or transformation to colorless species.
B.4.4.3 Oxygen and hydrogen peroxide. Oxygen may cause degradation of

anthocyanins by a direct oxidation mechanism and/or by indirect
I and
Oh oh
OxIdized an1hocyanln -
Degradatton
products
h OH
~and
Enzymattc Nonenzymattc
~AO:A
__ 00
Figure 8.6 Coupled oxidation mechanism leading to discoloration and degradation of

anthocyanins (from Peng and Markakis [228] with permission).
oxidation whereby oxidized constituents of the medium react with the

anthocyanins (Figure 8.6). Ascorbic acid and oxygen have been shown to
act synergistically in anthocyanin degradation [229]. Maximum pigment
losses occur under conditions most favorable to ascorbic acid oxidation,
that is, at high levels or concentrations of both oxygen and ascorbic acid
[55, 230]. Ascorbic acid-induced destruction of anthocyanins results from
indirect oxidation by hydrogen peroxide (HzOz), formed during aerobic
oxidation of the ascorbic acid. Significant bleaching of anthocyanin-
containing juices occurs when HzOz is added [231]. Nucleophilic attack at
the C-2 position of anthocyanins by HzOz cleaves the pyrylium ring to
yield a chalcone, which subsequently breaks down to various colorful
esters and coumarin derivatives [59, 232]. In acid solution, HzOz was
shown to oxidize acylated 3,5-diglycosides directly to acylated O-
benzylphenylacetic acid esters [233]. The oxidation products may take
part in further degradation and/or polymerization reactions [210, 230, 231].
Precipitate and haze development in fruit juices may result from direct
oxidation of hemiacetal or chalcone species [204].
8.4.4.4 Light. Anthocyanins are generally unstable when exposed to UV

or visible light or other sources of ionizing radiation [201, 212, 214, 234,
235]. Anthocyanins substituted at the C-5 hydroxyl group, which are known
to fluoresce [236], are more susceptible to photochemical decomposition
than those unsubstituted at this position [51, 201, 237]. Co-pigmentation
can accelerate or retard the decomposition depending on the nature of
the co-pigment [238]. Light causes an increase in the rates at which
anthocyanins undergo thermal degradation, via formation of a flavylium
cation excited state [212]. Photo-oxidation of anthocyanins yields the same
products as thermal degradation, whereby a C-4-hydroxy adduct
serves as an intermediate, which itself undergoes hydrolysis at position C-2

and consequent ring opening to yield chalcone. The chalcone then readily
undergoes further degradation to the usual anthocyanin degradation
products, eg 2,4,6-trihydroxybenzaldehyde and substituted benzoic acids
[212-214].
8.4.4.5 Enzymes. A number of enzymes, endogenous in many plant tissues,

have been implicated in anthocyanin degradation and concomitant loss of
color. These enzymes have been generally termed 'anthocyanases', but
based on their activity two distinct groups of enzymes have been identified.
1. Glycosidases, which hydrolyse the glycosidic bond(s) of anthocyanins

to yield free sugar and aglycone, the instability of the latter chromophores
resulting in their spontaneous degradation via the colorful chalcone
[213,239-241];
2. Polyphenoloxidases (PPO) which act on anthocyanins in the presence of
o-diphenols via a coupled oxidation mechanism (Figure 8.6) [228, 242-
248]. In addition to glycosidases and PPO, Grommeck and Markakis
[249] have reported a perioxidase-catalysed anthocyanin degradation.
PPO are virtually ubiquitous in the plant kingdom, catalysing the oxidative
transformation of catechol and other o-dihydroxyphenols to 0-
quinones, which may subsequently either react with each other, with amino
acids or proteins, and/or with other phenolic compounds including
anthocyanins, to yield brown colored higher molecular weight polymers; or
oxidize compounds of lower oxidation-reduction potential [250].
Anthocyanins are poor substrates for PPO [246, 247]. The quinonoidal
base may be more susceptible to oxidative degradation by PPO than the
flavylium cation [245]; the rate of decolorization mediated by PPO may also
be dependent on the substitution pattern in the B-ring and extent of
glycosylation [251].
Various commercial enzyme preparations, usually obtained from fungal
sources, have been shown to contain glycosidases [213,239,241,252] and/
or PPO [253]. In most cases, secondary activity in commercial enzyme
preparations is desirable, and the costs of purification often preclude total
removal of such activity. Fungal 'anthocyanase' preparations have been
used to remove excess anthocyanin from blackberry jams and jellies that
were too dark and unattractive [254]; and similar preparations have been
suggested for use in the manufacture of white wines from mature red
grapes [239]. However, anthocyanase activity, whether endogenous or
exogenous, can be problematic if maximum pigment retention is desired.
A preliminary steam blanch prior to subsequent processing and/or storage
[255] and packing in high concentrations (eg > 20%) of sugar/syrups
[256] have proven effective in destroying and inhibiting, respectively, endogenous

anthocyanase activity in fruits. Glucose, gluconic acid and glucono-5-lactone are
competitive inhibitors of glycosidases [257]. PPO activity in various anthocyanin-
containing fruit extracts has been effectively inhibited by S02 [258], bisulfite,
dithiothreitol, phenyl-hydrazine and cysteine [248, 259] and gallotannin [251]. The
stabilizing effect of gallotan-nin is also attributed to copigmentation, whereby
excess gallotannin complexes with anthocyanin via hydrophobic interactions.
Ascorbic acid also has a protective effect on anthocyanin degradation by PPO, by
being preferentially oxidized by the o-quinone formed from enzymatic oxidation of
the mediating phenol. Anthocyanins and their associated color are not destroyed
as long as ascorbic acid is present in the system [245].
8.4.4.6 Nucleophilic and electrophilic agents. In acidic media (eg < pH 3)

anthocyanins occur mainly as the phtavylium cation, which itself exists in as many
as six resonance species, the positive charge being delocalized over the entire
heterocyclic structure [260]. Since the highest partial positive charge occurs at the
C-2 and -4 positions [261], non- or monoacylated anthocyanins are particularly
prone to nucleophilic attack at these positions, generally yielding colorful species.
The reaction of ftavylium salts with nucleophiles occurs readily when the C-5
position is un-substituted [50, 51]. Nucleophilic attack by water occurs readily at
the C-2 position, yielding the colorful hemiacetal (Figure 8.4). Prevention of the
hydration reaction is essential to stabilization of anthocyanins. The C-4 position
has been considered less favorable for attack by water [53] and, thus, at equilibrium
the C-4 adduct occurs only as a minor species.
The C-4 position is more susceptible to attack by amino acids and carbon
nucleophiles such as catechin, phenol, phloroglucinol, 4-hydroxycoumarin and
dimedone, to yield 4-substituted ftav-2-enes [261-266]. These compounds are
highly reactive and may undergo further changes depending on the nature of the
4-substituent.
The decolorizing action of S02, an antiseptic agent used extensively in the wine
industry, also results from formation of a colorful C-4 adduct [267-270]. This
reaction is reversible; However, acidification to about pH 1 is required for
restoration of red color. The equilibrium constants and rates of reaction for
formation of anthocyanin-bisulfite complexes have been published elsewhere [268,
269]. The large values of these constants are consistent with observations that
only small amounts of free sulfur dioxide are required to decolorize significant
quantities of anthocyanins.
Kinetically, anthocyanin-bisulfite complexes are quite stable; the bisulfite moiety
presumably deactivates the C-3 glycosidic bond, thereby preventing its hydrolysis
and consequent degradation reactions [202, 211]. Steric factors also contribute to
the stability of anthocyanin-bisulfite complexes.
Noteworthy, is that phenyl, methoxyphenyl, carboxyl and, especially,
methyl substitution at the C-4 position markedly stabilizes the flavylium

chromophore: these C-4 substituted anthocyanins are virtually resistant to
nucleophilic attack [48,191-195,271]. Such C-4 substitution may therefore
provide a practical means to stabilize anthocyanins for use as food
colorants. The only natural C-4 substituted anthocyanin known is purpur-
inidin fructo-glucoside extracted from willow bark [272].
Anthocyanins have been shown to react with flavan-3-0Is, such as
catechin, in the presence of acetaldehyde to yield a covalently linked
product(s) of enhanced color [273-275]. The reaction, which is accelerated
at reduced pH, has been suggested to occur via positions 6 and/or 8 of
both the flavan-3-01 and anthocyanin. Acetaldehyde alone, and other
aldehydes, readily cause fading of crude anthocyanin extracts through
electrophilic attack at these positions. Glycosyl substitution at C-5 reduces
the nucleophilic character of the C-6 and C-8 positions of the flavonoid
nucleus; Thus, anthocyanidin 3,5-diglycosides are less prone to electrophilic
attack than 3-glycosides [273].
B.4.4.7 Sugars and their degradation products. The use of high sugar
concentrations (ie >20%) or syrups to preserve fruits and fruit products has
an overall protective effect on anthocyanin chromophores [256], presumably
by lowering water activity (aw )' Reduced aw is associated with a reduced
rate of anthocyanin degradation [217]: the hydration of antho-cyanin
chromophores to colorful species becomes less favorable as water
becomes limiting. Indeed, dried anthocyanin powders (aw ::; 0.3) are
relatively stable at room temperature for several years when held in
hermetically sealed containers [276-278].
Above a threshold level (eg 100 ppm), sugars and their degradation
products accelerate the degradation of anthocyanins [207]. Fructose,
arabinose, lactose and sorbose have greater degradative effects on antho-
cyanins than glucose, sucrose or maltose [205, 206, 279]. The rate of
anthocyanin degradation is associated with the rate at which the sugar
itself is degraded to furfural-type compounds [210, 280]. These compounds,
furfural (formed mainly from aldo-pentoses) and t-hydroxymethylfurfural
(HMF; formed from keto-hexoses), derive from the Maillard reaction [281]
or from oxidation of ascorbic acid [282, 283], polyuronic acids [55 ] or
anthocyan ins themselves. These degradation products readily condense
and/or react with anthocyanins, possibly via electrophilic attack, ultimately
leading to formation of colorful or complex brown colored compounds.
The advanced sugar degradation products, levulinic and formic acids,
which are readily formed from HMF and furfural in the acidic environments
of most anthocyanin-containing fruits and their juices, do not react nearly
as rapidly with anthocyanins as their parent furyl aldehydes [206, 210 ,
279, 280]. Anthocyanin degradation in the presence of furfural and HMF is
directly temperature dependent and more pronounced in natural systems,
eg fruit juice. Oxygen enhances the degradative effects of all sugars and sugar
derivatives.
8.4.4.8 Co-pigmentation. Harborne [57, 284] suggested that all antho-cyanins

may be ionically bound in the cell vacuole to aliphatic organic acids such as
malonic, malic or citric acid. Such interactions could provide a mechanism of color
stabilization in vivo. Physical of the flavylium cation and/or neutral or anionic
quinonoidal bases onto a suitable surface could also stabilize anthocyanin
chromophores by taking them out of the bulk absorption solution, thereby
preventing color loss caused by hydration reactions [70]. It is possible that the
stability of anthocyanins associated with pectin arises from such interaction [285,
286]. Similarly, the high stability of anthocyanin extracts from the dried flowers of
Clitorea ternatia used to color Malaysian rice-cakes was attributed to adsorption of
the chromophores onto the starch of the glutinous rice [287].
In aqueous media anthocyanins are known to form weak complexes with

numerous compounds such as proteins, tannins, other flavonoids, organic acids,
nucleic acids, alkaloids, polysaccharides and metal ions through which is referred
to as intermolecular co-pigmentation [27, 30 , 31 , 53, 70, 288]. The most effective
copigments have a planar 'IT-electron-rich moiety and are colourless, but when
complexed with anthocyanins they increase the color and stability of the
chromophore. Thus, the flavonols quercitin and rutin, the aurone aureusidin and C-
glycosyl flavones such as swertisin are effective copigments (Table 8.3). A double
bond in the 2,3-position of the C-ring is generally required of flavonoids for efficient
co-pigmentation [289]. C- and O-glycosylated flavones are also effective copigments
[51], as well as flavonolsulfonic acids [238]. Color augmentation, that is, a
bathochromic shift or increase in the wavelength of maximum absorption in the
visible range, results from a reduction in the local charge-distribution or polarity of
the anthocyanin upon complexation with copigment [197].
Both the flavylium cation and quinonoidal base may participate in co-pigmentation
[27, 290, 291]. Co-pigmentation involving both anthocyanin species results in a
bathochromic shift in the visible wavelength of maximum absorption, from red to a
stable blue or purple color; However, increases in maximum absorption or tinctorial
strength may occur only when complex formation involves the quinonoidal base
[290]. Colored anthocyanin species (flavylium cation and quinonoidal bases) are
almost planar with a strongly localized system of 'IT-electrons [260] that facilitates
good molecular contact with a copigment possessing similar struc-tural features.
The extent of the bathochromic shift and increase in maximum absorption

(Table 8.3) are empirical and qualitative parameters, and provide no quantitative
information regarding the co-pigmentation reaction itself. The stability of a given
complex is reflected in the value of the equilibrium
ANTHOCY ANINS AND BETALAINS 267
Table 8.3 Co-pigmentation of cyanidin 3,5-diglucoside (2 x 10-3 M) at pH 3.32
Copigments (6 x 10-3M ) Amax <1.Amax % Absorbance

(nm) (nm) increase at
Amax (nm)
None 508
Auron
Aureusidine 540 32 327
Alkaloids
Caffeine 513 18
Brucine 512 54 122
amino acids
Alanine 508 0 5
Arginine 508 0 20
Aspartic acid 508 0 3

glutamic acid 508 0 6
Glycine 508 0 9
Histidine 508 0 19
Proline 508 0 25
Benzoic acids
Benzoic acid o- 509 18
Hydroxybenzoic acid p- 509 9
Hydroxybenzoic acid 510 1 19

Protocatechuic acid 510 122 23
Coumarin
Esculin 514 6 66
Cinnamic acids m-
Hydroxycinnamic acid p- 513 5 44
Hydroxycinnamic acid 513 5 32
Caffeic acid 515 7 56
Ferulic acid 517 9 60
Sinapic acid 519 11 117
Chlorogenic acid 513 5 75
Dihydrochaicone
Phloridzin 517 9 101
Flavan 3-ols
( + )-Catechin 514 6 78
Flavone
Apigenin 7-glucosidea 517 9 68
C-Glycosyl flavones
8-C-Glucosylapigenin (vitexin) 517 9 238
6-C-Glucosylapigenin (isovitexin) 537 29 241
6-C-Glucosylgenkwanin (swertisin) 541 33 467
Flavonones
Hesperidin 521 13 119
Naringin 518 10 97
Flavonols
Kaempferol 3-glucoside 530 22 239
Kaempferol 3-robinobioside-7-rhamnoside (robinin) 524 16 185
Quercetin 3-glucoside (isoquercitrin) 527 19 188
Quercetin 3-rhamnoside (quercitrin) 527 19 217
Quercetin 3-galactoside (hyperin) 531 23 282
Quercetin 3-rutinoside (rutin) 528 20 228
Quercetin 7-glucoside (quercimeritrin) 518 10 173
7-0-Methylquercetin-3-rhamnoside (xanthorhamnin) 530 22 215
a Formed a slight precipitate. (From Asen et al. [289] by permission of the authors and Pergamon Press.)
binding constant, K, for the complexation reaction [292]. Thus, for co-
pigmentation involving the flavylium cation:
(8.4)
where [AH(CP)n +] represents the concentration of copigment-flavylium cation

complex; [AH+]o and [CP]o the equilibrium concentrations of flavylium cation
and copigment, respectively; and n is the stoichiometric constant, that is, the
number of copigment molecules linked to the flavylium cation in the complex. At
a wavelength close to Amax for ab-sorption of the free flavylium cation AH+,
the equilibrium absorbance is given by:
(8.5)
where EAH + is the molar absorption coefficient of AH+ and l is the optical
pathlength. In the presence of copigment, the absorbance of a given anthocyanin-
containing solution is expressed according to:
A = EAH+ [AH+]ol + EAH(CP)n+ + [AH(CP)n+]ol (8.6)

where EAH(CP)n + is the molar absorption coefficient of AH+ in the copigment
complex. The magnitude of the co-pigmentation effect under a given set of
experimental conditions is then generally described by the relationship:
(A - Ao)/Ao = Kr[CP]on (8.7)
The value of r (= EAH(CP) +/EAH+) is dependent on the wavelength of

absorbance measurement and is often estimated from the ratio A/Ao, where A
is the absorbance associated with an entirely complexed flavylium cation, that
is, the absorbance of a strongly acidic anthocyanin-containing solution to which
excess copigment is added. Plots of In{(A - Ao)/Ao} versus In{[CP]o} are
generally linear with slope n and intercept In(Kr). Such plots have proven useful
in quantifying the co-pigmentation reaction; Values of K and n for a number of
anthocyanin-copigment complexes have been reported [197, 291-294]. The
magnitude of the co-pigmentation effect has been shown to be influenced by pH,
copigment and anthocyanin structure, as well as the molar ratio of copigment to
anthocyanin.
Quantitation of the co-pigmentation effect using equation (8.7) has confirmed
the hypothesis of Goto and coworkers [27, 31, 295] that complex formation is
mediated largely by hydrophobic forces [292, 293]. In the absence of water, the
co-pigmentation effect does not exist: the copigment competes with water for
association with the anthocyanin. Thus, stability is rendered through displacement
of the hydration-dehydration equilibrium (Figure 8.4) towards the chromophores;
for example, with quercitin acting as copigment the hydration constant Kh ' for
cyanin is reduced from 10-2 to 7 X 10-4 M [53]. Association of anthocyanin with
copigment occurs via a
vertical stacking process similar to that which occurs in intramolecular co-

pigmentation (Figure 8.2) [27, 31, 53, 70, 188, 197,291,295]. The chiral
stacking provides an efficient means of protecting the anthocyanin chromo-
phore against nucleophilic attack and, specifically, from hydration and
associated loss of color. Intermolecular co-pigmentation would appear to
be less efficient in the stabilization of anthocyanin chromophores than
intramolecular co-pigmentation, that is, acylation; yet, at the molecular level,
the characteristics of both phenomena are similar. Hydrogen bonding is
likely involved in some co-pigmentation complexes, especially when the
anthocyanin and/or copigment contain sugar moieties [296]; However,
hydrogen bonding is not likely to contribute much to complex stabilization if
only because water itself serves as an excellent hydrogen bond donor and
acceptor. The co-pigmentation effect is greater for anthocyanin 3,5-
diglycosides than for 3-monoglycosides [197] and enhanced, also, with
increased acyl and, especially, methyl substitution [294]. These structural
effects on co-pigmentation reflect differences in the thermodynamics of the
hydration process [197]. Variation in magnitude of the co-pigmentation
effect with pH also reflects the displacement of the hydration equilibrium
towards colored anthocyanin species [293, 294].
Co pigments have been shown to associate with colorful forms of
anthocyanins [197] and with cyclodextrins [291, 297, 298], to result in what
has been referred to as 'anti-co-pigmentation'. Such associations may have
a significant influence on the magnitude of the co-pigmentation effect via
competition of colorful anthocyanin species with the flavylium cation for
copigment, complex formation with colorful species being favored as the
molar ratio of copigment/pigment increases. Generally, co-pigmentation is
more pronounced as anthocyanin concentration and the ratio of copigment
to anthocyanin increase [30, 293, 294, 299-301].
However, there appears to be an optimal molar ratio of pigment to
copigment at which color intensity and stability are maximized, and beyond
which anti-co-pigmentation has a significant effect.
When pigment concentrations become relatively high, anthocyanins
themselves may act as copigments and participate in self-association
reactions [298, 302]. Schewffeldt and Hrazdina [301] noted that competition
can take place between co-pigmentation and self-association re-actions.
They demonstrated that with low concentrations of anthocyanin from Vitis
spp., color augmentation by co-pigmentation with rutin was intensified, but
decreased with corresponding increases in anthocyanin concentration. The
net result of competing co-pigmentation and self-association reactions is
that color may increase more than proportionally to pigment concentration.
4'-Hydroxyl and 5-glycosyl groups appear to be essential structural elements
for self-association [303, 304]. Urea and dimethylsulfoxide disrupt self-
associated aggregates, while sodium salts and magnesium chloride
promote self-association [303-304]. Figueiredo
and Pina [305] have alternatively suggested that ionic salts render color
enhancement through formation of an ion-pair between the charged
flavylium cation and the anion, which displaces the equilibrium towards the
flavylium cation. Self-association constants for the flavylium cation and
quinonoidal base forms of the common anthocyanins have been reported
by Hoshino [306, 307]. Generally, the strength of mutual interaction
increases with substitution of hydroxyl or methoxyl groups in the B-ring,
and is greater for the anionic quinonoidal base forms relative to self-
association of neutral quinonoidal bases or of flavylium cations. Self-
association occurs via chiral stacking (Figure 8.2) with the configuration of
a left-handed screw [27].
The co-pigmentation effect, both intra- and intermolecular, has been
considered primarily responsible for the coloration of flower and fruit
tissues, fruit juices and red wines [292] since anthocyanins alone are
known to be virtually colorless at the pH of these products. That juices
obtained from enzyme-treated fruit mashes are more highly colored than
nonenzyme-treated press-juices can be attributed to decompartmentalization
of various cellular constituents, including flavonoids, alkaloids, amino acids
and nucleosides, that may participate in co-pigmentation with anthocyanins
to varying degrees. Flavonoids, as copigments, are always found in
conjunction with anthocyanins, likely because of their similar biosynthetic
path-ways. Polymeric flavonoids and anthocyanins have been shown to
play an important role in the coloration of grapes and red wines [223, 225,
226, 308]. The constituent tannins (condensed flavonoids) have a protective
effect on anthocyanins. During aging of red wines, monomeric anthocya-
nins are progressively and irreversibly replaced by polymeric pigments
through self-association reactions. Such polymeric material is less pH-
sensitive and relatively resistant to discoloration by S02, ascorbic acid and
light [142, 289, 309].
8.4.5 Extraction and analysis

The choice of a method to extract anthocyanins from any of their various
sources depends largely on the purpose of the extraction, and also on the
nature of the constituent anthocyanins. A knowledge of the factors that
influence anthocyanin structure and stability is vital. If the extracted
pigments are to be subsequently analyzed, qualitatively or quantitatively, it
is desirable that a method be chosen that retains the pigments as close to
their natural state (ie in vivo) as possible. On the other hand, if the extracted
pigments are to be used as a colorant/food ingredient then maximum
pigment yield, tinctorial strength and stability are of greater concern. It is
also important that the extraction and cleanup procedures are not too
complex, time-consuming or costly. Methods of anthocyanin extraction and
purification, and of their subsequent analysis, have been the
subject of several comprehensive reviews [27, 29, 32, 310-312], to which

the reader is referred.
8.4.5.1 Extraction. Anthocyanins are not stable in neutral or alkaline

solution. Thus, extraction procedures have generally involved the use of
acidic solvents which disrupt plant cell membranes and simultaneously
dissolve the water-soluble pigments. The traditional and most common
means of anthocyanin extraction involves maceration or soaking of plant
material in a low boiling point alcohol containing a small amount of mineral
acid (eg ::; 1% HCl) [311]. Methanol is most often used; however; Due to
its toxicity, acidified ethanol may be preferred with food sources, although
it is a less effective extractant and more difficult to concentrate due to its
higher boiling point [310]. Acidification with HCl serves to maintain a low
pH, thereby providing a favorable medium for the formation of flavylium
chloride salts from simple anthocyanins. The use of mineral acids such as
HCl may, however, alter the native form of complex pigments by breaking
associations with metals, copigments, and so on [313-315]. The loss of
labile acyl and sugar residues may also occur during subsequent
concentration [202]. Thus, to obtain anthocyanins closer to their natural
state several neutral solvents have been suggested for initial pigment
extraction, including 60% methanol, n-butanol, ethy-lene glycol, propylene
glycol, cold acetone, acetone/methanol/water mix-tures and , simply,
boiling water [202, 276, 287, 316]. In addition, weaker organic acids-mainly
formic acid, but also acetic, citric and tartaric acids--or stronger, more
volatile acids such as trifluoroacetic acid have become popular for use in
extraction solvents, especially for extraction of complex, polyacylated
anthocyanins [277 , 313-315, 317]. Extraction of anthocyanins with ethanolic
or aqueous S02 or bisulfite solutions has led to increased pigment yields
and to concentrates of higher purity, color intensity and pigment stability
than those obtained via hot water extraction [234, 318-320]. The initial
decanted or filtered anthocyanin extracts are usually quite dilute and
require concentration prior to subsequent purification or use as a food
colorant. Depending on the means of extraction, decreasing the ratio of
extraction solvent to plant material, as in continuous counter-current
extraction. [321], could avoid the need for a concentration step. To minimize
pigment degradation, concentration is best carried out in vacuum at
temperatures below 30°C [310]. The acidic aqueous anthocyanin
concentrates thus obtained can be used as such, frozen [276], freeze-dried
[234, 278, 322, 323] or spray-dried [277].
8.4.5.2 Purification. Purification of anthocyanin-containing extracts is often

necessary, since none of the solvent systems commonly used for extraction
is specific for the anthocyanins: there may be considerable quantities of
extraneous material (eg other polyphenolic substances,
pectin) that could influence the stability and/or analysis of these pigments.
Co-extracted flavonoids, which react similarly with the common reagents
used for phenolic analysis, are commonly removed via chromatographic
techniques employing insoluble polyvinylpyrrolidone (PVP) [324-326],
polyamide [327], combination polyamide-PVP [59, 328, 329 ]. Sephadex
G- 25 [101] or LH-20 [101, 330, 331], octadecylsilane [101, 331, 332], weak
anion exchange (eg Amberlite CG-50) [111, 333, 334], polyethylene glycol
dimethacrylate [ 335] and cellulose-type resins [120, 127,240,336].
Droplet counter-current chromatography (DCCC) using n-butanol-glacial
acetic acid-water (BA W) as the solvent system [337-339], preparative thin-
layer chromatography (PTLC) [340--343], solvent-solvent extraction with n-
butanol [108], and precipitation with basic lead acetate [l08] or via the
spherical agglomeration technique employing a benzene-methanol-aqueous
hydrochloric acid solvent mixture [344] have also been used to separate
and/or purify anthocyanins from their crude or concentrated extracts. If
appreciable quantities of lipid, chlorophyll or unwanted polyphenols are
suspected to be present in anthocyanin-containing extracts, these materials
may be removed by washing with petroleum ether, ethyl ether, diethyl ether
or ethyl acetate [310, 311, 345]. This may improve purification but is not
always necessary.
Purification of anthocyanins for analytical purposes was traditionally
carried out using paper or thin-layer chromatographic techniques [45, 310,
345]. However, more rapid and efficient separation of complex mixtures is
achieved with reversed-phase high-performance liquid chromatography
(HPLC) [225, 226, 330, 332, 338, 339, 346-350]. The technique is non-
destructive and, therefore, separated peaks are readily collected for
subsequent analyses. With appropriate selection of eluent, column type and
length, flow rate and temperature, HPLC can be used to resolve and
quantitate microgram quantities of anthocyanin without the need for
preliminary purification of extracts [351-355]. A major application of HPLC
is, therefore, the simultaneous qualitative and quantitative analysis of
anthocyanins [356].
8.4.5.3 Qualitative analysis. Current analytical approaches that allow for

unambiguous identification of individual anthocyanins are based on proven
in vitro techniques. Well developed chromatographic and spectro-scopic
methods have been applied for rapid and accurate identification of
anthocyanins. However, absolute characterization of anthocyanins cannot
generally be established by chromatographic or spectral methods alone.
Structural characterization generally involves identification of the aglycone,
sugar moieties and acyl groups, if present, and the position(s) of attach-
ment of the sugar and acyl groups. Such is currently facilitated by a
combination of different spectroscopic techniques, including infra-red (IR)
[357], Raman resonance [358, 359], ultraviolet/visible (UVMS) [45,312,
360], circular dichroism (CD) [31, 295, 303, 304, 306, 307, 361], nuclear
magnetic resonance (NMR) [31, 92, 306, 307, 347, 361-372] and mass
spectrometry (MS ) [347, 366, 369, 372-376]. The complete structural
characterization of anthocyanins is possible using current one- and two-
dimensional (lD and 2D) NMR techniques; However, relatively large
quantities of purified material are required for resolution of proton signals
associated with the glycosyl moieties [369] and positions C-6 and C-8 of
the flavylium nucleus [376]. When only small amounts of material are
available, MS techniques are the preferred methods for analysis of
anthocyanin structure,[312]. These techniques are often interfaced with
liquid chro-matography or HPLC to facilitate pigment purification, and
employ such ionization methods as fast-atom bombardment (FAB) [31,
366, 369], electrospray [374, 375] and thermospray [377]. While the
sophisticated NMR and MS techniques have become indispensable for
the structural characterization of complex, polyacylated anthocyanins, the
equipment is costly and not always readily available. Thus, the classical
hydrolysis techniques for structural elucidation of anthocyanins will
continue to be important; they can be quickly and efficiently carried out
when interfaced with HPLC and gas-liquid chromatography (GLC) [378, 379].
Much information regarding the identity of a given anthocyanin and its
aglycone can be derived simply by observing its color in aqueous solution
or on paper. The color generally changes from orange-red to bluish-red as
the structure is modified through pelargonidin, cyanidin, peonidin, mal-
vidin, petunidin and delphinidin [310]. In addition, under UV-light
anthocyanins substituted at the C-5 position usually fluoresce [236]. The
spectral characteristics of most of the known anthocyanidins and many of
the anthocyanins in 0.1% HCl-methanol have been published by Har-
borne [45, 312, 360]. In acid solution anthocyanins and their aglycones
exhibit two characteristic maximum absorption; one in the visible region
between 465 and 550 nm and a smaller one in the UV region at about 275
nm. The wavelength of maximum absorption can be used to tentatively
identify the aglycone; However, confirmation by other methods is required.
A bathochromic shift of 15-35 nm from the visible maximum upon addition
of 5% alcoholic aluminum chloride (AICI3) to pigments in 0.1 % HCI-
methanol indicates the presence of a free o-dihydroxyl group in the
aglycone, a structural characteristic of cyanidin , delphinidin and petunidin
[380]. Glycosylation of anthocyanidins at the C-3 position generally results
in a bathochromic shift in the wavelength of maximum absorption, while
glycosyl substitution at C-5 produces a shoulder on the absorption curve
at 440 nm . The ratio of absorbance at the UV maximum to that at the
visible maximum, and the ratio of absorbance at 440 nm to that at the
visible maximum provide valuable information regarding the extent and
position of glycosyl substitution [50, 360]. Acylated anthocyanins exhibit
an additional weak absorption maximum in the 310 to 335 nm region, the
current maximum indicating the type of aromatic acylation involved [59,

310]. The complex acylated anthocyanins exhibit yet an additional
absorption maximum at 560---600 and/or 600---640 nm at pH > 4.0 [381].
The traditional means of anthocyanin characterization generally involves
(controlled) acid, alkali, enzyme and/or peroxide hydrolysis and subsequent
chromatography of hydrolysis products to facilitate their identification [45,
310, 382]. Analytical HPLC has become the standard chromatographic
procedure, especially since the advent of the photo diode array detector
[351, 352, 356, 383-385]. General anthocyanin structure-retention
relationships have been reviewed by Strack and Wray [32, 312).
The chromatographic mobility (Rf value) of anthocyanins on paper [45,
310, 382] or cellulose strips (ie TLC) [126, 202, 343, 386] provide an
alternative useful parameter for identification purposes. Pigment char-
acterization by all chromatographic methods requires comparison with
authentic anthocyanin, aglycone and/or sugar standards. Although antho-
cyanins and their aglycones can be purchased from various suppliers, they
often require purification prior to their use as reference pigments.
Acid hydrolysis of anthocyan ins generally yields aglycones and sugars,
the sugars being cleaved from the pigment in a more or less random
fashion [120,310]. The course of hydrolysis can be followed, and the
glycosylation pattern determined, by the production of intermediate pigments [387].
Alkaline hydrolysis, while it too may be used to determine the nature of the
aglycone, is more commonly employed for acyl group determination.
Under nitrogen alkaline hydrolysis specifically removes acyl groups,
generally leaving the glycoside intact [120]. The removal of oxygen is
necessary since anthocyanins possessing o-dihydroxy groups are unstable
in alkali [310]. Enzymatic hydrolysis procedures generally involve the use
of f3-glycosidases of fungal origin [239, 257]. The activity of f3-glycosidase
is generally not influenced by the nature of the aglycone [239]. The glycosyl
moieties of simpler glycosides (eg 3-0-f3-glycosides) are rapidly hydro-
lysed, thereby allowing relatively easy identification and differentiation from
more complex anthocyanins: since ex-glycosides are hydrolysed more
slowly, if at all, by f3- glucosidase, acylated pigments are virtually
unaffected during enzymatic hydrolyses. Oxidation of anthocyanins with
peroxide liberates the C-3 sugar [388]. Subsequent permanganate
treatment releases sugars attached to the readily ruptured heterocyclic
ring, and ozonolysis releases sugars attached to enolic and phenolic hydroxyl groups [38
Peroxidation of anthocyanidin 3,5-diglycosides under neutral conditions
yields coumarin derivatives, while peroxidation of anthocyanidin 3-
glycosides does not [209, 232].
8.4.5.4 Quantitative analysis. Quantitative methods may be conveniently

divided into three groups depending on the needs of the analysis [310,
389]:
1. Determination of total anthocyanin concentration in systems containing

little or no interfering substances,
2. Determination of total anthocyanin concentration in systems that contain
interfering material, 3.
Quantitation of individual pigments.
In fresh plant extracts/juices there are generally few interfering
compounds that absorb energy in the region of maximum absorption of
anthocyanins (465-550 nm). Total anthocyanin concentration may thus be
determined by measuring the absorbance of the sample (diluted with
acidified alcohol) at the appropriate wavelength. It is important that Beer's
Law be obeyed in the concentration range under study; self-association
and co-pigmentation effects cause deviations in Beer's Law and can lead
to inaccurate estimates of total anthocyanin concentration [390].
Considerable sample dilution may be necessary to counteract these effects.
Adams [202] suggested that absorbance measurements be carried out at
a single pH as low as possible to ensure maximum color development and
low response to changes in pH which may occur (Figure 8.7). Comparative
measurements of total anthocyanin may be made at pH 1.0 for samples
containing predominantly 3-glycosides [202] and at pH less than 1.0 for
samples containing mainly 3,5-diglycosides [268].
For samples containing a mixture of anthocyanins, absorbance
measurement at a single low pH value are proportional to their total
concentration [392]. Estimates of the concentrations of individual
anthocyanins in the mixture can only be obtained with prior knowledge of
the proportions of individual pigments therein. Absolute concentrations
may then be estimated by the use of weighted average absorptivities and
absorbance measurement at weighted average wavelengths [323]. More
accurate estimates are obtained with samples in which a single pigment is
present or predominates. Molar extinction coefficients (absorptivities) of a
number of anthocyanins have been reported [137, 310, 393]. It is important
that the same solvent and pH be used for absorbance measurement as
were used for determination of molar absorptivities [53, 137, 323, 382]. The
single pH method is subject to interference by and, therefore, cannot be
used in the presence of brown colored degradation products, for example,
from sugar or pigment breakdown. Unpurified pigment extracts may also
contain compounds that at low pH absorb in the range of maximum
absorption of the anthocyanins [202]. Differential or subtractive absorption
methods [389] may be used to determine the total anthocyanin concentration
in those samples that contain interfering substances.
The subtractive method involves measurement of sample absorbance at
the visible maximum, followed by bleaching and remeasurement to give a
blank reading. Subtraction gives the absorbance due to anthocyanin, which
can be converted to anthocyanin concentration with reference to a
0.7 ~max 468
0.6
Q)
() 0.5
c
C'il ..0
~
0 0.4
~ max 373
(J) ..0 <:
0.3
0.2
0.1
350 400 450 500

Wavelength (nm)
Figure 8.7 Effect of pH on the visible spectrum of 3'-methoxy-4',7-dihydroxyftavylium chloride

(5.2 x 10-3 gil) in aqueous solution after standing for 2 h (redrawn from Jurd (391) with
permission). pH: 1.0.63; 2, 2.30; 3, 2.65; 4,2.94; 5, 3.11; 6,3.40; 7,3.67; 8, 3.81; 9, 4.10;
10,4.36.
calibration curve prepared using a standard pigment [202]' The most

common bleaching agents have been sodium sulfite [389, 394, 395] and
hydrogen peroxide [396]. The subtractive method has come under
criticism since the bleaching agents used may cause a decrease in
absorbance of some interfering substances, resulting in erroneously high
values of total pigment concentration [310].
The differential method relies on the structural transformations of
anthocyanin chromophores as a function of pH (Figures 8.5 and 8.7). It is
also based on the observation that the spectral characteristics of
interfering brown (degradation) products are generally not altered with
changes in pH [223]. The difference in absorbance at two pH values and same wavelen
(eg the anthocyanin visible maximum or at 550-600 nm for polyacylated

pigments [381]) is assumed to provide a measure of anthocyanin
concentration since absorbance due to interfering substances cancels in the
subtraction. A number of different pH values have been used in the differential
method, but measurement at pH 1.0 and 4.5 is most common [397].
Noteworthy, is that an insufficient time for equilibration between the different
anthocyanin species upon pH adjustment can yield erroneous results.
An advantage of the differential method is that data can be used to

calculate an anthocyanin degradation index (DI), defined as the ratio of total
anthocyanin determined at a single pH to that determined by the differential
method [310]. The DI is a useful measure of browning products that absorb
at the visible maxima of anthocyanins. However, it cannot be used with such
products as aged red wines which contain condensed/polymeric anthocyanins
that are relatively pH insensitive and absorb strongly at the visible maxima
of anthocyanins [223]. They cannot be considered as anthocyanin
degradation products since they contribute to the total anthocyanin
concentration and color of the product. The DI has been used as an indicator
of anthocyanin color and overall quality deterioration in a number of berry
fruit products [55, 398-401]. The simple ratio of absorbance of red (eg
anthocyanin) pigments measured in the 465 to 550 nm region to that of
brown (eg degradation) products measured in the 400 to 440 nm region also
provides an index of anthocyanin or, more accurately, color deterioration
[389, 402, 403].
Measurements of anthocyanin concentration rarely correlate with the
actual color of a given product or sample; since the pH of analysis and
endogenous pH of the sample usually differ, the distribution and
concentration of anthocyanin chromophores also differ. Wrolstad [389]
reviewed methods for determining color density, polymeric colour, percentage
contribution by tannin and monomeric anthocyanin colour, calculated from
only a few absorbance readings at the native sample pH. A two point index—
the difference in absorbance at the visible maximum and at 420 nm—has
been shown to correlate well with visual appearance [404].
However, for those with available equipment, tristimulus measurements have
been shown to correlate much better with human visual responses [310,
403-405].
The quantitative analysis of individual anthocyanins requires their prior
separation/purification from a mixture, usually carried out by chromatog-
raphy. Spectral measurements are usually of limited use since the absorption
spectra of most anthocyanins are so similar [360]; yet, as previously
mentioned, molar absorptivities have been reported and used for quantita-
tion of a number of anthocyanins. Quantitation of individual anthocyanins
has traditionally involved their separation by paper or thin layer chroma-
tography, followed by photometric determination of pigments in situ by
reflectance or transmittance densitometry [55, 111, 139, 406]. Reversed-

phase HPLC is currently the method of choice for quantitation of
anthocyanins [32, 312, 356]. The speed, resolving power and ability to
quantitatively separate pigments has revolutionized the qualitative and
quantitative analysis of anthocyanins.
8.4.6 Current and potential sources and uses

Several synthetic flavylium salts have been suggested as potential food
color additives [48, 271,191, 193, 195,407]. The production of synthetic
anthocyanins identical to their natural counterparts (ie nature-identical) is
also possible [51], although yields via chemical synthesis are generally
poor [408]. These latter products provide a source of food color additives
with consistent quality/purity and known properties. However, all syn-thetic
flavylia, including the nature-identicals, currently require extensive
toxicological testing prior to clearance as safe food additives. Public concern
over the safety of food additives has restricted the sources of commercial
anthocyanin preparations to food plants naturally containing these pigments;
this is not likely to change in the very near future.
The potential of various food plants as commercial sources of antho-
cyanins is limited by availability of raw materials and overall economic
considerations. Numerous plants and plant parts have been suggested as
potential commercial sources (reviewed by Francis [9]), including cran-
berry [277, 334, 409, 410], blueberry [321], red cabbage [321, 322], roselle
( Hibiscus sabdariffa) calyces [276, 316, 411], miracle fruit [133, 412],
berries of Vibernum dentatum [413], bilberries (Vaccinium myrtillus) [414],
duhat [Sysyium cumini Linn.) [415], black olives [416], flowers of Clitorea
ternatia [63, 290] and Ipomea tricolor [417,418], and leaves of cherry-plum
[419,420], Perilla ocimoides (ie shiso) [233] and Tradescantia pallida [69,
198, 379]. The anthocyanins of interest from most of these sources are di-,
tri- or polyacylated, and many exist in more complex, supra-molecular forms
[27, 31, 421, 422]. They, therefore, display good stability in aqueous
systems at slightly acidic or neutral pH; yet, their yields are often poor and
source materials are not generally readily available in quantities that may
be necessary for commercial extraction. Grapes provide the greatest
quantity of anthocyanins for use as food colorants: production of grapes
constitutes about a quarter of the annual fruit crop worldwide [9, 239].
Both a spray-dried powder and a concentrated solution containing grape
anthocyanins have been marketed for years from Italy under the trade
name of Enocolor (formerly Enocyanina) [423].
Economically, the best potential commercial sources of anthocyanins are
those from which the pigment is a byproduct of manufacturing of other
value-added products [424]. The extraction of grape anthocyan into juice or
wine is an inefficient process and large quantities of extractable
pigment are available in grape pomaces and waste material [9]. Antho-
cyanin extracts have been produced from these sources [207, 234, 277,
317, 318] and used to successfully color a wide range of commercial
products [425]. Anthocyanin extracts have also been prepared from
cranberry presscake [277, 334, 409]. Approximately 40% of the anthocyanins
from cranberries remains in the presscake following juice extraction [404].
The tropical red berry known as miracle fruit contains a taste modifier,
Miraculin, that has potential as a sweetener; anthocyanin extracts have
been obtained as a by-product of taste modifier production [133, 411]. The
production of expensive food or other plant crops for their pigment content
alone is not economically feasible. Yet, the availability of highly pigmented
inexpensive crops such as red cabbage [321, 322] and bilberries [413]
makes the use of these crops as potential sources of commercial
anthocyanin preparations viable.
Anthocyanin preparations from natural sources may vary considerably in
quality [426] since they may contain partially degraded and/or con-densed
pigments, tannins, copigments and various impurities which are co-extracted
with the anthocyanins. Stable, relatively pure anthocyanin preparations
have been produced in relatively large quantities (eg up to about 5% on a
dry basis) by suspension cultures of cells of Populus spp.
[427,428], Daucus carota [429, 430], Vitis spp. [431] and Euphorbia millii
[432]. Anthocyanin production by cell cultures from numerous other sources
has been reported; Studies have been directed not only towards improving
productivity for large scale pigment production, but also towards identifying
those factors that influence anthocyanin accumulation.
One of the greatest limitations to commercial production of anthocyanins
via cell or tissue culture is the requirement for light. Only a few species of
plant cell culture have been reported to produce anthocyanins in the dark,
but levels of production have been generally low. The exception is a highly
productive cell line of Aralia cordata, obtained by continuous cell-aggregate
cloning, which has been reported to yield anthocyanins in concentrations
as high as 17% on a dry weight basis [433-435]. While an effective scaled-
up process has been documented [433], costs of production must be
competitive with conventional anthocyanin recovery processes.
Costs could be reduced considerably should an inexpensive alternative to
refined sugars be made available and found to be suitable, eg milk whey
[436]. Time may yet see the commercial production of anthocyanin
preparations via application of biotechnologies.
Natural anthocyanin preparations used as food colorants suffer the same
fate as endogenous pigments; their addition to food systems may
encourage reactions with endogenous constituents that can lead to their
stabilization or destabilization, and thereby influence product quality. With
careful formulation/selection of certain ingredients, choice of appropriate
stages during formulation and processing at which the colorant and/or other
ingredients are added, and control of processing and storage conditions, a wide
range of high quality products can be attractively colored with anthocyanins.
Counsel et al. [436] have reviewed numerous food applications for anthocyanins
and other natural pigments, including chewing gums, hard candies, fruit chews,
dry beverage powders, cream fillings, icings and fruit coloration. Anthocyanins
may also be successfully used in high acid foods such as soft drinks, jams and
jellies, and in red wines where they contribute to the overall aging process [408].
For those food products for which endogenous pigments are to serve as the only
or major source of coloration, procurement of intensely colored raw material is
necessary to ensure that if some pigment is destroyed during processing/storage,
a sufficient portion remains to impart the characteristic product color [389].
Few practical stabilizing agents have been found for the anthocyanins [424].
Of nineteen different additives, only thiourea, propyl gallate, and quercetin
demonstrated a stabilizing effect on color retention in straw-berry juice and
buffered pigment solutions [210]. Cysteine, which may act as a reducing agent
and/or inhibitor of PPO, was shown to inhibit anthocyanin degradation in Concord
grape juice below a temperature of 75°C [244]. Ascorbic acid can render a
stabilizing effect on one hand, by being preferentially oxidized by PPO, but a
destabilizing effect on the other, by indirectly oxidizing the anthocyanins via its
oxidation products [51]. Tartaric acid and glutathione have been shown to have
a protective effect on anthocyanins by acting as mildly acidic and antioxidant
agents, respectively [437]. The addition of phenolic compounds, such as rutin
and caffeic acid, also markedly stabilized the color of blood orange fruit juice,
presumably by means of intermolecular co-pigmentation [437]. Indeed, in view
of the factors that influence pigment stability, the most practical means of
stabilizing the color of anthocyanins within the bounds of maintaining their
'natural' status is via complex formation (eg surface adsorption), intermolecular
co-pigmentation and/or condensation re-actions (eg with proteins, tannins or
other polyphenols).
8.5 Betalains
B.5.1 Structure
The structure of the betalain chromophore may be described as a protonated 1,7-
diazaheptamethin system (Figure 8.8); betaxanthins and betacyanins are
distinguished by substitution on the dihydropyridine moiety by specific Rand R'
groups. The yellow betaxanthins are character-ized by having Rand R' groups
that do not extend the conjugation of the 1,7-diazaheptamethin system. However,
if conjugation is extended, where-by Rand R' comprise a substituted aromatic
ring (ie cyclodopa), the
h
COOH HOOC .... COOH
Figure 8.8 Structural transformations of the betalain chromophore.
chromophore is red and characterized as a betacyanin [3, 4]. All beta-

cyanins are glycosylated [3] and derive mainly from the aglycones betanidin
or isobetanidin (the C-15 epimer of betanidin). 2-Decarboxybetanidin has
also been reported, as a minor pigment in Carpobrotus ancinaciformus
[438], and a glycoside of neobetanidin, 14,15-dehydrobetanidin, has also
been identified as a natural constituent of some plants [439, 440]. The most
common betacyanin is betanin, the 5-0-J3-glucoside of betanidin (Table
8.4). The most common glycosyl moiety is glucose; sophorose and rhamnose
occur much less frequently. Acylation of pigments may also occur whereby
the acyl group is attached via an ester linkage to the sugar moiety. The most
common acyl groups include sulfuric, malonic, 3-hydroxy-3-methylglutaric,
citric, p-coumaric, ferulic, caffeic and sinapic acids. The structures of many
acylated betacyanins have yet to be fully characterized [4].
In betaxanthins the cyclodopa unit of betacyanins is displaced by either

an amine or amino acid, suggesting the potential for existence of over 200
different betaxanthin pigments. For example, the R' group of vulgaxanthin-I
from Beta vulgaris is a residue derived from glutamic acid; that of
indicaxanthin from Opuntia fiscus-indica joins with the R group to give a
proline moiety (Table 8.5). Other R' groups of the betaxanthins include
glutamine, methionine sulfoxide, tyramine, DOPA and 5-hydroxy-norvaline.
The R group of betaxanthins is usually a hydrogen. Like the betacyanins,
the structures of many of the betaxanthins have yet to be fully elucidated [4,
441, 442].
8.5.2 Distribution
Unlike the more widely distributed anthocyanins, betalains have been

detected only in red-violet, orange and yellow-pigmented botanical species
belonging to closely related families of the order Caryophyllales [3, 4, 33].
Table
.o0N
0
N
LC
laceinca
rutob
s ecnerefeR
dnuopmoC noitun
tirtsebttuaS
p
"" t
sR 6R
rehtie"
ninateB 6 7 3, 0
8 58
7
8
9 73
9
0 4
5
vulgaris
Beta
esocu-3
lgj h ti:"(
Chenopodium
nihtnaramA cinor-o)cd-u
i-O
3
cl3
'a
2
g
j(-j h
-ninaisoleCI -)lyoram-uO
--op
03
'2
c(-[j h seY
rum
Chenopodium
rub
-ninaisoleC
II -)ly-sonlu
-aO
-r-r0
e3
't2(-f[j h
-niniserI
-c3in
-yo
'6
xro
-u)rd
cd-u
i-O
3
c3l'h
y 2
g
a
j(- h
-)ly-)ralyteu
nslogollacym
u
h--03
lt'6
ge(-j-m
3j
ninateberP 98
0
1
2 73
1 5
Phyllocactus
vulgaris
Beta
sanderiana
-)H30--S
03
'6(-j h
nitcacollyhP
h
ninainiviR -)H30--S
03
'3(-j h
-nihtnarpmaLI -) lyoramu--op3
O
'6
c(j h
Lampranthus
-nihtnarpmaILI -lyolur-e
O
3
'6fj h
Bougainvillea
-niellivniaguo-3B
Irj
h
19
3
714
85
3
rubra
-ninerhpmoG
HI
-ninerhpmoG
H
II -)Iyoramu--O
-op3
'6
c(-j
Basella Gomphrena
-ninerhpmoIG
H
II -)ly-sonluar-re
O
3
't6(fj
Basella
-ninerhpmoG
V h -)ly-sonluar-re
O
3
't6(fj
Table 8.5 Structures of some known betaxanthins
R'O~I .H
~ +.'
R,O N eOOH
H~~COOH
Yo
h
Compound Substitution pattern botanical source Reference
R R'
Indicaxanthin Rand R' together form proline Opuntia ficus-indica 388

Portulacaxanthin-I Rand R' together form Portulaca grandiflora 389
hydroxyproline
Portulacaxanthin-II H tyrosine Portulaca grandiflora 492
Portulacaxanthin-III H glycine Portulaca grandiflora 492
Vulgaxanthin-I H glutamine Beta vulgaris 390
Vulgaxanthin-II H glutamic acid Beta vulgaris 390
Miraxanthin-I H methionine sulphoxide M irabilis jalapa 391
Miraxanthin-II H aspartic acid Mirabilis jalapa 391
Miraxanthin-III H tyramine Mirabilis jalapa 391
Miraxanthin-V H dopamine Mirabilis jalapa 391
Dopaxanthin h L-DOPA Glottiphyllum longum 392
Humilixanthin H hydroxynorvaline Rivinia humilis 393
Muscaaurin-I H ibotenic acid Amanita muscaria 394
Muscaaurin-II H stizolobic acid Amanita muscaria 394
Muscaaurin-VII H histidine Amanita muscaria 394
Many of these species have developed the ability to carry out C4-
photosynthesis and typically contain unusual sieve-element plastids and
relatively large concentrations of ferulic acid in their cell walls [3].
Although remaining a point of phylotaxonomic contention, ten betalain-
producing families have been identified: Aizoaceae, Amaranthaceae,
BaseJla-ceae, Cactaceae, Chenopodaceae, Didiereaceae, Holophytaceae,
Nyctagina-ceae, Phytolaccaceae (including Stegnospermaceae) and Portulacaceae.
Betalains of fungal origin have also been found. A violet betacyanin,
muscapurpurin, and seven yellow betaxanthins, muscaaurins-I to -VII, have
been isolated from the poisonous mushroom Amanita muscaria (fly agaric;
Order Agaricinales) [5, 6]. The unusual amino acid stizolobic acid and a
cyclized form of this amino acid are found in muscaaurin-II and muscapurpurin,
respectively. Some of the muscaaurins have identical structures to those of
known betaxanthins produced by members of the Caryophyllales, ie
indicaxanthin, vulgaxanthin-I and -II, miraxanthin-II1
[4-6]. The yellow pigment muscaflavin, possessing a dihydropyridine structure

characteristic of the betalains, has also been found in pigment extracts of Amanita
muscaria [443, 444]. Muscaflavin also occurs naturally in Hygrocybe toadstools
accompanied by pigments that are muscaflavin analogs of the betaxanthins,
commonly referred to as hygroaurins [6, 445].
Betalains, as with the anthocyanins, accumulate in cell vacuoles of the flowers,
fruits and leaves of the plants that synthesize them, mainly in epidermal and/or
subepidermal tissues. Furthermore, betalains often accumulate late in plant stalks
and are found in high concentrations in underground parts of the common red
table beet [5]. Of the numerous natural sources of the betalains (eg red beet, cacti,
cockscomb, pokeberry), the red beet and prickly pear are the only food products
containing this class of pigments [33, 446]. The major betalain in beets, betanin,
accounts for 75 to 95% of the total betacyanin content; the remainder is comprised
of isobetanin, prebetanin and isoprebetanin. These latter two pigments are sulphate
monoesters of betanin and isobetanin, respectively. The major yellow pigments in
the red beet are vulgaxanthin-I and -II.
8.5.3 Biosynthesis
The biosynthesis of betalains arises from arogenate of the shikimate pathway, and
its conversion to tyrosine via arogenate dehydrogenase [447].
The dihydropyridine moiety present in all betalains is synthesized in vivo from two
molecules of L-5,6-dihydroxyphenylalanine (L-DOPA), one of which must first
undergo 4,5-extradiol oxidative cleavage and recyclization [448] through seca-
DOPA [449, 450] to betalamic acid (Figure 8.9).
Alternatively, in toadstools L-DOPA undergoes 2,3-extradiol cleavage to form
muscaflavin [6]. Betalamic acid constitutes an essential structural feature of all
natural betalains, but accumulates as a natural constituent only in those plants that
produce betaxanthins [451-453]. The conversion of L-DOPA to betalamic acid is
catalyzed by a dioxygenase [454, 455]. Its subsequent condensation with
cyclodopa or a derivative of cyclodopa leads to betacyanidins; Condensation with
other amines or amino acids results in betaxanthins. Glycosylation of betacyanidins
occurs late in the biosynthetic pathway [456], via uridine-5-diphosphate (UDP)
linked sugars as mediated by nucleoside-diphosphate-sugar-dependent glycosyl
transferases [457].
Alternatively glycosylation of cyclodopa may occur prior to its condensation with
betalamic acid [458, 459]. Acylation takes place subsequent to glycosylation, and
most often involves hydroxycinnamic acid transferases with 1-0-hydroxycinnamic
acid-acylglycosides as the acyl donors [460-
462].
The use of betalain-producing cell cultures has facilitated the elucidation of
enzymes involved in betalin biosynthesis and the influence of various environmental
factors such as light, temperature, precursor availability,
.H
Yo
HO '" ~
. COOH
Tyrosine
HO~.H
_____ > HOWH

HOV H,NA COOH HO '" ~
COOH
h
L-DOPA
5-Cyclodopus
1
HO~'"
I, ·.H J HO~ .H Ho~~Acoo
"0 A ~;: DO I / eoo:;9-c~-_OG-;~_c:-on_atlo_n---.. Betacyanlns
h Yo
Betanldln
HOOC" N COOH
Yo
h
5-Betalamlc acid
Amino acid or amine

other than cyclodopa 1
.1
Betaxanthins
Figure 8.9 Biosynthesis of betalains.
presence of cytokinins and auxins, and availability of nutrients. A key

regulatory step in the biosynthesis and accumulation of betalains appears
to be the hydroxylation of tyrosine [463] which may be under auxin and/or
cytokinin control [464-466]. The ratio of auxin to cytokinin is an important
factor in establishing and stabilizing in vitro cell culture lines of specific
colored phenotypes [467], and may be important in regulating the type and
accumulation of betalains in vivo. In contrast to anthocyanin accumulation
in both plant and cell culture systems, the accumulation of betalain pigments
is greatest during growth or cell division [468]. Light can serve as a powerful
stimulant of betalain biosynthesis, although its effect appears to be species-
dependent; while light is an absolute requirement for pigment synthesis in
some plants (eg Amaranthus and Phytalacca spp.), it is not for others (eg
Beta vulgaris). UV- and red-light-induced betalain synthe-sis appears to be
mediated by phytochrome via gene activation [469].
Cytokinins have also been shown to promote betalain biosynthesis,
presumably also via gene activation, even in the dark [470, 471]. The
mechanisms of light- and cytokinin-induction, while synergistic, appear to be

independent of one another [472].
8.5.4 Color and structural stability
The stability of betalains is influenced by numerous confounding factors (eg pH,

temperature, oxygen, water activity, light) that have limited the use of these
pigments as food colorants. The majority of work to date has focused on the
influence of these various factors on betanin. Comparat-ively fewer studies have
been reported on the effects of these factors on the betaxanthins. To date, no
systematic work has been carried out to investigate the effects of acylation and/or
betalain substitution on the structural stability of these pigments.
8.5.4.1 pH. Generally, the red color of betanin solutions remains unchanged from
pH 3.0 to 7.0, exhibiting maximum absorption at 537-538 nm [446]. Below pH 3.0
the color changes to violet as the absorption maximum is shifted to 534 to 536 nm
and its intensity decreases; a slight increase in absorbance at 570-640 nm is also
observed. Above pH 7.0 the color of betanin solutions also becomes bluer, due to
a bathochro-mic shift in the wavelength of maximum absorption. The greatest
blueing effect occurs at pH 9.0, with maximum absorption at 543 to 544 nm. Above
pH 10.0 a decrease in intensity at the absorption maximum of 540-550 nm is
accompanied by an increase in absorption at 400-460 nm due to release of
betalamic acid, which is yellow; pigmentation thus changes from blue to yellow as
a result of alkaline hydrolysis of betanin to betalamic acid and cycIodopa-5-0-
glycoside [473] (Figure 8.10).
8.5.4.2 Temperature. The thermolability of the betalains is probably the most

restrictive factor in their popular use as food colorants. The thermal degradation of
both betanin and vulgaxanthin-I have been shown to follow first-order kinetics over
a pH range 3.0 to 7.0 under aerobic conditions [446, 474-477], but to deviate from
first-order kinetics under anaerobic conditions [478 ]. The activation energy (Ea)
for betanin deg-radation in the presence of oxygen is approximately 84 kJ mol-I
at pH 3.0-
7.0 [476, 477,479,480]. Under similar conditions, vulgaxanthin-I has an Ea of
around 67 kJ mol-I [476], and is therefore more sensitive than betanin to thermal
degradation. The thermal stability of the betalain pigments is greatest between pH
5.0 and 6.0 in the presence of oxygen [474, 476, 481] and between pH 4.0 and
5.0 in its absence [474, 477]. The rate of degradation of betanin and vulgaxanthin-
I is more rapid in model systems, suggesting a protective effect conferred by
constituents in the natural system (eg by polyphenols, antioxidants). In both natural
and model systems betanin may be regenerated via Schiff's base condensation
G~WH
00 ~ \0=
WZJlCOOH
Yo
h
lsobetonln
1~~~
GuOWH
HO::-- 6+ 'COOH ~~OH
GIuOW
::-- +
•
Yo
+ "H
H.O. NCOOH ~t~
h
Yo
Yo
HOOC COOH ~
h
Betolamlc Acid
Yo
h Cyciodopa-5-O-glucoside
Betonin
l~
GluO~
HO::-- 6COOH +
~~H
CO,
Yo
h
h COOH
Yo
Decorboxylated Betonln
Figure S.lO Mechanisms of betanin degradation,
of betalamic acid with cyclodopa-SO-glycoside (Figure 8.10) [474, 481-

483]. Betanin may also yield isobetanin and/or decarboxylated betanin,
their formation being favored upon heating at pH 3.0-4.0. These latter
compounds do not differ significantly from betanin in their absorption/color
properties and they yield similar thermal and pH degradation products (ie
betalamic acid derivatives and cyclodopa-SO-glycoside) [473].
The primary steps in betanin degradation, as mediated by temperature

and/or pH, involve nucleophilic attack (eg by water) at the C-ll position [484],
ie at the carbon atom adjacent to the quaternary amino nitrogen (Figure
8.10). As already alluded to, this reaction is reversible, the regeneration of
betanin being greatest in the pH range in which the pigment is most stable
[477, 482, 48S]. While maximum stability of betalamic acid occurs at pH
6.0-8.0 [486], that of cyclodopa-SO-glycoside occurs at pH 3.0 [477]. The
Ea for regeneration of pigment ranges from 2.S
to 14.7 kJ mol-I at pH 3.0 to SO [483]. Thus, provided that betalamic acid

and cyclodopa-SO-glycoside are present, regeneration of betanin is
favoured, especially at lower temperatures. However, betalamic acid is
heat sensitive; it may undergo aldol condensation or participate in Maillard
reactions thereby making it unavailable for the regeneration reaction [477].
At temperatures approaching 130aC under alkaline conditions betalamic
acid yields formic acid and 4-methylpyridine-2,6-dicarboxylic acid [487].
The glycosidic moiety of cyclodopa-SO-glycoside may also be cleaved at
high temperatures. It is also very susceptible to oxidation reactions,
initiating polymerization to melanin-type compounds [477]. Thus, as
temperature increases, particularly in the presence of oxygen (viz. during
storage or processing), irreversible betanin degradation is promoted. The
quantity of betanin degradation during and regeneration after thermal
treatment is dependent on, among other things, the presence of
nucleophiles, temperature, pH, and also initial betanin concentration; as
initial betanin concentration increases so it does color stability [483].
8.5.4.3 Light. Like the anthocyanins, betalains are susceptible to

degradation by various sources of radiation. The photochemical destruction
of these pigments involves molecular oxygen and follows first-order kinetics
[480]. Photooxidation of red beet pigments is also pH-dependent, greater
degradation occurring at pH 3.0 than at pH SO [488]. Exposure of betanin
solutions to UV/visible light increases the rate of pigment degradation by
as much as lS% [446]. Absorbed light in the UV or visible range presumably
acts to excite 1T electrons of the pigment chromophore to a more energetic
state (ie 1T*). This causes a higher reactivity or lowered activation energy
for the molecule. The result is a decrease in the dependence of degradation
rate on temperature [480]. Gamma irradiation also enhances the rate of
betanin degradation; doses exceeding 100 krad result in total loss of color
[489].
8.5.4.4 Oxygen. Involvement of molecular oxygen in pigment degradation

is not restricted to photooxidation; it has been implicated in thermal
degradation as well. Pigment losses are reduced through nitrogen purging
of purified betanin solutions [474] and through application of glucose
oxidase (an oxygen scavenger) to red beet concentrates [490]. Metal ions,
common food constituents or contaminants from processing equipment,
may function as pro-oxidants. Metal cations (eg iron, copper, tin, alu-
minium) accelerate the rate of betanin degradation [491, 492]. As electron
donors or acceptors, depending on their oxidation state, metal ions may
stabilize the electrophilic center of betalains resulting in rearrangement of
associated bonds, destruction of the chromophore and concomitant loss
of color [491]. No protective effect is provided by ascorbic acid or a-
tocopherol, commonly added to food systems as antioxidants;
Extremely elevated concentrations of ascorbic acid (ie 1000 ppm) decrease

pigment shelf-life since it then acts as a pro-oxidant. High concentrations of
citric acid and EDTA (eg 10 000 ppm) have been found to markedly improve
betanin stability, possibly by partially neutralizing the electrophilic center (ie
they may sequester betanin) and/or by sequestering metal ions that
otherwise act as pro-oxidants [491, 492]. The brunt of evidence suggests
that betanin oxidation does not occur via a free radical mechanism.
8.5.4.5 Water activity (aw). The effect of aw on betanin stability has also
been documented [475, 493, 494]. First-order rate constants for betanin
degradation have generally varied exponentially with respect to aw, a four-
fold increase in pigment stability being observed as aw decreases from 1.0
to 0.37. Reduced aw may increase betalain stability by reducing the
nucleophile (ie water) concentration, by limiting reactant mobility and/or by
decreasing oxygen solubility. Using various water/alcohol model systems
and correction for the appearance of degradation products, Simon et al.
[494] noted that there was a maximum in the dependence of the rate
constant on aw. Their results were explained in terms of the propensity of
the electrophilic center of betalains to nucleophilic attack. Increasing
amounts of water in alcohol increased the rate constant since the water
was better able to transfer protons than alcohol. However, for a certain aw
or water/alcohol ratio, the contribution of the proton transfer rate to the
overall rate constant approached a maximum. Further increases in aw were
associated with a decrease in concentration of the stronger nucleophile (ie
alcohol), and the rate constant thus decreased. Their results [494]
demonstrate the important role of solvolysis in betanin degradation.
8.5.4.6 Enzymes. As with other natural pigments, specific decolorizing

enzymes also influence betalain stability. Decolorizing activity has generally
been found concentrated where most pigment also accumulates, that is, in
subcellular tissue separated from the epidermal layers [495, 496].
However, while betalains are localized in cell vacuoles, the majority of
decolorizing activity (ie 70--76%) occurs in leucoplasts; additional activity is
found mainly associated with microsomal fractions [42]. This different
intracellular compartmentation of enzyme and substrate may be regarded
as an adaptation of betalain-producing plants to function in pigment storage.
Much of the decolorizing activity becomes strongly associated with cell wall
material during extraction, requiring high salt concentrations (eg 2: 1 M
NaCl) for its recovery [43,497]. The presence of decolorizing activity is of
consequence during extraction/purification, and in subsequent use of the
extract as a food dye, since pigment yields may be significantly reduced
[496, 497]. Inactivation, for example through mild thermal treatment, may
be required to prevent or reduce enzyme catalyzed fading of pigment
extracts.
The enzymatic conversion of betalains to colorful species is associated

with the consumption of molecular oxygen, but does not involve hydrogen
peroxide [42]; the enzyme has thus been referred to as betalain oxidase.
An enzyme with similar properties has been isolated from several sources
[43, 495, 496, 498]. It decolorizes both betacyanins and betaxanthins, and
exhibits optimum activity at pH 3.4-3.5 and 38-40°C. Its activity is inhibited
by NH4N03 , KN03 , KCI and NaCl. Further inhibition of the enzyme by
sodium azide, sodium diethyldithiocarbamate and thiourea has led to the
suggestion that its activity may be dependent on the presence of a metal
ion (ie iron) in the active site and on disulfide linkages [495].
Enzymatic reaction products are reportedly identical to those initially
appearing as a consequence of thermal treatment or alkaline hydrolysis
(Figure 8.10) [498]; However, in the acidic conditions of the reaction,
betalamic acid is unstable and is readily converted to 2-hydroxy-3-
hydrobetalamic acid [42]. Three additional betalain oxidases were partially
purified from tissues of Phytolacca Americana, with pH optima between
5.0 and 5.5 [43]. These particular enzymes were suggested to have a
different function in vivo from that with a pH optimum at pH 3.5, since they
were not inhibited by various salts. Further work is required to more fully
characterize the various betalain oxidases, to develop effective strategies
by which their activity can be controlled.
8.5.5 Extraction and analysis
8.5.5.1 Extraction and purification. The availability of commercial

preparations of betalains is currently restricted by legislation in most
countries to beet juice concentrates produced by concentrating juice under
vacuum to 60-65% total solids, or beet powders produced by spray- or
freeze-drying the concentrate [10, 11 ]. Beet juices have traditionally been
prepared by hydraulic press operations in which raw beets are blanched,
chopped, pressed and the expressed juice filtered. Less than 50% recovery
of betalains can be expected from most conventional press operations
unless macerating enzymes are used to facilitate pressing. However, up
to 90% recovery of betalains from beet roots has been reported with use
of a continuous diffusion apparatus [499, 500]. Since commercial
preparations are not pure they vary in color, depending on the proportions
of betacyanin and betaxanthin extracted, and often possess beet-like odor
and taste due to the presence of geosmin. Purification of the betalains is
possible on both small and large scale, although the methods used
generally give poor yields.
Separation and purification of betalains from crude plant extracts is
usually necessary if qualitative and/or quantitative analyzes are to be
carried out. A number of different approaches have been reviewed [3, 4].
Preliminary separation of betalains in a variety of crude aqueous plant

extracts has been accomplished by non ionic absorption on strongly acid
resins (eg Dowex SOW) followed by polyamide column chromatography,
using increasing concentrations of methanol in aqueous citric acid as the
developing solvent [501, 502]. Citric acid is removed from resolved betalains
by resin treatment. Sequential chromatography on series of Sephadex ion
exchangers has also provided good separation of betalains [5, 443, 459,
503]. Adams and von Elbe [504] reported that gel filtration column
chromatography on Sephadex or polyacrylamide (Bio-Gel P-6) gels could
be used to rapidly and efficiently separate the betalains from beet juice.
Numerous other chromatographic and electrophoretic pro-cedures have
been used over the years to separate/purify betalains from a variety of
sources. However, most of these procedures are time-consuming. Since
their initial application for analysis of the betalains [505], HPLC methods
have become widely adopted as a fast and efficient means of betalain
separation and analysis [440, 441, 506-511].
8.5.5.2 Qualitative analysis. Structural characterization and identification of

betalains generally involves direct comparison of spectroscopic,
chromatographic and electrophoretic properties with authentic standards,
before and after controlled hydrolysis [4, 33, 512]. Since the betalains differ
from anthocyanins in being more sensitive to acid hydrolysis, in their colors
with changes in pH, and in their chromatographic and electrophoretic
properties, they can be easily differentiated by simple color tests [3, 311].
Since the occurrence of these two pigment types is mutually exclusive,
color tests can usually be performed using crude plant extracts [311 ].
Betacyanins and betaxanthins possess similar chromatographic and

electrophoretic properties; Thus, similar techniques are used for their
isolation/purification. However, polyamide chromatography is only moderately
successful for the separation of betaxanthins. Preparative paper
electrophoresis and chromatography using alternate supports are commonly
used for separation of individual betaxanthins from co-occurring betacyanins
and other plant constituents [512]. HPLC is also becoming an invaluable
means by which to separate and analyze the betalains; the number of
betaxanthin structures was found to be considerably greater using HPLC
than was known from more traditional separation techniques [441]. The
spectral properties and RP-ion pair HPLC retention characteristics of a
number of natural and semisynthetic betaxanthins were recently reported
[442].
Tentative identification of betalains can be deduced from their chroma-
tographic or electrophoretic behavior [512]. For example, on polyamide or
cellulose-based resins:
1. The retention of betacyanins decreases with increased glycosyl substitu-

tion.
2. The retention of 6-glycosides exceeds that of the 5-glycosides.
3. Iso-derivatives are retained slightly longer than their corresponding C-15
epimers.
4. Acyl groups, aromatic more than aliphatic, increase the retention of
pigments.
Each pigment possesses characteristic electrophoretic mobility between
0.30 and 1.78, relative to betanin, usually determined at both pH 4.5 and
2.4 [502, 513].
Corroborative data may be provided by analysis of absorption spectra.
All betacyanins exhibit visible maxima between 534 and 552 nm [502],
while all betaxanthins have absorption maxima in the range 474 to 486 nm
[513, 514]. Acylated betalains generally exhibit a second absorption
maximum in the UV region of 260 to 320 nm, where the absorption of non-
acylated pigments is weak [512, 515]. As with the anthocyanins, the
number of acyl residues in betalains can be estimated from the ratio of
absorption at the visible maximum to that at the UV maximum [512].
Further valuable structural information may be obtained from lH-NMR
spectra [439, 441, 515]. Indeed, the current sensitivity of NMR spectral
ethers allows complete structural characterization of betalain pigments,
even when sample size is small [4]. Corroborative molecular weight data
have been obtained by FAB MS and/or GC/MS [439, 441]. The former
technique has the advantage that the betalain pigments do not require
derivatization beforehand. The IR spectra of betalains have been found to
provide little useful information [515].
Betacyanins occur naturally as C-15 epimers, the betanidin derivatives
generally being present in greater quantities than their corresponding
stereoisomers [512]. Strong acid hydrolysis of betanin gives, along with
glucose, a mixture of both aglycones [516], while milder controlled acid
hydrolysis [517], or ~-glucosidase hydrolysis [518], yields only betanidin.
Acylation often renders the sugar moiety resistant to enzymatic cleavage.
Acid hydrolysis of isobetanidin derivatives yields only the isobetanidin
stereoisomers [501]. It is thus possible to establish whether two betacyanins
of unknown structure are C-15 epimers; if this is the case, subsequent
analyzes can be conveniently carried out using a mixture of both pigments
[512]. In addition to acid and enzyme treatments, oxidation using alkaline
hydrogen peroxide can liberate constituent glycosyl moieties [519]. The
cleaved sugar group(s) may be identified by conventional paper chroma-
tography or HPLC; the aglycone and its degradation products may be
identified by conventional methods, for example by comparison with
authentic standards using paper chromatography, electrophoresis or HPLC.
The position(s) of glycosyl attachment may be determined by treatment
with excess diazomethane that converts the aglycone into an O-methyl

neobetacyanidin trimethylester glycoside in which the originally free phenolic
hydroxyl group of the aglycone has been methylated and thus labeled for
identification [512]. In acidic media, the neobetacyanidin exhibits a
bathochromic shift of about 100 nm relative to the parent glycoside [520,
521]. Diazomethane methylation similarly converts beta-xanthins into neo-
pigments which exhibit absorption maxima at 340 to 360 nm and show a
bathochromic shift of 80-100 nm in acid solution [522].
Alkaline fusion of the neo-pigments yields 4-methylpyridine-2,6-
dicarboxylic acid [3, 523].
The identity and position of sugar groups, as well as of acyl or sulfate
groups linked to the glycosyl moieties, are now most often determined by
NMR techniques. Traditionally, acyl and/or sulfate residues are removed by
cold alkaline hydrolysis under nitrogen (ie oxygen must be absent), isolated
and identified by chromatographic techniques [515]. Their position of
attachment has been determined by:
1. Mild periodate oxidation, subsequent borohydride reduction, mild acid
hydrolysis, a second borohydride reaction, then chromatographic
identification of the resulting polyols; or
2. Pigment methylation with Mel/AgO in dimethylformamide and subsequent
identification of the products released after acid hydrolysis of the
permethylated compound [512, 515].
8.5.5.3 Quantitative analysis. In the absence of interfering substances (eg

fresh extracts or juices, purified samples), the quantitation of betalains can
be accomplished using spectrophotometry, whereby absorption-ance at
the visible maximum is expressed in terms of concentration by use of
appropriate absorptivities [449, 502 , 516, 524-526]. Nilsson [527] developed
a spectrophotometric method that directly determines beta-cyanin and
betaxanthin pigments in beets without their initial separation.
The method is based on the observation that while vulgaxanthin-I absorbs
only at 476-478 nm, betanin exhibits a maximum at 535-540 nm but also
absorbs at the visible maximum of vulgaxanthin-1. Calculation of the ratio
of absorbance at 538 nm to that at 477 nm as a function of concentration
(ie dilution) leads to determination of the measurable concentration of
vulgaxanthin-1. Absorbance of the sample at 538 nm is due only to betanin,
allowing its direct determination. Results are expressed in terms of total
betacyanin and total betaxanthin concentrations since minor constituent
betalains also contribute to measured absorbances.
If interfering substances are present in the sample, for example due to
betalain degradation, the betalain pigments must first be purified. The
procedures generally used, usually paper or column chromatography [504]
or electrophoresis [528], not only purify but also separate pigments,
thereby allowing for quantitation of total and individual betalains simultaneously.

Due to its speed and high resolving power, and the fact that preliminary
purification is not always necessary, HPLC is the method of choice for quantitative
analysis of individual and total betalains [437, 480, 491,507,510,511].
8.5.6 Current and potential sources and uses
The use of betalain pigments as food colorants dates back to at least the turn of
the century when juice from pokeberries, which contain betanin, was added to
wine to impart a more desirable red color [529]. With the passing of legislation
against the adulteration of foods, this practice was eventually prohibited. Current
legislation restricts betalain colorants to concentrates or powders obtained from
aqueous extracts of red beets (Beta vulgaris). Because of the relatively high
pigment concentrations therein, red beets are the only plant food source from
which extraction of betalains for commercial use as a colorant is economically
feasible. Commercial beet colorants typically contain 0.4-1.0% pigment (expressed
as betanin), 80% sugar, 8% ash and 10% protein [10]. Their color can vary
considerably, depending on the content of yellow betaxanthins; thus their color
varies with beet variety, beet root quality and age at harvest, and method of
pigment extraction.
Improvements in the pigment content of table beets have been reported

through traditional breeding procedures [530]. Selection of high pigment yielding
populations was attributed to high realized heritability for total pigment; selection
may have increased the frequency of a dominant allele of the multiple allelic
series at the R locus that is associated with pigment concentration [531].
Increased pigment content of commercial beet color-ants could also be achieved
if legislative restrictions on the use of purified pigments were lifted. Purification of
beet extracts can be carried out via industrial-scale chromatography. Although
yields have not generally been good to date, purification of crude extracts by ion-
exchange and gel-filtration chromatography has been proven successful on a
laboratory scale [532]. Further developments could make chromatographic
systems very useful for large-scale production of purified betalain dyes.
Purification of red beet extracts has also been carried out by fermentation using
Aspergillus niger [533, 534], Candida utilis [535, 536] and Saccharomyces
oviformis [511], resulting in products with improved stability and no off-flavors, (ie
geosmin is removed in the fermentation process). The practical application of
microbial purification for large-scale preparation of commercial betalain dyes is
uncertain.
Similarly, the accumulation of betalains in beetroot and other cell suspension

and tissue cultures has been reported, but the practical application of this or
other biotechnologies to the production of highly purified
betalain preparations for use as food colorants have yet to be demonstrated

[537]. In vitro cell suspension or tissue cultures can be more efficient and
yield greater levels of pigment than whole plant cultivation.
The in vitro production system has several advantages over conventional
beet cropping and extraction for commercial betalain production [538]:
1. High culture growth rates allow fermentations to be carried out in the

time required for a single beetroot crop to be raised.
2. The compound responsible for the characteristic beetroot odor (ie
geosmin) is not formed.
3. Growth conditions can be established that eliminate the requirement for
secondary fermentations that may otherwise be necessary for purification.
4. One can be selective with respect to the nature of the betalain pigment(s)
produced.
Yet, conventional red beet extraction production is still less expensive than
comparable biotechnological processes, or than chemical syntheses that
tend to be plagued by low product yields. Further increases in pigment
yields are deemed necessary for biotechnological processes to become
economically viable [538]. Alternatively, world market demands for these
natural dyes in more highly purified forms than are currently available from
conventional processes could render in vitro processes more viable in the
future.
Commercial and purified betalain preparations have been shown to be
effective colorants of foods with compatible chemical and/or physical
properties. Microbiologically purified beetroot preparations have demon-
strated suitable stability for use in various pharmaceutical forms [539-541].
Beetroot colorants may be used alone to produce hues resembling raspberry
or cherry, or in combination with other colorants such as annatto to obtain
strawberry shades [10, 436]. Considering the factors that influence pigment
stability beet root preparations may be successfully used to color products
with short shelf-lives that are marketed in a dry state, that are packaged to
reduce exposure to light, oxygen and humidity, and/or that do not receive
high or prolonged heat treatment. If thermal processing is required pigment
degradation can be minimized by adding the colorant after the heat
treatment or as near the end of the heating cycle as possible.
As reviewed by Counsell et al. [436] and von Elbe [542], beetroot colorant
can be effectively used to color hard candies and fruit chews, dairy products
such as yogurt and ice cream [543], salad dressing, frostings, cake mixes,
gelatin desserts, starch-based puddings [544], meat substitutes [446],
poultry meat sausages [545, 546], gravy mixes, soft drinks and powdered
drink mixes [547].
8.6 Conclusions
The successful use of anthocyanins and betalains (especially betacyanins) to color

a variety of foods and pharmaceuticals has been clearly demonstrated. Although
these pigments may not replace the more stable synthetic red dyes, they do provide
suitable natural alternatives for the production of highly colored and high-quality
products. Their successful application is obviously limited to products with
compatible physicochemical properties. However, if current legislation restricting
the use of more highly purified extracts and sources other than grapes (and grape
by-products) and beets were lifted, wider application of anthocyanins and betalains
as food colorants could be realized.
At present, the anthocyanins would appear to be more stable than the

betacyanins. However, systematic research to investigate the stabilizing influence
of acylation, various condensation reactions and/or surface adsorption has not
been carried out on the betalains. Polyacylated anthocyanins, and those which
partake in condensation reactions (eg self-association and co-pigmentation) and/
or are adsorbed onto surfaces, demonstrate marked stability and offer great
promise as stable colorants.
With further technological advances, for example in the development of industry-
scale chromatographic purifications and/or separations and application of
biotechnology (eg cell suspension or tissue culture) for large-scale pigment
production, discovery of new pigment sources and structural variants with enhanced
stability, and modification of foods to stabilize constituent anthocyanins and
betalains, the potential of these natural pigments for wider application as colorants
would seem to be unlimited. A greater knowledge of the structural features
necessary for enhanced stability and color properties is required, however. Further
investigations into the physicochemistry of anthocyanins and betalains should lead
to increased application of these natural colorants in food products.
Acknowledgments
The authors gratefully appreciate the help of Cathy Young and Allen Mao in the
preparation of the chapter for the first edition of this volume, and the
Department of Food Science, University of Guelph, for financial assistance-
ance.
References
1. Timberlake. CF and Bridle. P. in Anthocyanins as Food Colors (P. Markakis. ed.).

Academic Press, New York (1982) Chapter 5.
2. Mazza, G. and Miniati, E. Anthocyanins in Fruits, Vegetables and Grains, CRC Press, Boca
Raton (1993).
3. Mabry, TJ in Secondary Plant Products (EA Bell and BV Charlwood, eds), Springer-Verlag,
Berlin (1980) Chapter 11.
4. Steglich, W. and Strack, D. in The Alkaloids: Chemistry and Pharmacology, Vol. 39 (A.
Brossi, ed.), Academic Press, New York (1990) Chapter 1.
5. Musso, H. Tetrahedron 35 (1979) 2843-2853.
6. Gill, M. and Steglich, W. Prog. Chern. Org. Nat. Prod. 51 (1987) 1-286.
7. Kimler, L., Mears, J., Mabry, TJ and RosIer, H. Taxon. 19 (1970) 875-878.
8. Meggos, HN Food Technol. 38 (1984) 70-74.
9. Francis FJ CRC Crit. Rev. Food Sci. Nutr. 28 (1989) 273-314.
10. Marmion, DM Handbook of us Colorants for Foods, Drugs and Cosmetics. 2nd edn.
John Wiley & Sons, New York (1984) Chapter 8.
11. Collins, P. and Timberlake, C. Inti. Food Ingred. 6 (1993) 32-38.
12. Singleton, VL and Esau, PL Phenolic Substances in Grapes and Wines and Their
Significance, Academic Press, New York (1969).
13. Horowitz, RM and Gentili, B. 1. Agric. Food Chern. 17 (1969) 696-700.
14. Haveland-Smith, R.B. Mut. Res. 91 (1981) 285-290. 15.
von Elbe, JH and Schwartz, SJ Arch. Toxicol. 49 (1981) 93-98.
16. Constantion, L., Albasini, A., Rastelli, G. and Benvenuti, S. Planta Med. 58 (1992)
342-344.
17. Frankel, E.N. Kanner, J., German, J.B., Parks, E. and Kinsella, J.E. Lancet 341 (1993)
454-457.
18. Tamura, H. and Yamagami, A. 1. Agric. Food Chern. 42 (1994) 1612-1615.
19. Tsuda, T., Ohshima, K., Kawakishi, S. and Osawa, T. 1. Agric. Food Chern. 42 (1994)
248-251.
20. Vlaskovska, M., Drenska, D. and Ovcharov, R. Probl. Vutr. Med. 18 (1990) 3-19.
21. Drenska, D., Bantutova, I. and Ovcharov, R. Farmatsiya (Sofia) 39 (1989) 33-40.
22. Igarashi, K., Abe, S. and Satoh, J. Agric. Bioi. Chern. 54 (1990) 171-175.
23. Pourrat, A. Lejeune, B., Grand, A., Bastide, P. and Bastide, J. Med. et Nut. 23 (1987)
166-17.
24. Igarashi, K. and Inagaki, K. Agric. Bioi. Chern. 55 (1991) 285-287.
25. Karaivanova, M.H., Drenska, D. and Ovcharov, R. Comptes Rendus de I'Academie Bulgare
des Sciences 42 (1989) 119-21.
26. Markakis, P. (ed.) Anthocyanins as Food Colors. Academic Press, New York (1982).
27. Goto, T. Progr. Chern. Org. Nat. Prod. 52 (1987) 113-158.
28. Jackman, RL, Yada, RY, Tung, MA and Speers, RA 1. Food Biochemistry. 11 (1987)
201-247.
29. Jackman, RL, Yada, RY and Tung, MA 1. Food Biochemistry. 11 (1987) 279-308.
30. Mazza, G. and Brouillard, R. Food Chern. 25 (1991) 17-33.
31. Goto, T. and Kondo, T. Angew Chern. Int. Ed. Engl. 30 (1991) 17-33.
32. Strack, D. and Wray, V. in The Flavonoids: Advances in Research Since 1986 (JB
Harborne, ed.), Chapman & Hall, London (1993) Chapter 1.
33. Piattelli, M. in The Biochemistry of Plants. Vol. 7. Secondary Plant Products (PK
Stumpf and EE Conn, eds), Academic Press, New York (1981) Chapter 18.
34. Liebisch, HW in Biochemistry of Alkaloids (K. Mothes, HR Schutte and M.
Luckner, eds), VEB Deutscher Verlag der Wissenschaften, Berlin (1985) Chapter 14.
35. Stafford, HA Plant Sci. 101 (1994) 91-98.
36. McClure, JW in The Flavonoids (JB Harborne, TJ Mabry and H. Mabry, eds),
Chapman & Hall, London (1975) Chapter 18.
37. Sosnova, V. Bioi. Plant. 12 (1970) 424-427.
38. Kimler, L.M. Abstr. Bot. Soc. Am. 70th Annu. Meet. (1975) 36.
39. Stenlid, G. Phytochem. 15 (1976) 661-663.
40. Hrazdina, G. in The Flavonoids: Advances in Research (JB Harborne and TJ Mabry,
eds), Chapman & Hall, London (1982) Chapter 3.
41a. Takahashi, A., Takeda, K. and Ohnishi, T. Plant Cell Physiol. 32 (1991) 541-547. 41b.
Koes, R.E., Quattracchio, F. and Mol, JNM BioEssays 16 (1994) 123-132.
42. Mabry, TJ in Comparative Phytochemistry (T. Swain, ed.), Academic Press, London (1966)
Chapter 14.
43. Zakharova, NS, Petrova, TA and Bokuchava, MA Soviet Plant Physiol. 36 (1989)
273-277.
44. Kumon, K., Sasaki, J., Sejimi, M., Takeuchi, Y. and Hayashi, Y. Plant Cell Physiol. 31
(1990) 233-240.
45. Harborne. JB in The Comparative Biochemistry of the F1avonoids, Academic Press,
New York (1967) Chapter 1.
46. Grisebach, H. in Anthocyanins as Food Colors (P. Markakis, ed.), Academic Press,
47. Kunz, S., Burkhardt, G. and Becker, H. Phytochemistry. 35 (1994) 233--235.
48. Sweeny, JG and Iacobucci, GA 1. Agric. Food Chem. 31 (1983) 531-533.
49. Arnie, D. and Trinajstic, N. 1. Chem. Soc. Perkin Trans. 2 (1991) 891-895.
50. Timberlake, CF and Bridle, P. 1. Sci. Food Agric. 18 (1967) 473--478.
51. Iacobucci, GA and Sweeny, JG Tetrahedron 39 (1983) 3005-3038.
52. Jurd, L. Adv. Food Res., Supp. 3 (1972) 123-142.
53. Brouillard, R. in Anthocyanins as Food Colors (P. Markakis, ed.), Academic Press,
54. Harborne, JB in Chemical Plant Taxonomy (T. Swain, ed.), Academic Press, New York
(1963) Chapter 13.
55. Starr, MS and Francis, FJ Food Technol. 22 (1968) 1293--1295.
56. Martinelli, E.M., Scilingo, A. and Pifferi, G. Anal. Chim. Acta 259 (1992) 109-113.
57. Harborne, JB Phytochem. 3 (1964) 151-160.
58. Gueffroy, DE, Kepner, RE and Webb, AD Phytochem. 10 (1971) 813-819.
59. Hrazdina, G. and Franzese, AJ Phytochem. 13 (1974) 225-229.
60. Kondo, T., Suzuki, Yoshida, K., Oki, K., Veda, M., Isobe, M. and Goto, T.
Tetrahedron Lett. 32 (1991) 6375-6378.
61. Terahara, N., Kenjiro, T. and Honda, TZ Naturforsch. 48 (1993) 430-435.
62. Yoshitama, K., Kaneshige, M., Ishikura, N., Araki, F., Yahara, S. and Abe, K. 1. Plant Res.
107 (1994) 209--214.
63. Kondo, T., Veda, M. and Goto, T. Tetrahedron 46 (1990) 4749--4756.
64. Terahara, N., Saito, N., Honda, T., Toki, K. and Osajima, Y. Tetrahedron Lett. 30 (1989)
5305-5308.
65. Terahara, N., Saito, N., Honda, T., Toki, K. and Osajima, Y. Tetrahedron Lett. 31 (1990)
2921-2924.
66. Terahara, N., Saito, N., Honda, T., Toki, K. and Osajima, Y. Heterocycles 31 (1990)
1773--1776.
67. Terahara, N., Saito, N., Honda, T., Toki, K. and Osajima, Y. Phytochemistry. 29 (1990)
949-953.
68. Kondo, T., Yamashiki, J., Kawahori, K. and Goto, T. Tetrahedron Lett. 30 (1989)
6055-6058.
69. Shi, Z., Daun, H. and Francis, F.J. 1. Food Sci. 58 (1993) 1068--1069.
70. Brouillard, R. Phytochem. 22 (1983) 1311-1323.
71. Brouillard, R. Phytochemistry. 20 (1981) 143--145.
72. Yoshitama, K. Bot. Mag. Tokyo 91 (1978) 207-212.
73. Dangles, 0., Saito, N. and Brouillard, R. Phytochem. 34 (1993) 119--124.
74. Yoshitama, K. and Hayashi, K. Bot. Mag. Tokyo 87 (1974) 33--40.
75. Lu, T.S., Saito, N., Yokoi, M., Shigihara, A. and Honda, T. Phytochemistry. 30 (1992) 289-
295.
76. Stewart, RN, Norris, KH and Asen, S. Phytochemistry. 14 (1975) 937-942.
77. Asen, S. Acta Hortic. 63 (1976) 217-223.
78. Timberlake, CF and Bridle, P. in The Flavonoids (JB Harborne, TJ Mabry and H.
Mabry, eds), Chapman & Hall, London (1975) Chapter 5.
79. Harborne, JB and Grayer, RJ in The F1avonoids: Advances in Research Since 1980 (JB
80. Fuleki, T. 1. Food Sci. 36 (1971) 101-104.
81. Du, CT, Wang, PL and Francis, FJ 1. Food Sci. 39 (1974) 1265-1266.
82. Terahara, N., Yamaguchi, M. and Honda, T. Biosci. Biotech. Biochern. 58 (1994) 1324-
1325.
83. Tanchev, SS and Timberlake, CF Phytochern. 8 (1969) 1825-1827.
84. Hrazdina, G., Iredale, H. and Mattick, L.R. Phytochern. 16 (1977) 297-299.
85. Idaka, E., Suzuki, K., Yamakita, H., Ogawa, T., Kondo, T. and Goto, T. Chern. Lett.
(1987) 145-148.
86. Jdaka, E., Yamakita, H., Ogawa, T., Kondo, T., Yamamoto, M. and Goto, T. Chern.
Lett. (1987) 1213-1216.
87. Chandler, B.V. Nature 182 (1958) 933.
88. Wrolstad, RE and Heatherbell, DA l. Sci. Food Agric. 35 (1974) 1221-1228.
89. Rasper, V. and Coursey, DC Experientia 23 (1967) 611--612.
90. Imbert, MP and Seaforth, C. Experientia 24 (1968) 445-447.
91. Shoyama, Y., Nishioka, I., Kerath, W., Uemoto, S., Fujieda, K. and Okuba, H.
Phytochern. 29 (1990) 2999-3001.
92. Yoshida, K., Kondo, T., Kameda, K., Kawakishi, S., Lubag, A.J.M., Mendoza, EMT and Goto,
T. Tetrahedron, Lett. 32 (1991) 5575-5578.
93. Ishikura, N. and Sugahara, K. Bot. Mag. Tokyo 92 (1979) 57--61.
94. Puech, AA, Rebeiz, CA, Catlin, PB and Crane, JC l. Food Sci. 40 (1975) 775-
779.
95. Co, H. and Markakis, P. l. Food Sci. 33 (1968) 281-283.
96. Wrolstad, R.E., Hildrum, K.1. and Amos, JF l. Chronatogr. 50 (1970) 311-318.
97. Ishikura, N. Bot. Mag. Tokyo 88 (1975) 41-45.
98. Yoshikura, K. and Hamaguchi, Y. l. lap. Soc. Food Nutr. 22 (1969) 367-370.
99. Odake, T., Terahara, N., Saito, N., Toki, K. and Honda, T. Phytochern. 31 (1992)
2127-2130.
100. Shi, Z., Bassa, LA., Gabriel, SL and Francis, FJ l. Food Sci. 57 (1992) 755-757, 770.
101. Lee, HS and Wicker, L.l. Food Sci. 56 (1991) 466-468, 483.
102. Sun, BH and Francis, FJ l. Food Sci. 32 (1967) 647--649.
103. Timberlake, C.F. and Bridle, P. l. Sci. Food Agric. 22 (1971) 509-513.
104. Mazza, G. and Velioglu, YS Food Chern. 43 (1992) 113-117.
105. Proctor, JT and Creasy, LL Phytochern. 8 (1969) 2108.
106. Pruthi, J.S., Susheela, R. and Lal, G. l. Food Sci. 26 (1961) 385-388.
107. Yoshikura, K. and Hamaguchi, Y. l. lap. Soc. Food Nutr. 24 (1971) 275-278.
108. Lynn, DY and Luh, BS l. Food Sci. 29 (1964) 735-743.
109. Li, K. and Wagenknecht, AC Nature 182 (1958) 657.
110. Harborne, JB and Hall, E. Phytochern. 3 (1964) 453-463.
111. Dekazos, ED l. Food Sci. 35 (1970) 237-241.
112. Shrikhande, AJ and Francis, FJ 1. Food Sci. 38 (1973) 649--651.
113. Chandra, H., Nair, M.G. and Iezzoni, A. l. Agric. Food Chern. 40 (1992) 967-969.
114. Tanchev, SS and Vasilev, VN Gradinar. Lozar. Nauka 10 (1973) 23-28.
115. Du, cT, Wang, PL and Francis, FJ l. Food Sci. 40 (1975) 417-418.
116. Ishikura, N. and Hayashi, K. Bot. Mag. Tokyo 78 (1965) 91-96.
117. Fuleki, T.l. Food Sci. 34 (1969) 365-369.
118. Gallop, RA Variety, Composition and Color in Canned Fruits, Particularly Rhubarb.
Fruit & Veg. Canning Res. Assoc., Chipping Campden, Glos. Sci. Bull. No.5 (1965).
119. Wrolstad, RE and Heatherbell, DA 1. Food Sci. 33 (1968) 592-594.
120. Chandler, BV and Harper, KA Austral. l. Chern. IS (1962) 114-120.
121. Casoli, U., Cultrera, R. and Gherardi, S. Ind. Conserve 42 (1967) 255.
122. Nybom, N. Frukt. Baer. (1970) 106-118.
123. Oeydin, J. Hortic. Res. 14 (1974) 1-7.
124. Barritt, BH and Torre, Lc l. Chronatogr. 75 (1973) 151-155.
125. Jennings, D.L. and Carmichael, E. New Phytol. 84 (1980) 505-513.
126. Nybom, N. l. Chronatogr. 38 (1968) 382-387.
127. Daravingas, G. and Cain, RF l. Food Sci. 31 (1966) 927-936.
128. Daravingas, G. and Cain, RF l. Food Sci. 33 (1968) 138-142.
129. Watanabe, S., Sakamura, S. and Obata, Y. Agric. Bioi. Chern. 30 (1966) 420-422.
130. Tanchev, SS, Ruskov, PJ and Timberlake, CF Phytochern. 9 (1970) 1681-1682.
131. Saito, N., Hotta, R., Imai, K. and Hayashi, K. Proc. lap. Academic 41 (1965) 593-598.
132. Francis, F.J. and Harborne, J.B. 1. Food Sci. 31 (1966) 524--528.
133. Buckmire, R.E. and Francis, F.J. 1. Food Sci. 41 (1976) 1363-1365.
134. Francis, FJ, Harborne, JB and Barker, WG 1. Food Sci. 31 (1966) 583-587.
135. Ballinger, WE, Maness, EP and Kushman, LJ 1. Am. Soc. Hort. Sci. 95 (1970) 283-
285.
136. Ballinger, WE, Maness, EP, Galletta, GJ and Kushman, LJ 1. Am. Soc. Hort.
Sci. 97 (1972) 381-384.
137. Zapsalis, C. and Francis, FJ 1. Food Sci. 30 (1965) 396-399.
138. Fuleki, T. and Francis, F.J. Phytochemistry. 6 (1967) 1705-1708.
139. Fuleki, T. and Francis, FJ 1. Food Sci. 33 (1968) 471-478.
140. Hicks, KB, Sondey, SM, Hargrave, D., Sapers, GM and Bilyk, A. LC Mag. 3
(1985) 981-982, 984.
141. Andersen, 0.M. 1. Food Sci. 54 (1989) 383-384, 387.
142. Ribereau-Gayon, P. in Anthocyanins as Food Colors (P. Markakis, ed.), Academic Press,
143. Harborne, JB and Gavazzi, G. Phytochem. 8 (1969) 999-1001.
144. Lawanson, AO and Osude, BA Z. PJianzenphysiol. 67 (1972) 460-463.
145. Nakatani, N., Fukuda, H. and Fuwa, H. Agric. BioI. Chem. 43 (1979) 389-391.
146. Lancaster, JE, Grant, JE, Lister, CE and Taylor, MC 1. Am. Soc. Hort. Sci. 119
(1994) 63-69.
147. Grisebach, H. in The Biochemistry of Plant Phenolics (CF van Sumere and PJ Lea,
eds), Clarendon Press, Oxford (1985) Chapter 10.
148. Heller, W. and Forkmann, G. in The Flavonoids: Advances in Research Since 1980 (JB
149. Mol, J. Stuitje, A., Gerats, A., van der Krol, A. and Jorgensen, R. TIBTECH7 (1989)
148-153.
150. Forkmann, G. Plant Breeding 106 (1991) 1-26.
151. Holton, TA and Tanaka, Y. TlBTECH 12 (1994) 4042.
152. Stafford, HA Flavonoid Metabolism, CRC Press, Boca Raton (1990).
153. Dooner, HK, Robbins, TP and Jorgensen, RA Annu. Rev. Genet. 25 (1991) 173-
199.
154. van der Meer, IM, Stuitje, AR and Mol, JNM in Control of Plant Gene Expression (DPS
Verma, ed.), CRC Press, Boca Raton (1993).
155. Quattracchio, F., Wing, JF, Leppen, HTC, Mol, JNM and Koes, RE Plant Cell
5 (1993) 1497-1512.
156. Ludwig, SR, Habera, LF, Dellaporta, SL and Wessler, SR Proc. Natl. Academic Sci.
USA 86 (1989) 7092-7096.
157. Radicella, JP, Turks, D. and Chandler, VL Plant Mol. BioI. 17 (1991) 127-130.
158. Jain, VK and Guruprusad, KN Physiol. Plant. 75 (1989) 233-236.
159. Weiss, D., van Blokland, R., Kooter, JM, Mol, JNM and van Tunen, AJ Plant
Physiol. 98 (1992) 191-197.
160. Hattori, T., Vasil, V., Rosenkrans, L., Hannah, L.C., McCarty, D.R. and Vasil, I.K.
Genes Dev. 6 (1992) 609-618.
161. Ilan, A., Zanewich, K.P., Rood, S.B. and Dooner, D.K. Physiol. Plant. 92 (1994) 47-
52.
162. Mancinelli, A.L., Rossi, F. and Moroni, A. Plant Physiol. 96 (1991) 1079-1085.
163. Jain, VK and Guruprusad, KN 1. Exp. Bot. 41 (1990) 53-58.
164. Khare, M. and Guruprusad, KN Plant Sci. 91 (1993) 1-5.
165. Galbiati, M., Chiusi, A., Peteriongo, P., Mancinelli, A. and Gavazzi, G. Maydica 39
(1994) 89-95.
166. Shichijo, C., Hamada, T., Hiraoka, M., Johnson, CB and Hashimoto, T. Planta 191
(1993) 283-245.
167. Egin-Buyler, B., Loyal, R. and Ebel, J. Arch. Biochem. Biophys. 203 (1980) 90-100.
168. Forkmann, G. and Stotz, G.Z. Naturforsch. 36c (1981) 411-416.
169. Stotz, G. and Forkmann, G.Z. Naturforsch. 37c (1982) 19-23.
170. Menting, JGT, Scopes, RK and Stevensen, TW Plant Physiol. 106 (1994) 633-642.
171. Heller, W., Britsch, L., Forkmann, G. and Grisebach, H. Planta 163 (1985) 191-196.
172. Stich, K. and Forkmann, G. Phytochem. 27 (1988) 785-789.
173. Stich, K. and Forkmann, GZ Naturforsch. 43c (1988) 311-314.

174. Timberlake, C. F. and Bridle, P. Nature 212 (1966) 158-159.
175. Teusch, M., Forkmann, G. and Syffert, W. Phytochemistry. 26 (1987) 991-994.
176. Ino,1. and Yamaguchi, M.-A. Phytochem. 33 (1993) 1415-1417.
177. Ino, 1., Nishiyama, H. and Yamaguchi, M.-A. Phytochem. 33 (1993) 1425-1426.
178. Glassgen, WE and Seitz, HU Planta 186 (1992) 582-585.
179. Hrazdina, G., Zobel, AM and Hoch, Hc Proc. Natl. Academic Sci. USA 84 (1988) 8966-
8970.
180. Hrazdina, G. and Wagner, GJ in The Biochemistry of Plant Phenolics (CF van Sumere and
PJ Lea, eds), Clarendon Press, Oxford (1985) Chapter 7.
181. Hrazdina, G. and Wagner, GJ Arch. Biochem. Biophys. 237 (1985) 88-100.
182. Jonsson, L.M., Donker-Koopman, W.E., Uitslager, P. and Schram, A.W. Plant
Physiol. 72 (1983) 287-290.
183. Joanny, K. and Reddy, AR Maydica 37 (1992) 165-167.
184. Hopp, W. and Seitz, HV Planta 170 (1987) 74-85.
185. Seitz, HU and Hinderer, W. in Cell Culture and Somatic Cell Genetics of Plants, Vol.5 (F.
Constabel and 1.K. Vasil, eds), Academic Press, New York (1988) Chapter 3.
186. Nozzolillo, C. and Ishikura, N. Plant Cell Rep. 7 (1988) 389-392.
187. Nozue, M., Kubo, H., Nishimura, M., Katou, A., Hattori, C., Usuda, N., Nagata, T. and
Yasuda, H. Plant Cell Physiol. 34 (1993) 803-808.
188. Cormier, F., Crevier, H. and Do, CB Can. 1. Bot. 68 (1990) 1822-1825.
189. Do, CB and Cormier, F. Plant Cell Rep. 9 (1991) 500-504.
190. Hirasuna, TJ, Shuler, ML, Lackney, VK and Spanswick, RM Plant Sci. 78 (1991)
107-120.
191. Mazza, G. and Brouillard, R.I. Agric. Food Chem. 35 (1987) 422-426.
192. Brouillard, R. and Lang, J. Can. 1. Chem. 68 (1990) 755-761.
193. Baranac, J., Amic, D. and Vukadinovic, VI Agric. Food Chem. 38 (1990) 932-936.
194. McClelland, RA and McGall, GH 1. Org. Chem. 47 (1982) 3730-3736.
195. Friends D., Baranac, J. and Vukadinovic, VI Agric. Food. Chem. 38 (1990) 936-940.
196. Brouillard, R. and Dubois, JE 1. Am. Chem. Soc. 99 (1977) 1359-1364.
197. Dangles, O. and Elhajji, N. Helv. Chim. Acta 77 (1994) 1595-1610.
198. Francis, FJ Trends Food Sci. Technol. 3 (1992) 27-30.
199. Hrazdina, G., Borzell, AJ and Robinson, WB Am. 1. Enol. Vitic. 21 (1970) 201-204.
200. Robinson, WD, Weiers, LD, Bertino, JJ and Mattick, LR Am. 1. Enol. Vitic. 17
(1966) 178-183.
201. Van Buren, JP, Bertino, JJ and Robinson, WB Am. f. Enol. Vitic. 19 (1968) 147-
154.
202. Adams, JB Changes in the Polyphenols of Red Fruits During Processing-The Kinetics and
Mechanism of Anthocyanin Degradation. Campden Food Pres. Res. Assoc., Chipping
Campden, Glos. Tech. Bull. P. 2. (1972).
203. Adams, JB f. Sci. Food Agric. 24 (1973) 747-762.
204. Lukton, A., Chichester, CO and MacKinney, G. Food Techno!. 10 (1956) 427-432.
205. Meschter, EE f. Agric. Food Chem. 1 (1953) 574-579.
206. Tinsley, 1.1. and Bockian, A. H. Food Res. 25 (1960) 161-173.
207. Calvi, JP and Francis, FJ 1. Food Sci. 43 (1978) 1448-1456.
208. Brouillard, R. and Delaporte, B. 1. Am. Chem. Soc. 99 (1977) 8461-8468.
209. Hrazdina, G. Phytochem. 10 (1971) 1125-1130.
210. Markakis, P., Livingston, GE and Fellers, CR Food Res. 22 (1957) 117-129.
211. Adams, JB Food Manuf. (1973) (Feb.) 19-20,41.
212. Furtado, P., Figueiredo, P., Chaves des Neves, C. and Pina, F. f. Photochem.
Photobiol. A: Chem. 75 (1993) 113-118.
213. Piffaut, B., Kader, F., Girardin, M. and Metche, M. Food Chem. 50 (1994) 115-120.
214. Macarone, E., Ferrigno, V., Longo, LL and Rapisarda, P. Annal. Chim. 77 (1987)
499-508.
215. Tanchev, SS and Ioncheva, N. Nahrung 20 (1976) 889-893.
216. Markakis, P. in Anthocyanins as Food Colors (P. Markakis, ed.), Academic Press, New York
(1982) Chapter 6.
217. Erlandson, JA and Wrolstad, RE f. Food Sci. 37 (1~·72) 592-595.
218. Abers, JE and Wrolstad, RE J. Food Sci. 44 (1979) 75-78, 81.

219. Tanchev, S. Proc. 6th Int. Congr. Food Sci. Techno!. (1983) 96.
220. Mishkin, M. and Saguy, I. Z. Lebensrn. or. Forsch. 175 (1982) 410-412.
221. Havlikova, L. and Mikova, K. Z. Lebensrn. or. Forsch. 181 (1985) 427-432.
222. Cemeroglu, B., Velioglu, S. and Isik, S. J. Food Sci. 59 (1994) 1216-1218.
223. Somers, TC Phytochern. 10 (1971) 2175-2186.
224. Adams, JB and Ongley, MJ J. Food Technol. 8 (1973) 139-145.
225. McCloskey, LP and Yengoyan, LS Am. J. Eno!. Vitic. 32 (1981) 257-261.
226. Bakker, J. and Timberlake, CF J. Sci. Food Agric. 37 (1986) 288-292.
227. Rommel, A., Wrolstad, RE and Heatherbell, DA J. Food Sci. 57 (1992) 385-391,
410.
228. Peng, cY and Markakis, P. Nature 199 (1963) 597-598.
229. Keith, E. S. and Powers, J. J. Agric. Food Chern. 13 (1965) 577-579.
230. Sondheimer, E. and Kertesz, AI Food Res. 18 (1953) 475-479.
231. Sondheimer, E. and Kertesz, AI Food Res. 18 (1952) 288-297.
232. Hrazdina, G. Phytochern. 9 (1970) 1647-1652.
233. Yoshida, K., Kondo, T., Kameda, K. and Goto, T. Agric. Bioi. Chern. 54 (1990) 1745-
1751.
234. Palamidis, N. and Markakis, P. J. Food Sci. 40 (1975) 1047-1049.
235. Markakis, P., Livingston, GE and Fagerson, JS Food Res. 24 (1959) 520-528.
236. Pellegrino, F., Sekular, P. and Alfano, R.R. Photobiochern. Photobiophys. 2 (1981)
15-20.
237. Santhanam, M., Hautala, RR, Sweeny, JG and Iacobucci, GA Photochern.
Photobiol. 38 (1984) 477-480.
238. Sweeny, JG, Wilkinson, MM and Iacobucci, GA J. Agric. Food Chern. 29 (1981)
563-567.
239. Huang, H. T. J. Agric. Food Chern. 3 (1955) 141-146.
240. Forsyth, WGc and Quesnel, VC Biochern. J. 65 (1957) 177-179.
241. Blom, H. Food Chern. 12 (1983) 197-204.
242. van Buren, JP, Scheiner, DM and Wagenknecht, AC Nature 185 (1960) 165-166.
243. Wagenknecht, AC, Scheiner, DM and van Buren, JP Food Technol. 14 (1960) 47-49.
244. Skalski, C. and Sistrunk, W. A. J. Food Sci. 38 (1973) 1060-1062.
245. Pifferi, PG and Cultrera, R. J. Food Sci. 39 (1974) 786-791.
246. Wesche-Ebeling, P. and Montgomery, MW J. Food Sci. 55 (1990) 731-734, 745.
247. Oszmianski, J. and Lee, C.Y. J. Agric. Food Chern. 39 (1991) 1050-1052.
248. Siddiq, M., Arnold, JF, Sinha, NK and Cash, JN J. Food Process. Pres. 18 (1994)
75-84.
249. Grommeck, R. and Markakis, P. J. Food Sci. 29 (1964) 53-57.
250. Matthew, AG and Parpia, HAB Adv. Food Res. 19 (1971) 75-145.
251. Sakabura, H. and Ichinose, H. Agric. Chern. Soc. Jap. 56 (1982) 517-524.
252. Jiang, J., Paterson, A. and Piggott, JR Inti. J. Food Sci. Technol. 25 (1990) 596-600.
253. Tanchev, S., Vladimirov, G. and Ioncheva, N. Nauchni Trudove, Vissh Inst. Khranit.
Vkusova Prornyshl. 16 (1969) 77-82.
254. Yang, HY and Steele, WF Food Technol. 12 (1958) 517-519.
255. Siegel, A., Markakis, P. and Bedford, cL J. Food Sci. 36 (1971) 962-963.
256. Wrolstad, R.E., Skrede, G., Lea, P. and Enersen, G. J. Food Sci. 55 (1990) 1064-1065,
1072.
257. Blom, H. and Thomassen, MS Food Chern. 17 (1985) 157-168.
258. Goodman, L.P. and Markakis, P. J. Food Sci. 30 (1965) 135-137.
259. Cash, JN, Sistrunk, WA and Stotte, CA J. Food Sci. 41 (1976) 1398-1402.
260. Ingold, CK Structure and Mechanism in Organic Chemistry. Cornell University Press,
Ithica (1969).
261. Bendz, G., Martensson, O. and Nilsson, E. Ark. Kerni 27 (1967) 65-77.
262. Shriner, R.L. and Sutton, R. J. Am. Chern. Soc. 85 (1963) 3989-3991.
263. Jurd, L. Tetrahedron 21 (1965) 3707-3714.
264. Jurd, L. Tetrahedron 23 (1967) 1057-1064.
265. Jurd, L. J. Heterocycl. Chern. 18 (19981) 429-430.
266. Jurd, L. and Waiss, A.C. Jr. Tetrahedron 21 (1965) 1471-1483.
267. Jurd, L. J. Food Sci. 29 (1964) 16-19.

268. Timberlake, CF and Bridle, P. J. Sci. Food Agric. 18 (1967) 479-485.
269. Brouillard, R. and El Hage Chahine, JM Bull. Liaison-Groupe Polyphenols 9 (1980)
77-78.
270. Brouillard, R. and El Hage Chahine, JM Am. Chem. Soc. 102 (1980) 5375-5378.
271. Timberlake, CF and Bridle, P. Chem. Ind. Oct. (1968) 1489.
272. Bridle, P., Scott, KG and Timberlake, C.F. Phytochemistry. 12 (1973) 1103-1106.
273. Timberlake, C. F. and Bridle, P. J. Sci. Food Agric. 28 (1977) 539-544.
274. Bakker, J., Picinelli, A. and Bridle, P. Vitis 32 (1993) 111-118.
275. Garcia-Viguera, C., Bridle, P. and Bakker, J. Vitis 33 (1994) 37-40.
276. Esselen, WB and Sammy, GM Food Prod. Dev. 9(8) (1975) 37-38, 40.
277. Main, JH, Clydesdale, FM and Francis, FJ J. Food Sci. 43 (1978) 1693-1694, 1697.
278. Bronnum-Hansen, K. and Flink, J.M. J. Food Technol. 20 (1985) 725-733.
279. MacKinney, G., Lukton, A. and Chichester, CO Food Technol. 9 (1955) 324-326.
280. Debicki-Pospisil, J., Lovric, T., Trinajstic, N. and Sabljic, A. J. Food Sci. 48 (1983)
411-416.
281. Hodge, JE J. Agric. Food Chem. 1 (1953) 928-943.
282. Kurata, T. and Sakurai, Y. Agric. Bioi. Chem. 31 (1967) 170-176.
283. Sloan, JL, Bills, DD and Libbey, LM J. Agric. Food Chem. 17 (1969) 1370-1372.
284. Harborne, JB Recent Adv. Phytochem. 12 (1979) 457-474.
285. Asen, S., Stewart, R.N., Norris, K.H. and Massie, D.R. Phytochemistry. 9 (1970) 619-627.
286. Bayer, F., Fink, A., Nether, K. and Wegmann, K. Angew Chem. Inter. Ed. Engl. 5
(1966) 791-798.
287. Lowry, J.B. and Chew, L. Econ. Bot. 28 (1974) 61-62.
288. Osawa, Y. in Anthocyanins as Food Colors (P. Markakis, ed.), Academic Press, New York
(1982) Chapter 2.
289. Asen, S., Stewart, R.N. and Norris, K.H. Phytochemistry. 11 (1972) 1139-1144.
290. Hoshino, T., Matsumoto, U. and Goto, T. Phytochemistry. 19 (1980) 663-667.
291. Mistry, TV, Cai, Y., Lilley, TH and Haslam, E. J. Chem. Soc., Perkin Trans. 2 (1991) 1287-1296.
292. Brouillard, R., Mazza, G., Saad, Z., Albrecht-Gray, A.M. and Cheminat, A. J. Am.
Chem. Soc. 111 (1989) 2604-2610.
293. Mazza, G. and Brouillard, R. Phytochem. 29 (1990) 1097-1102.
294. Davies, A.J. and Mazza, G.J. Agric . Food Chem. 41 (1993) 716-720.
295. Goto, T., Hoshino, T. and Takase, S. Tetrahedron Lett. 20 (1979) 2905-2908.
296. Wigand, M.C., Dangles, O. and Brouillard, R. Phytochem. 31 (1992) 4317-4324.
297. Dangles, 0., Wigand, M.C. and Brouillard, R. Phytochem. 31 (1992) 3811-3812.
298. Dangles, O. and Brouillard, R. J. Chem. Soc., Perkin Trans. 2 (1992) 247-257.
301. Schewffeldt, P. and Hrazdina, G. J. Food Sci. 43 (1978) 517-520.
302. Nakayama, TOM and Powers, JJ Adv. Food Res., Supp. 3 (1972) 193-199.
303. Hoshino, T., Matsumoto, U. and Goto, T. Phytochemistry. 20 (1981) 1971-1976.
304. Hoshino, T., Matsumoto, U., Harada, N. and Goto, T. Tetrahedron Lett. 22 (1981)
3621-3624.
305. Figueiredo, P. and Pina, F. J. Chem. Soc., Perkin Trans. 2 (1994) 775-778.
306. Hoshino, T. Phytochemistry. 30 (1991) 2049-2055.
308. Singleton, VL and Trousdale, EK Am. J. Enol. Vitic. 43 (1992) 63-70.
309. Jurd, L. and Asen, S. Phytochemistry. 5 (1966) 1263-1271.
310. Francis, F.J. in Anthocyanins as Food Colors (P. Markakis, ed.), Academic Press, New York
(1982) Chapter 7.
311. Harborne, JB Phytochemical Methods. A Guide to Modern Techniques of Plant Analysis, 2nd
edn. Chapman & Hall, London (1984).
312. Strack, D. and Wray, V. in Methods in Plant Biochemistry, VoU (JB Harborne, ed.), Academic
Press, London (1989).
313. Anderson, DW, Julian, EA, Kepner, RE and Webb, AD Phytochem. 9 (1970)
1569-1578.
314. Moore, AB, Francis, FJ and Clydesdale, FM 1. Food Protect. 45 (1982) 738-743.
315. Moore, AB, Francis, FJ and Jason, ME J. Food Protect. 45 (1982) 590-593.
316. Esselen, WB and Sammy, GM Food Prod. Dev. 7 (1973) 80,82,86.
317. Philip, T. J. Food Sci. 39 (1974) 859.
318. Langston, MSK US Patent 4 500 556 (1985).
319. Pouget, M.P., Lejeune, B., Vennat, B. and Pourrat, A. Lebensm.-Wiss. or. Technol.23
(1990) 103-105.
320. Dallas, C. and Laureano, O. Vitis 33 (1994) 41-47.
321. Weisenborn, D., Glowacki, A., Hettiarchchy, N. and Zander, L. Trans. ASAE 36 (1993)
1411-1416.
322. Shewfelt, R.L. and Ahmed. EM Food Prod. Dev. 11(4) (1977) 52, 57-59, 62.
323. Shewfelt, RL and Ahmed, EM J. Food Sci. 43 (1978) 435-438.
324. Hrazdina, G. J. Agric. Food Chem. 18 (1970) 243-245.
325. Wrolstad, R.E. and Struthers, B.J. J. Chromatogr. 55 (1971) 405-408. 326.
van Teeling, eG, Cansfield, PE and Gallop, RA J. Chromatogr. Sci. 9 (1971) 505-
509.
327. Strack, D. and Mansell, R.L. J. Chromatogr. 109 (1975) 325-331.
328. Hrazdina, G. Lebensm.-Wiss. or. Technol. 8 (1975) 111-113.
329. Manley, C. H. and Shubiak, P. Can. Inst. Food Sci. Technol. 8 (1975) 35-39.
330. Davies, A.J. and Mazza, G.J. Agric . Food Chem. 40 (1992) 1341-1345.
331. Kim, J.H., Nonaka, G.I., Fujieda, K. and Uemoto, S. Phytochemistry. 28 (1989) 1503.
332. Oszmianski, J. and Sapis, Je J. Food Sci. 53 (1988) 1241-1242.
333. Fuleki, T. and Francis, FJ J. Food Sci. 33 (1968) 266-274.
334. Chiriboga, C. and Francis, FJ J. Amer. Soc. Hort. Sci. 95 (1970) 233-236.
335. Goldstein, G. J. Chromatogr. 129 (1976) 466-468.
336. Spagna, G. and Pifferi, PG Food Chem. 44 (1992) 185-188.
337. Francis, GW and Andersen, 0.M. J. Chromatogr. 283 (1984) 445-448.
338. Andersen, 0.M. J. Food Sci. 50 (1985) 1230-1232.
339. Andersen, 0.M. J. Food Sci. 52 (1987) 665--666, 680.
340. Asen, S. J. Chromatogr. 18 (1965) 602--603.
341. Quarmby, C. J. Chromatogr. 34 (1968) 52-58.
342. Barritt, BH and Torre, Le J. Chromatogr. 75 (1973) 151-155.
343. Andersen, 0.M. and Francis, GW J. Chromatogr. 318 (1985) 450-455.
344. Lasagabaster, A., Martin, e. and Goni, MM J. Chem. Tech. Biotech. 60 (1994) 397-
403.
345. Francis, FJ HortSci. 2 (1967) 170-171.
346. Wulf, LW and Nagel, eW Am. J. Enol. Vitic. 29 (1978) 42-49.
347. Bakker, J. and Timberlake, eF J. Sci. Food Agric. 36 (1985) 1315-1324.
348. Sapers, GM, Hicks, KB, Burgher, AM, Hargrave, DL, Son dey, SM and Bilyk,
A. J. Am. Soc. Hort. Sci. 111 (1986) 945-950.
349. Mazza, G. J. Food Sci. 51 (1986) 1260-1264.
350. Baj, A., Bombardelli, E., Gabetta, B. and Martinelli, EM J. Chromatogr. 279 (1983)
265-372.
351. Hong, V. and Wrolstad, RE J. Agric. Food Chem. 38 (1990) 698-708.
352. Hong, V. and Wrolstad, RE J. Agric. Food Chem. 38 (1990) 708-715.
353. Drdak, M., DauCik, P., Simko, P., Karovicova, J. and Rajniakova, A. Nahrung 36
(1992) 411-413.
354. Drdak, M., DauCik, P., Karovicova, J., Simko, P. Rajniakova, A. and Morova, E.
Nahrung 36 (1992) 411-413.
355. Lamuela-Raventos, RM and Waterhouse, AL Am. J. Enol. Vitic. 45 (1994) 1-5.
356. Lee, HS and Hong, VJ J. Chromatogr. 624 (1992) 221-234.
357. Ribereau-Gayon, PR and Josien, ML Bull. Soc. Chim. Fr. (1960) 934-937.
358. Statoua, A., Merlin, Je, Del Haye, M. and Brouillard, R. Raman Spectrosc., Proc.
Int. Conf 8th (1982) 629--630.
359. Merlin, J.-e., Statoua, A., Cornard, J.-P., Saidi-Idrissi, M. and Brouillard, R.
Phytochem. 35 (1993) 227-232.
360. Harborne, JB Biochem. J. 70 (1958) 22-28.
362. Hoshino, T., Matsumoto, U., Goto, T. and Harada, N. Tetrahedron Lett. 23 (1982)
433-436.
363. Goto, T., Takase, S. and Kondo, T. Tetrahederon Lett. 19 (1978) 2413-2416.
364. Goto, T., Kondo, T., Imagawa, H. and Miura, I. Tetrahedron Lett. 22 (1981) 3213-
3216.
365. Goto, T., Kondo, T., Tamura, H., Imagawa, H., Iiono, A. and Takeda, T. Tetrahedron
Lett. 23 (1982) 3695-3698.
366. Timberlake, CF, Bakker, J., Bridle, P., Loeffler, RST, Saito, N. and Self, R. Bull.
Liaison-groupe Polyphenols 13 (1986) 96-101.
367. Andersen, 0.M., Aksnes, D.W., Nerdal, W. and Johansen, O. Phytochemistry. Anal. 2
(1991) 175-183.
368. Nerdal, W., Pedersen, A.T. and Andersen, 0.M. Acta Chem. Scand. 46 (1992) 872-
876.
369. Tamura, H., Hayashi, Y., Sugisawa, H. and Kondo, T. Phytochemistry. Anal. 5 (1994) 190-
196.
370. Love, GD, Snape, eE, Jarvis, MC and Morrison, IM Phytochem. 35 (1994) 489-
491.
371. Comus, G., Wyler, H. and Lauterwein, J. Phytochem. 20 (1981) 1461-1462.
372. Takura, H., Kumaoka, Y. and Sugisawa, H. Agric. Bioi. Chem. 53 (1989) 1969-1970.
373. Saito, N., Timberlake, C.F., Tucknott, O.G. and Lewis, IAS Phytochem. 22 (1983)
1007-1009.
374. Glassgen, WE, Seitz, HU and Metzger, JW Bioi. Mass Spectrom. 21 (1992) 271-
277.
375. Glassgen, WE, Wray, Y., Strack, D., Metzger, JW and Seitz, HU Phytochemistry. 31 (1992)
1593-1601.
376. Pedersen, A.T., Andersen, 0.M., Aksnes, D.W. and Nerdal, W. Magn. Reson. Chem. 31
(1993) 972-976.
377. Wolfender, J.-L. Maillard, M. and Hostettmann, K. Phytochem. Anal. 5 (1994) 153-
182.
378. Gao, L. and Mazza, G. J. Agric. Food Chem. 42 (1994) 118-125.
379. Baublis, AJ and Berber-Jimenez, MD J. Agric. Food Chem. 43 (1995) 640-646.
380. Geissman, TA, Jorgenson, EC and Harborne, JB Chem. Ind. 1953) (Dec.) 1389.
381. Lin, M., Shi, Z. and Francis, FJ J. Food Sci. 57 (1992) 766-767.
382. Harborne, JB J. Chromatogr. 1 (1958) 473-488.
383. Werner, DJ, Maness, EP and Ballinger, WE HortSci. 24 (1989) 488-489.
384. Hebrero, E., Santos-Buelga, e. and Rivas-Gonzalo, JC Am. J. Enol. Vitic. 39 (1988)
227-233.
385. Hebrero, E., Garcia-Rodriguez, C., Santos-Buelga, e. and Rivas-Gonzalo, Je Am. J.
Enol. Vitic. 40 (1989) 283-291.
386. Lees, DH and Francis, FJ J. Food Sci. 36 (1971) 1056-1060.
387. BIom. H. Rev. Food Sci. Technol. 7 (1983) 587-589.
388. Chandler, BY and Harper, KA Aust. J. Chem. 14 (1961) 586-595.
389. Wrolstad, RE Color and Pigment Analyzes in Fruit Products. Bull. 624. Oregon Agric.
Exp. St., Corvallis (1976).
390. Staples, L.C. and Francis. FJ Food Technol. 22 (1968) 611--614.
391. Jurd, L.J. Org . Chem. 28 (1963) 987-991.
392. Ponting, JD, Sanshuck, WD and Brekke, JE Food Res. 25 (1960) 471-478.
393. Niketic-Aleksic, GK and Hrazdina, G. Lebensm.-Wiss. or. Techno! 5 (1972) 163-165.
394. Dickinson, D. and Gawler, JH J. Sci. Food Agric. 7 (1956) 699-705.
395. Somers, Te and Evans, ME J. Sci. Food Agric. 25 (1974) 1369-1379.
396. Swain, T. and Hillis, WE J. Sci. Food Agric. 10 (1959) 63--68.
397. Fuleki, T. and Francis, FJ J. Food Sci. 33 (1968) 78-83.
398. Tanchev, S. Z. Lebensm. U. Forsch. 150 (1972) 28-30.
399. Tanchev. S. Nahrung 18 (1974) 303-308.
400. Lees, DH and Francis, FJ HortSci. 7 (1972) 83-84.
401. Godek, S. Przemysl Fermentacyjny i Owocowo-Warzywyny 25(5/6) (1981) 27-30.
402. Speers, RA, Tung, MA and Jackman, RL Can. Inst. Food Sci. Technol. J. 20
(1987) 15-18.
403. Spayd, SE and Morris, JR 1. Food Sci. 46 (1981) 414--418.

404. Staples, Lc and Francis, FJ Food Technol. 22 (1968) 611-614. 405. van
Buren, JP, Hrazdina, G. and Robinson, WB J. Food Sci. 39 (1974) 325-328.
406. Torre, Lc and Barritt, BH J. Food Sci. 42 (1977) 488-490.
407. Jurd, L. Food Technol. 18 (1964) 559-561.
408. Hrazdina, G. Z. Lebensm.-Wiss. or. Technol. 14 (1981) 283-286.
409. Chiriboga, C. and Francis, FJ J. Food Sci. 38 (1973) 464--467.
410. Clydesdale, FM, Main, JH and Francis, FJ J. Food Protect. 42 (1979) 196-201.
411. Clydesdale, FM, Main, JH and Francis, FJ J. Food Protect. 42 (1979) 204-207.
412. Buckmire, R.E. and Francis, F.J. J. Food Sci. 43 (1978) 908-911.
413. Francis, F.J. Food Technol. 29 (1975) 52, 54.
414. Anon. Food Chem. 5 (1975) 69-80.
415. Martinez, SB and Del Valle, MJ UP Home Econ. J. 9 (1981) 7-10.
416. Coudonis, M., Katsaboxakis, K. and Papanicolaou, D. Rev. Food Sci. Technol. 7 (1983)
567-572.
417. Asen, S., Stewart, R.N. and Norris, K.H. Phytochemistry. 16 (1977) 1118-1119.
418. Asen, S., Stewart, RN and Norris, KH US Patent No.4 172 902 (1979).
419. Baker, cH, Johnston, MR and Barber, WD Food Prod. Dev. 8 (1974) (3) 83-84,
86-87.
420. Baker, CH, Johnston, MR and Barber, WD Food Prod. Dev. 8 (1974) (4) 65-70.
421. Kondo, T., Yoshida, K., Nakagawa, A., Kawai, T., Tamura, H. and Goto, T. Nature
358 (1992) 515-518.
422. Kondo, T., Veda, M., Tamura, H., Yoshida, K., Isobe, M. and Goto, T. Angew.
Chem. Int. Ed. Engl. 33 (1994) 978-979.
423. Anon, Enocolor. The Importance of Natural Colour. Reggiana Antociani, Industria Coloranti Naturali,
Italy (1981).
424. Taylor, AJ in Developments in Food Colors-2 (J. Walford, ed.), Applied Science Pub., London
(1984) Chapter 5.
425. Clydesdale, FM, Main, JH, Francis, FJ and Damon, RA Jr. J. Food Sci. 43 (1978) 1687-1692, 1697.
426. Lancrenon, X. Process Biochem. 16 (1978) (10) 16.

427. Matsumoto, T., Nishida, K., Noguchi, M. and Tamaki, E. Agric. Bioi. Chem. 37 (1973)
561-567.
428. Tholakalabavi, A., Zwiazek, JJ and Thorpe, TA In Vitro Cell. Dev. Bioi. 30P (1994)
164-170.
429. Dougal, DK and Weyrauch, KW Biotech. Bioeng. 22 (1980) 337-352.
430. Nagarajan, R.P., Keshavarz, E. and Gerson, D.F. J. Ferment. Bioeng. 68 (1989) 102-
106.
431. Yamakawa, T., Kato, S., Ishida, K., Kodama, T. and Minoda, Y. Agric. Bioi. Chem. 47 (1983)
2185-2192.
432. Yamamoto, Y., Knoshita, Y., Watanabe, S. and Yamada, Y. Agric. Bioi. Chem. 53
(1989) 417-423.
433. Kobayashi, Y., Akita, M., Sakamoto, K., Liu, H., Shigeoka, T., Koyano, T.,
Kawamura, M. and Furuya, T. Appl. Microbiol. Biotech. 40 (1993) 215-218.
434. Sakamoto, K., Iida, K., Sawamura, K., Hajiro, K., Asada, Y., Yoshikawa, T. and
Furuya, T. Phytochem. 33 (1993) 357-360.
435. Sakamoto, K., Iida, K., Sawamura, K., Hajiro, K., Asada, Y., Yoshikawa, T. and Furuya, T. Plant
Cell Tiss. Org. Cult. 36 (1994) 21-26.
436. Counsell, JN, Jeffries, GS and Knewstubb, cJ in Natural Colors for Food and Other Uses (JN
Counsell, ed.), Applied Science Pub., New York (1981) Chapter 7.
437. Maccarone, E., Maccarone, A. and Rapisarda, P. J. Food Sci. 50 (1985) 901-904.
438. Piattelli, M. and Impellizzeri, G. Phytochemistry. 9 (1970) 2553-2556.
439. Alard, D., Wray, V., Grotjahn, L., Reznik, H. and Strack, D. Phytochemistry. 24 (1985)
2383-2385.
440. Strack, D., Engel, V. and Wray, V. Phytochemistry. 26 (1987) 2399-2400.
441. Strack, D., Schmitt, D., Reznik, H., Boland, W., Grotjahn, L. and Wray, V.
Phytochem. 26 (1987) 2285-2287.
442. Trezzini, GF and Zryd, 1.-P. Phytochern. 30 (1991) 1901-1903.

443. Dopp, H. and Musso, H. Naturwissenschaften 60 (1973) 477--478.
444. Dopp, H. and Musso, H. Chern. Ber. 106 (1973) 3473-3482. 445.
von Ardenne, R., Dopp, H., Musso, H. and Steglich, W.Z. Naturforsch. 29C (1974)
637-{)39.
446. von Elbe, 1.H. and Maing, I.-v. Cereal Sci. Today 18 (1973) 263-264. 447.
lensen, RA in The Shikirnic Acid Pathway (EE Conn, ed.), Plenum, New York (1986) Chapter 3.
448. ImpeJlizzeri, G. and PiatteJli, M. Phytochern. 11 (1972) 2499-2502.

449. Chang, c., Kimler, L. and Mabry, TJ Phytochern. 13 (1974) 2771-2775.
450. Terradas, F. and Wyler, H. Phytochern. 30 (1991) 3251-3253.
451. Kimler, L., Larson, RA, Messenger, L., Moore, JB and Mabry, Tl 1. Chern. Soc., Chern.
Cornrnun. (1971) 1329--1330.
452. Mabry, Tl, Kimler, L and Larson, RA Hoppe-Seyler's Z. Physiol. Chern. (1972)
3532.
453. Reznick, H. Z. Pjlanzenphysiol. 87 (1978) 95-102.
454. Girod, P.-A. and Zryd, J.-P. Phytochern. 30 (1991) 169-174.
455. Terradas, F. and Wyler, H. Helv. Chirn. Acta 74 (1991) 124-140.
456. Sciuto, S, Oriente, G. and PiatteJli, M. Phytochern. 11 (1972) 2259--2262.
457. Heuer, S. and Strack, D. Planta 186 (1992) 626-{)28.
458. Sciuto, S., Oriente, G., PiatteJli, M., ImpeJlizzeri, G. and Amico, V. Phytochern. 13
(1974) 947-951.
459. Wyler, H., Meuer, U., Bauer, 1. and Stravs-MombeJli, L. Helv. Chirn. Act 67 (1984)
1348.
460. Bockern, M. and Strack, D. Planta 174 (1988) 101-105.
461. Bockern, M., Wray, V. and Strack, D. Planta 184 (1991) 261-270.
462. Bockern, M., Heuer, S. and Strack, D. Bot. Act 105 (1992) 146--151.
463. Hirano, H. and Komamine, A. Physiol. Plant. 90 (1994) 239-245.
464. Sakuta, M., Hirano, H. and Komamine, A. Physiol. Plant. 83 (1991) 154-158.
465. Hirano, H., Sakuta, M. and Komamine, AZ Naturforsch. 47C (1992) 705-710.
466. Sweet, SS and Guruprasad, KN Indian 1. Exp. Bioi. 31 (1993) 168-172.
467. Girod, P.-A. and Zryd, 1.-P. Plant Cell Tissue Organ Cui. 25 (1991) 13-16.
468. Hirose, H., Yamakawa, T., Kodama, T. and Komamine, A. Plant Cell Physiol. 31
(1990) 267-271.
469. Obrenovic, S. Ann. Bot. 66 (1990) 245-248.
470. PiattelJi, M., Giudici de Nicola, M. and Castogiovanni, V. Phytochern. 10 (1971) 289-
293.
471. Bianco-Colomas, 1. and Hugues, M. 1. Plant Physiol. 136 (1990) 734-739.
472. Bianco-Colomas, 1., Lenoel, cP and Bulard, C. Plant Growth Regulators 7 (1988) 19-
27.
473. Schwartz, Sl and von Elbe, 1.H. Z. Lebensrn. or. Forsch. 176 (1983) 448--453. 474.
von Elbe, 1.H., Maing, I.-V. and Amundsen, CH 1. Food Sci. 39 (1974) 334-337.
475. Pasch, 1.H. and von Elbe, JH 1. Food Sci. 40 (1975) 1145-1146.
476. Saguy, 1.1. 1. Food Sci. 44 (1979) 1554-1555.
477. Huang, AS and von Elbe, 1.H. 1. Food Sci. 52 (1987) 1689-1693.
478. Attoe, EL and von Elbe, 1.H. 1. Agric. Food Chern. 30 (1982) 708-712.
479. Saguy, 1., Kopelman, 1.1. and Mizrahi, S. 1. Agric. Food Chern. 26 (1978) 360-362.
480. Attoe, EL and von Elbe, 1.H. 1. Food Sci. 46 (1981) 1934-1937.
481. Savolainen, K. and Kuusi, T. Z. Lehensrn. or. Forsch. 166 (1978) 19-22. 482.
von Elbe, 1.H., Schwartz, Sl and Hildenbrand, BE 1. Food Sci. 46 (1981) 1713-1715.
483. Huang, AS and von Elbe, 1.H. 1. Food Sci. 50 (1985) 1115-1120, 1129.
484. Altamirano, R.C., Drdak, M., Simon, P., Rajniakova, A., Karovicova, 1. and PrecJik, L. Food
Chern. 46 (1993) 73-75.
485. Bilyk, A. and Howard, M. 1. Agric. Food Chern. 30 (1982) 906--908.
486. Attoe. EL and von Elbe, 1.H. Z. Lebensrn. or. Forsch. 179 (1984) 232-236.
487. Wyler, H. and Dreiding, AS Helv. Chirn. Acta 45 (1962) 638-{)40.
488. Sapers, GM and Hornstein, 1.S. J. Food Sci. 44 (1979) 1245-1248.
489. Aurstad, K. and Dahle, HK Z. Lebensrn. or. Forsch. 151 (1972) 171-174.
490. Mikova, K. and Kyzlink, V. Sbornik Vysokeskoly Chemicko-Technologicke v. Praze, E.

58 (1985) 9-15.
491. Pasch, JH and von Elbe, JH f. Food Sci. 44 (1979) 72-74.
492. Czapski, JZ Lebensm. or. Forsch. 191 (1990) 275-278.
493. Drd:ik, M., Vallov:i, M., Greif, G., Simko, P. and Kusy, PZ Lebensm. or. Forsch. 190
(1990) 121-122.
494. Simon, P., Drd:ik, M. and Altamirano, RC Food Chem. 46 (1993) 155-158.
495. Soboleva, GA, UI'yanova, MS, Zakharova, NS and Bokuchava, MA Biokhimiya 41 (1976) 968-973.
496. Shih, ee and Wiley, RC f. Food Sci. 47 (1981) 164--166, 172.

497. Lashley, D. and Wiley, RC f. Food Sci. 44 (1979) 1568-1569.
498. Elliott, DC, Schultz, eG and Cassar, RA Phytochemistry. 22 (1983) 383-387.
499. Wiley, Re and Lee, Y.-N. F. Food Sci. 43 (1978) 1056-1058.
500. Wiley, Re, Lee, Y.-N., Saladini, JJ, Wyss, RC and Topalain, HH f. Food Sci. 44
(1979) 208-212.
501. Piattelli, M. and Minale, L. Phytochemistry. 3 (1964) 307-311.
502. Piattelli, M. and Minale, L. Phytochem. 3 (1964) 547-557.
503. D6pp, H. and Musso, H. Z Naturforsch. 29C (1974) 640-642.
504. Adams, JP and von Elbe, JH f. Food Sci. 42 (1977) 410-414.
505. Vincent, KR and Scholz, RG f. Agric. Food Chem. 26 (1978) 812-816.
506. Singer, JW and von Elbe, JH f. Food Sci. 45 (1980) 489-491.
507. Schwartz, SJ and von Elbe, JH f. Agric. Food Chem. 28 (1980) 540-543.
508. Strack, D. and Reznik, HZ Pjianzenphysiol. 94 (1979) 163-167.
509. Strack, D., Engel, D. and Reznik, HZ Pjianzenphysiol. 101 (1981) 215-222.
510. Poumit, A., Lejeune, B., Grand, A. and Pourrat, H. f. Food Sci. 53 (1988) 294--295.
511. Drd:ik, M., Vallova, M., Daucik, P. and Greif, GZ Lebensm. or. Forsch. 188 (1989)
547-550.
512. Piattelli, M. in Chemistry and Biochemistry of Plant Pigments, 2nd edn. Vol. 1. (TW
Goodwin, ed.), Academic Press, New York (1976) Chapter 11.
513. Mabry, TJ and Dreiding, AS Recent Adv. Phytochem. 1 (1968) 145-160.
514. Piattelli, M., Minale, L. and Nicholaus, RA Rend. Accad. Sci. Phys. Mat., Naples 32
(1965) 55-56.
515. Minale, L., Piattelli, M., de Stefano, S. and Nicolaus, RA Phytochem. 5 (1966) 1037-
1052.
516. Wyler, H. and Dreiding, A.S. Helv. Chim. Acta 42 (1959) 1699-1702.
517. Schmidt, O.Th., Becher, P. and Hubnerr, M. Chem. Ber. 93 (1960) 1296-1304.
518. Piattelli, M., Minale, L. and Prota, G. Tetrahedron 20 (1964) 2325-2329.
519. Piattelli, M. and Minale, L. Ann. Chim. (Rome) 56 (1966) 1060-1064.
520. Mabry, TJ, Wyler, H., Sassy, G., Mercier, M., Parikh, I. and Dreiding, AS Helv.
Chim. Acta 45 (1962) 640--647.
521. Mabry, TJ, Wyler, H., Parikh, I. and Dreiding, AS Tetrahedron 23 (1967) 3111-
3127.
522. Piattelli, M., Minale, L. and Nicolaus, RA Phytochem. 4 (1965) 817-823.
523. Piattelli, M., Minale, L. and Prota, G. Ann. Chim. (Rome) 54 (1964) 955-962.
524. Wilcox, M.E., Wyler, H., Mabry, T.J. and Dreiding, A.S. Helv. Chim. Act 48 (1965)
252-258.
525. Piattelli, M., Minale, L. and Prota, G. Phytochem. 4 (1965) 121-125.
526. Piattelli, M., Giudici de Nicola, M. and Castrogiovanni, V. Phytochem. 8 (1969)
731-736.
527. Nilsson, T. Lantbrukshogskolans Annaler 36 (1970) 179-219. 528.
von Elbe, JH, Sy, SH, Maing, I.-Y. and Gabelman, WH f. Food Sci. 37 (1972)
932-934.
529. Wyler, H. and Dreiding, A.S. Helv. Chim. Acta 44 (1961) 249-257.
530. Wolyn, DJ and Gabelman, WH f. Amer. Soc. Hort. Sci. 115 (1990) 165-169.
531. Wolyn, DJ and Gabelman, WH f. inherited. 80 (1989) 33-38.
532. Wilkins, eK Inter. F. Food Sci. Technol. 22 (1987) 571-573.
533. Pourrat, A., Lejeune, B., Jean, D. and Pourrat, H. Ann. Pharm. Fr. 38 (1980) 261-266.
534. Pourrat, H., Lejeune, B., Regerat, F. and Pourrat, A. Biotech. Lett. 5 (1983) 381-384.
535. Adams, JP, von Elbe, JH and Amundson, CH I. Food Sci. 41 (1976) 78-81.
536. Avila, A., Garcia-Hernandez, F. and Santos, E. Dev. Ind. Microbiol. 24 (1983) 553-
561.
537. Bell, ERJ and White, EB Inter. Biotech Ind. 9(3) (1989) 20-26.
538. Leathers, R.R., Davin, C. and Zryd, J.-P. In Vitro Cell. Dev. Bioi. 18P (1992) 39-45.
539. Lejeune, B., Pouget, M.P. and Pourrat, A. Labo-Pharma 334 (1983) 638-643.
540. Lejeune, B., Pourrat, A. and Pouget, M.P. Ann. Pharm. Fr. 44 (1986) 461.
541. Lejeune, B., Grand, A. and Pourrat, A. S. TP Pharma 3 (1987) 400. 542.
von Elbe, JH in Current Aspects of Food Colorants (TE Furia, ed.), CRC Press,
Cleveland (1977) Chapter 3.
543. Pasch, JH, von Elbe, JH and Sell, RJ I. Milk Food Technol. 38 (1975) 25-28.
544. Altamirano, RC, Drdak, M., Simon, P., Smelik, A. and Simko, P. I. Sci. Food Agric. 58
(1992) 595-596.
545. von Elbe, JH, Klement, JT, Amundson, CH, Cassens, RG and Lindsay, RC I.
Food Sci. 39 (1974) 128-132.
546. Dhillon, AS and Mauer, AJ Poultry Sci. 54 (1975) 1272-1277.
547. Kopelman, 1.1. and Saguy, I. I. Food Proc. Pres. 1 (1977) 217-224.

Jackman 1996

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Jackman 1996

Uploaded by

Copyright:

Available Formats

Machine Translated by Google

8 Anthocyanins and betalains

Anthocyanins occur widely in plants, being responsible for their blue,

GAF Hendry et al. (eds.), Natural Food Colorants

ANTHOCYANINS AND BETALAINS 245

some fungal species, eg toadstools [5,6], often accumulate in the stalk of

246 NATURAL FOOD COLORANTS

The mutually exclusive occurrence of anthocyanins and betalains, and similarity in

Anthocyanins possess the characteristic C6C3C6 carbon skeleton of other natural

ANTHOCYANINS AND BETALAINS 247

Table 8.1 Structure of naturally occurring anthocyanidins

Anthocyanidin Substitution pattern (R) Color

3 Yes 6 7 3' S'

Pelargonidin (Pg) OH OH H OH HH Orange

248 NATURAL FOOD COLORANTS

occurring anthocyanidin in which the Band C rings are connected; the

Figure 8.1 Structure of the anthocyanidin riccionidin A.

ANTHOCYANINS AND BETALAINS 249

Although further investigation in this regard is required: anthocyanin

250 NATURAL FOOD COLORANTS

Figure 8.2 Schematic presentation of mechanisms of anthocyanin stabilization.

Methoxylation causes the chromophores to become more red [2]. Thus,

ANTHOCYANINS AND BETALAINS 251

Hydroxyl groups also generally increase perceived redness. Acylation of

A thorough survey of the distribution of anthocyanins in nature has

The biosynthesis of anthocyanins follows a pathway common to other

252 NATURAL FOOD COLORANTS

Table 8.2 Anthocyanins in selected food plants

Botanical name Common name Anthocyanins present References

Alium cepa Red onions Cy 3-glucoside and 3- 80-82

gentiobioside acylated with

Fragaria spp. Strawberries Pg and Cy 3-glucosides 95-97

Glycine maxima Soybean (seedcoat) Cy and Dp 3-glucosides 98

Punica granatum Pomegranate Cy, Dp and Pg 3-glucosides and 115 3,5-

ANTHOCYANINS AND BETALAINS 253

Table 8.2 continued

Botanical name Common name Anthocyanins present References

Raphanus sativus Red radish (root) Pg and Cy 3-sophoroside-5- 57, 116,

Zea mays purple corn Cy, Pg and Pn 3-glucosides, and 143-145

a Abbreviations: Cy = cyanidin; Pg = pelargonidin; Pn = peonidin; Dp = delphinidin; Pt = petunidin; Mv =

254 NATURAL FOOD COLORANTS

Anthocyanin biosynthesis proceeds according to the scheme in Figure 8.3. Initial

F3H is a 2-oxoglutarate-dependent dioxygenase requiring Fe2+, oxygen and

ANTHOCY ANINS AND BETALAINS 255

ShIdmI< Acid Pathway 3 MaIonyf-CoA

256 NATURAL FOOD COLORANTS

deoxyanthocyanidins (Table 8.1) [172, 173]. Subsequent dehydration of

ANTHOCYANINS AND BETALAINS 257

8.4.4 Factors influencing anthocyanin color and stability

As with most natural colorants, anthocyanins suffer from inherent instability;

K': = «[B] + [C])/[AH+])aH+ (8.2)

258 NATURAL FOOD COLORANTS

ANTHOCYANINS AND BETALAINS 259

Figure 8.5 pH-distribution of anthocyanin species and predominant chromophores at equilibrium

260 NATURAL FOOD COLORANTS

However, the cis-trans equilibrium of CE with the corresponding (Z)-form occurs

8.4.4.2 Temperature. As with most chemical reactions the stability of anthocyanins

ANTHOCYANINS AND BETALAINS 261

Coumarin glycoside has been identified as a common product of thermal

B.4.4.3 Oxygen and hydrogen peroxide. Oxygen may cause degradation of

262 NATURAL FOOD COLORANTS

Figure 8.6 Coupled oxidation mechanism leading to discoloration and degradation of

oxidation whereby oxidized constituents of the medium react with the