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Anthocyanins-nature, occurrence and dietary

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Journal of the Science of Food and Agriculture

J Sci Food Agric 80:10631072 (2000)

Review
Anthocyanins nature, occurrence and dietary
burden
Michael N Clifford*
School of Biological Sciences, University of Surrey, Guildford GU2 5XH, UK

Abstract: This paper reviews the literature on the occurrence of anthocyanins in foods and their
transformation during processing, including the formation of adducts and derived tannins. Data
describing the safety of anthocyanins and possible dietary effects are examined. Attention is drawn to
some misquotations in the literature and to some serious gaps in our knowledge, in particular, the lack
of pharmacokinetic data in humans essential to an understanding of associated biological effects.
# 2000 Society of Chemical Industry

Keywords: absorption; anthocyanidins; anthocyanins; burden; cyanidin; delphinidin; deoxyanthocyanidins;

derived tannins; malvidin; malvones; metabolism; pelargonidin; peonidin; petunidin; vitisins; xanthylium salts

INTRODUCTION

It is generally accepted that anthocyanins are the most

important group of water-soluble pigments in plants.
There is a vast literature on their occurrence including
a substantial number of reviews.114 This paper will
focus on the occurrence of anthocyanins in foods and
beverages, their transformation during processing, and
their bioavailability in man.
In plant tissues the anthocyanins produce blue,
purple, red and intermediate hues, and appear `black'
in some commodities. Their hue and structure are
dependent on pH value and the presence of copigments.14,15 They are glycosides with an anthocyanidin
(avonoid) C6C3C6 skeleton. The aglycones are
rarely found in fresh plant material other than as
artefacts. The main exceptions are the 3-deoxy forms
which are rare in foods. Such compounds have been
reported in red-skinned bananas and sorghum,16,17
and tricetanidin is found in black tea, being formed
during processing by oxidative degallation of ()epigallocatechin gallate.18,19
Several hundred anthocyanins are known varying in:
. the basic anthocyanidin skeleton ie the number
and position of hydroxyl and methoxyl substituents
(there are six common variations Fig 1);
. the identity, number and position(s) at which sugars
are attached to the skeleton; and
. the extent of sugar acylation and the identity of the
acylating agent(s).
Glucose, galactose, rhamnose and arabinose are the
sugars most commonly encountered, usually as 3-

Figure 1. Anthocyanin skeleton.

R1

R2

Anthocyanin

H
OH
OH
OCH3
OCH3
OCH3

H
H
OH
H
OH
OCH3

Pelargonidin-3-glucoside
Cyanidin-3-glucoside
Delphinidin-3-glucoside
Peonidin-3-glucoside
Petunidin-3-glucoside
Malvidin-3-glucoside

glycosides or 3,5-diglycosides. Rutinosides (6-O-a-Lrhamnosyl-D-glucosides), sophorosides (2-O-b-D-glucosyl-D-glucosides) and sambubiosides (2-O-b-Dxylosyl-D-glucosides) also occur, as do some 3,7diglycosides and 3-triosides. More information on
the occurrence of particular glycosides is available
elsewhere.7 The commoner acylating agents include
cinnamic acids (caffeic, p-coumaric, ferulic and
sinapic), which may themselves bear glycosidic sugars,
and a range of aliphatic acids (eg acetic, malic,
malonic, oxalic, and succinic). Aromatic and aliphatic

* Correspondence to: Michael N Clifford, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK
E-mail: M.Clifford@Surrey.ac.uk
(Received 29 October 1999; revised version received 3 December 1999; accepted 4 January 2000)

# 2000 Society of Chemical Industry. J Sci Food Agric 00225142/2000/$17.50

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MN Clifford

Figure 2. Anthocyanin equilibria, based on Iversen.102

acylation may occur in the same molecule, and from

zero to at least three acylating residues may be
present.20 Those pigments bearing a dibasic carboxylic
acid are zwitterionic at appropriate pH values. The
presence of a particular sugar or of dicarboxylic acids
might inuence the bioavailability as discussed below.
Early studies may have overlooked the occurrence of
acylated anthocyanins in foodstuffs.21 They are
certainly found in basil, blood orange, elderberries,
red cabbage, fennel, red lettuce, grapes, garlic, redskinned potato and purple sweet potato2233 but this
list is expected to increase. Some commodities, eg
peach skin, contain a limited number of anthocyanin
pigments, whereas others, eg hybrid red grapes, may
contain a complex mixture of more than 20 and
listings are available.1,9 The HPLC prole of anthocyanins in a commodity may be characteristic and such
`ngerprints' may be useful in detecting adulteration
of fruit juices or jams,34,35 in establishing the
geographic origin of a wine,4 or identifying the variety
of grape used in its manufacture.36
The anthocyanins occur in the vacuole as an
equilibrium of four molecular species. These, as
illustrated in Fig 2, are the coloured basic avylium
cation and three secondary structures, the quinoidal
base(s), the carbinol pseudobase (syn chromenol or
hemiacetal) and the chalcone pseudobase.37 (It should
be noted that these are retrochalcones and have the
carbonyl adjacent to the `catechol' ring in contrast to
the avones, isoavones, dihydrochalcones (eg phloretin) and chalcones (eg isoliquiritigenin).) Each of
these four molecular species has a number of rapidly
interconverting tautomeric forms and the chalcone
may be present in both cis and trans forms. The form(s)
occurring in the gastro-intestinal tract are not known
with certainty. It is likely that the cation will exist only
in the stomach, and that other forms will predominate
lower down the GI tract (and in the tissues if
absorbed).
In intact plant tissue anthocyanins adopt a tertiary
structure which protects them from nucleophilic
attack by water and which produces hyperchromic
and bathochromic effects.37,38 Juice extraction disrupts this environment and it has been suggested that
1064

this disruption destroys the protective tertiary structures except in those cases where the anthocyanins are
acylated and intramolecular (as distinct from intermolecular) copigmentation occurs.28 However, it has also
been suggested38,39 that the copigmentation that
occurs in solution (as in fruit juice or wine for
example) aligns the pigments and copigments in such
a way as to facilitate the formation of anthocyanin
copigment transformation products. This seems eminently plausible, but it does not necessarily follow that
the nature of the copigmenation that occurs in intact
cells is identical to that which occurs in juices and
extracts.
Since it is the striking colour of the anthocyanin that
attracts attention there is a tendency to overlook the
existence of the other equilibrium forms. Such an
oversight would have implications for analysis, quality
control and possibly for understanding their biological
properties. The generalised effect of pH on these
equilibria for a non-acylated monoglucoside is illustrated in Fig 3. Under the same conditions the 3,5diglycosides have a smaller concentration of the
cationic form at any given pH value whereas acylated
forms have appreciably more cation especially above
pH 5.20,21 The deoxyanthocyanins2 are more strongly
coloured at higher pH values than the commoner
anthocyanins because the quinoidal bases dominate
above approximately pH 3.03.5 as these compounds
have a lesser tendency to form the colourless pseudobase. Some anthocyanin transformation products40
remain in the avylium form even above pH 7. It has
been observed that certain anthocyanins, particularly
those having only a single hydroxyl in the B-ring, are
more intensely coloured at alkaline pH values than
previously expected and that the dominant coloured
forms at these pH values (quinoidal base and anion
rather than cation) are more stable than previously
thought.41,42 The stability and colour intensity of the
quionoidal forms is also enhanced by acylation (pcoumaroyl) of the glucoside.43
The quantication of anthocyanins is difcult since
their molar absorptivity depends on the balance of

Figure 3. The generalised effect of pH value on anthocyanin equilibria.

J Sci Food Agric 80:10631072 (2000)

Anthocyanin nature, occurrence and dietary burden: a review

coloured and colourless forms, pH value and presence

of copigments, and considerable care is required if
food composition or dietary burden are to be
measured reliably. Solid commodities require extraction prior to analysis, preferably in near anhydrous
methanolethanol mixtures. Ideally this should involve temperatures at which enzymes will be denatured (>70 C) but severe heat treatments should be
avoided because of the risk of hydrolysis and other
artefacts in the case of acetic and formic acids. Note
also that chalcone formation (Fig 2) is endothermic
and the chalcones degrade rapidly at elevated temperatures. However, the extracts once prepared should
be made acidic (using PCA or TFA) about one hour
prior to analysis to give adequate time for the colourless chalcones to re-equilibrate and thus give maximum yield of the coloured avylium cations.
Anthocyanins in aqueous acidic solution below pH
2 show negative deviations from the BeerLambert
law,13 whereas at higher pH values, as in many
foodstuffs, a positive deviation may occur. For
example, at pH 3.16, a 100-fold increase in the
concentration of cyanidin-3,5-diglucosde produces a
300-fold increase in absorbance.5 CD spectroscopy
has established that at 5  105 M simple (non-acylated) anthocyanins exist in a `monomeric' form
whereas above 5  103 M concentration-dependent
Cotton effects suggest chiral stacking.24 To obtain
quantitative data that can be compared meaningfully
between occasions and laboratories requires careful
standardisation of operating conditions and careful
selection of a standard, though until recently, these
have rarely been available commercially. It has been
suggested44 that, if possible, concentrated solutions
should be measured without dilution using short
pathlength cells but at least for simple anthocyanins,
dilution below 5  105 M would seem to be a valid
alternative.
It has been recognised for many years that anthocyanins make a signicant contribution to the colour,
and hence acceptability, of many fruits, some vegetables and associated products, including beverages
and preserves. Subsequently it was recognised that
anthocyanin-rich extracts might have potential as food
additives, especially when suitable materials were
available as a waste product from, for example, wine
production. The methylated and acylated anthocyanins are favoured for this purpose due to their greater
stability.

disrupts membranes and allows the mixing of enzymes

and substrates previously in separate compartments.
The vacuolar contents of fruits are generally acidic.
Removal of the sugar residue(s) at C3 by acid or
enzyme hydrolysis destabilises the pigment because
the associated chalcone lacking the sugars is an
unstable a-diketone. This readily degrades to 2,4,6trihydroxyphenylacetaldehyde and a benzoic acid (for
example syringic acid from malvidin).
Such enzymes may be inactivated by heat processing, but elevated temperatures per se shift the
anthocyanin equilibria towards the chalcone and such
changes are likely during jam making. These transformations continue during jam storage at rates depending upon the conditions. For example, the half life of
anthocyanins in strawberry jam has been reported as
1300 h at 20 C, but only 240 h at 38 C.45 Although
early studies46 suggest that the anthocyanins may be
completely destroyed, more recent studies indicate
that sufcient survive jam and juice manufacture to
permit chromatographic ngerprinting of anthocyanins as a check on fruit authenticity.34,35 The nature of
the transformation products is not known but there is
clear evidence for the involvement of sugars and
ascorbic acid (or their thermal degradation products),
metal ions and hydrogen peroxide derived from
ascorbic acid. Hydrogen peroxide attacks 3-glucosides
at C2 and produces colourless benzoyloxy-phenylacetic acid esters (malvones) through a BayerVilliger
type oxidation see Fig 4.
Anthocyanins are not substrates for polyphenol
oxidases (PPO).47,48 However, anthocyanins possessing a dihydroxy B-ring can undergo coupled oxidations with quinones formed by the action of PPO on
other phenols. Anthocyanins lacking a dihydroxy Bring form adducts with such quinones (see below).
Sulphur dioxide and its analogues, used as antimicrobial agents and as inhibitors of PPO, cause
reversible decolorisation of anthocyanins. The identity
and stereochemistry of the adduct at C4 has recently
been unequivocally conrmed49 see Fig 5.
Anthocyanins undergo profound changes during the
maturation of wines and ports and it is in this context
that most of the recent work has taken place. The
content of anthocyanins gradually declines during
maturation and the formerly sharp peaks are replaced
by a `hump' on which the residual material `oats', eg

DERIVED TANNINS ANTHOCYANIN

TRANSFORMATION PRODUCTS

Anthocyanins are unstable, particularly once removed

from their native environment and the protection
afforded by copigmentation. Various transformations
occur during the processing or domestic cooking of
foods containing anthocyanins producing yellowish or
brownish pigments which are largely uncharacterised.
Physical damage such as peeling, cutting, slicing, etc
J Sci Food Agric 80:10631072 (2000)

Figure 4. Structure of malvone formed from malvidin-3-glucoside, based

on Wong.15

1065

MN Clifford

Figure 5. Structure of the anthocyaninbisulphite adduct as reported by

Berke et al. 49

Ref 4. Supercially similar changes occur in pigment

extracts and concentrated fruit juices, eg Ref 5. Studies
on wines and model systems have permitted the
characterisation of four types of transformation:
. direct condensation between anthocyanins and
avanol monomers or oligomers followed in some
cases by dehydration and protonation to form
orange xanthylium salts;39,50,51
. direct condensation between anthocyanins and
quinones;48,52,53
. aldehyde `bridging' of anthocyanins with avanol
monomers and dimers involving acetaldehyde (derived from ethanol by oxidation) and possibly other
aldehydes arising from thermal transformation of
sugars and ascorbate.5456 Glyoxylic acid from
Fe2-catalysed oxidation of tartaric acid has recently
been shown to form avanol homo-dimers and
might therefore form hetero-dimers between avanols and anthocyanins;57
. cycloaddition of acetaldehyde, vinylphenols (derived from cinnamate decarboxylation) or pyruvic
acid.40,5862 A similar product involving malvidin-3glucoside, acetaldehyde and a procyanidin dimer
has also been observed in model solutions.55 Unlike
native anthocyanins, these cycloaddition products
retain signicant red colour even at neutral pH
values, are resistant to attack by sulphur dioxide,
and may become the dominant `monomeric' pigment in some red wines and ports.40 However, they
seem to contribute comparatively little to the total
colour of wine.61
Some specimen structures are shown in Fig 6. The
avylium cation by virtue of its positive charge is
resistant to attack by electrophiles and, although it has
not always been made clear in the published gures
illustrating these transformations, those transformations involving an attack by aldehyde or quinone (vide
supra) must formally take part through one of the other
equilibrium species. The most likely candidate is the
hemiacetal since it usually predominates at wine pH.63
Indeed, anthocyanin-caftaric quinone addducts in the
hemiacetal form have been detected by LC-MS.53
1066

Figure 6. (a) Structure of the direct condensation product formed from

malvidin-3,5-diglucoside and ()-catechin as reported by Bishop and
Nagel.50 (b)Structure of xanthylium salt based upon Liao et al. 39 Note that
Haslam103 shows a different form of this salt. (c)Structure of ()-catechinacetaldehyde-malvidin-3-glucoside adduct, based on Refs 5456.
(d)Structure of malvidin-3-glucoside-vinylphenol adduct as reported by
Cheynier and colleagues.59,62

J Sci Food Agric 80:10631072 (2000)

Anthocyanin nature, occurrence and dietary burden: a review

Content (mg litre1 or mg kg1) Reference

Commodity
Blackberry
Blueberry
Boysenberry
Cherry
Chokeberry
Cranberry
Cowberry
Currant (black)
Elderberry
Grape (red)
Loganberry
Orange, Blood (juice)
Plum
Raspberry (black)
Raspberry (red)
Raspberry (red) single strength juice
Sloe
Strawberry

Table 1. Selected data for the content of

anthocyanins in foods and beverages

1150
8254200
1609
204500
506010 000
6002 000
1 000
13004 000
2 00010 000
3007500
774
2 000
20250
17004277
100600
41101
1600
150350

66
64, 66, 67
68
64, 69
13, 66
64
64
64
13, 66
13, 64
68
13
64
64, 68, 70
64, 68, 70
34
71
64

Cabbage (red)
Eggplant
Onion
Rhubarb

250
7500
up to 250
up to 2 000

64
72
64
64

Wines (red)
Wines (Port)

240350
1401100

73, 74
64

DIETARY CONSIDERATIONS
Consumption data

Anthocyanins are widespread in food plants, occurring

in 27 families64 and the world-wide annual consumption has been estimated as 10 000 tonnes of anthocyanins from black grapes alone. The anthocyanin
contents of many fruits and vegetables have been
estimated by various groups of workers, using various
methods, and several summaries have been published.
Bearing in mind the comments above about the
difculty of quantication it is impossible to judge
the reliability and true comparability of the data in
these compilations. However, these remain the best
available: they are probably of the correct order of
magnitude. A compilation from the literature is shown
in Table 1.
In the USA, average daily intakes of anthocyanins
have been estimated at 215 mg during the summer and
180 mg during the winter.65 However, as pointed out
by Timberlake,64 regular consumers of red wine are
likely to have signicantly higher intakes since
concentrations in red wines of 200 mg litre1 are not
exceptional although a signicant portion of these may
be the transformation products referred to above and
for which no systematic data are available.
Interaction with ascorbate

Scientic interest in the biological properties of anthocyanins seems to have originated in the early papers of
Szent-Gyorgyi and colleagues7577 which, according to
Kuhnau,65 led eventually to the conclusion that
Vitamin C activity in Guinea pigs and humans was
intensied in a rather specic way by avonoids. The
J Sci Food Agric 80:10631072 (2000)

composition of the experimental and control diets

used in these early studies was insufciently controlled
to allow easy interpretation of the data generated and
certainly does not give unequivocal evidence for an
essential interaction between Vitamin C and avonoids.
With reference specically to the possible benecial
properties of anthocyanins, another statement by
Kuhnau65 in which he refers to the early feeding trials
of Szent-Gyorgyi76,77 only adds to the confusion This
synergism between the avonoids and ascorbic acid, which
was discovered in studies with avanones (`citrin'), was
found later to be much more impressive when other
avonoid subgroups were used, eg , anthocyanins
(Harper et al. 1969) '. The study by Harper et al 78
referred to in vitro model system studies of ascorbate
retention in processed fruit products which led, inter
alia, to the conclusions that in the absence of copper
ions `Cyanidin-3-rhamnoglucoside and delphinidin-3glucoside accelerated the oxidation.', and that `Anthocyanins were shown to possess slight antioxidant activity in the
presence of copper ions.' There is no justication from
this study for extrapolating this observation to a dietary
advantage in man.
Kuhnau65 also refers to a study (Sturua et al. 1971),
published in Russian with an English abstract in Chem
Abs (1971, 76, 21575), which apparently demonstrated that Guinea pigs acquired greater concentrations of Vitamin C in the liver and adrenals after being
fed grape anthocyanins. Hughes and Jones79 obtained
similar results when they fed to Guinea pigs equal
amounts of pure Vitamin C in water, Vitamin C in
Acerola cherry juice (reconstituted from powder,
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MN Clifford

virtually avonoid-free) and Vitamin C in blackcurrant

juice. Although data for food consumption are not
given, they reported that Guinea pigs grew faster on
the avonoid-containing blackcurrant juice. These
animals also acquired greater (4247%) Vitamin C
concentrations in adrenals and spleen than on the
Acerola cherry preparation or pure ascorbate. In a later
study80 they showed that Guinea pigs receiving a
maintenance dose of ascorbate (1 mg kg1 bw) acquired signicantly greater ascorbate concentrations in
the adrenals, spleen and leukocytes (but not the liver
or brain), and achieved greater growth rates, when
supplemented with a avonoid-rich extract of orange
peel (50 mg kg1 bw) or hesperidin (50 mg kg1 bw).
The increased rate of weight gain was striking,
beginning a few days after supplementation began,
but only in the scorbutic animals and not in those on
adequate ascorbate.
However, a recent depletionrepletion study in 12
healthy male volunteers failed to detect any nutritionally signicant differences between water, orange juice
and blackcurrant juice as vehicles for supplying
ascorbate.81

ABSORPTION, METABOLISM AND ELIMINATION

The data on the biological activity of pure anthocyanins are very limited although there are more data for
crude commercial extracts rich in anthocyanins. Such
publications as do exist are not always readily
available, and some reviews which cite them contain
serious misquotations which have then been propagated. These shortcomings will be addressed in the
following sections.
Human studies

The recognition of the `French paradox'82 led to the

suggestion that some component(s) of red wine might
protect against coronary heart disease and again
focused attention on dietary anthocyanins. Despite
the considerable consumption of anthocyanins there is
no proof of their absorption intact in humans. There
have been reports of human anthocyaninuria but,
according to the Joint WHO/FAO Expert Committee
on Food Additives (JECFA),83 these were associated
with the consumption of beetroot and should have
been termed betaninuria. Since there is now evidence
that some avonol glucosides are absorbed from the
duodenum by interaction with the sodium-glucose
cotransporter (SGLTI)84,85 it is possible that some
anthocyanin glucosides are similarly absorbed.
More recently there have been tentative reports,
based upon spectral properties from DAD-HPLC, of
anthocyanin-like substances in plasma86,87 and urine
after acidication.88 However, the lmax observed for
the red components in urine after reacidication did
not exceed 505 nm (which is lower than would be
expected for native anthocyanins). At the pH value of
plasma one would not expect native anthocyanins to
generate a signicant amount of the avylium cation.
1068

Accordingly, these spectroscopic data do not provide

conclusive evidence for the absorption of intact
anthocyanins or even anthocyanidins. Since the
plasma constituents absorbed at visible wavelengths
even at plasma pH values,86 it is possible that they
were acid-stable anthocyanin (cycloaddition) transformation products, and it is interesting to note that two
peaks with similar spectral properties were evident in
the urine before reacidication.88 Even if these very
speculative suggestions were subsequently conrmed
it would still be uncertain whether the adducts were
absorbed as such.
Animal studies

Two reviews64,83 surprisingly give different accounts

of some of the early work, particularly that by
Horwitt.89 Many of the early studies used the presence
of red pigments in the urine as the sole evidence that
anthocyanins had been absorbed. Such studies indicated that rabbits given an oral dose of 500 mg
anthocyanins absorbed some 12% of the dose in an
unchanged form. The rabbit faeces were intensely red,
and whether recycling through coprophagy effectively
increased the dose is not clear. Recovery of red
pigments in the urine was also observed when rats
were given 50 mg by percutaneous injection but not
when rats were given 100 mg by gavage. According to
JECFA,83 there was no coloration of the urine when
dogs were given 500 mg by gastric stula but,
according to Timberlake,64 pigment was found in the
bile and urine within 20 min and the lymph within
50 min. A reproductive study in rats90 and a subchronic toxicity study in beagle dogs91 both reported
impaired body weight gain but failed to nd any other
deleterious effects when a commercial grape colour
extract was given at 15% of the diet.
p-Hydroxy-phenyllactic acid, generated by the gut
microora, has been detected92,93 in the urine of rats
fed pelargonidin-3-glucoside. Metabolites similarly
obtained by feeding delphinidin or malvidin-3,5diglucoside could not be identied. It was concluded
that anthocyanins in general, and cyanidin in particular, are much more resistant to metabolism by the
gut microora than the analogous avanols and
avonols, a conclusion that is consistent with intensely
red faeces when large doses have been given. Reports
that cultured human-derived gut microora, in contrast to rat microora in vivo, are incapable of
metabolising anthocyanins may reect the great
sensitivity of human gut microora to very low levels
of oxygen during isolation or culturing.
There have been several investigations using crude
commercial preparations of Vaccinium myrtilus (bilberry) anthocyanins. This preparation contains sugars,
aliphatic acids, cinnamic acids, avan-3-ols and
avonol glycosides as well as 15 anthocyanins which
account for 36% by weight. According to Colantuoni
et al 94 these are the 3-O-arabinosides, 3-O-glucosides
and 3-O-galactosides of delphinidin, cyanidin, petunidin, malvidin and peonidin (8:5:4:3:1 by weight of
J Sci Food Agric 80:10631072 (2000)

Anthocyanin nature, occurrence and dietary burden: a review

aglycone), but Wagnet95 describes them as the mono

and diglycosides of peonidin, pelargonidin, cyanidin
and delphinidin.
Intraperitoneal or intravenous dosing of such preparations to rats resulted in rapid tissue distribution.
Accumulation was primarily in the kidney, skin, liver,
heart and lung followed by excretion in urine and
bile.96 The preparation has been given variously in
saline, distilled water or phosphate buffer at pH 7.4,
and it is clear that in the latter case comparatively little
if any of the anthocyanins would be presented as
cations.
The pharmacokinetics of a commercial preparation
of Vaccinium myrtilus anthocyanins in male rats have
been studied.97 After a single oral dose (400 mg kg1
bw) to rats, the plasma concentrations reached peak
level (23 mg ml1) after 15 min and then rapidly
declined within two hours. Anthocyanin elimination
occurred mainly through the bile and reached about
4% of the dose after 24 h. The extent of cumulative
urinary and biliary elimination together with the
gastrointestinal recovery demonstrates an absorption
of about 5%. The low apparent bioavailability was
attributed partly to a high hepatic extraction ratio and
partly to an irreversible transformation to colourless
trans chalcones that were thus not detected by the
colorimetric assay employed. No hepatic rst-pass
effect was observed. Despite the modest gastrointestinal absorption and the low absolute bioavailability
(1.2% of the administered dose), the peak plasma
levels (23 mg ml1) measured after the oral treatment
are in the range of biological activity reported for these
substances. HPLC analysis of serum and urine, with
detection at 530 nm, suggested that the 15 anthocyanins were absorbed intact. They were accompanied by
several uncharacterised 530 nm-absorbing peaks
which eluted more rapidly and which might therefore
have been sulphate or glucuronide conjugates.
Some have argued that the term `Vitamin P'
adopted by Szent-Gyorgyi and colleagues related to
the ability of avonoids to reduce capillary permeability. Others have suggested that the `P' referred to
the occurrence of such substances in paprika. Whatever, there has since the mid-1930s been a belief that
such substances taken orally did indeed reduce
capillary fragility. Animal studies with very large oral
doses (up to 410 mg kg1 bw) of crude commercial
preparations of anthocyanins, or wines, have produced
results indicating some effects on the circulatory
system94,98,99 including increased production of a
Prostacyclin (PGI2) thought to inhibit the initial step

in thrombus formation.100 However, these reports do

little to advance our understanding because the effect
cannot be associated with any particular constituent,
and in any case may not be relevant to humans
consuming normal diets.
A recent review13 suggested that anthocyanins
protect against inammatory diseases, but the paper
cited101 presents no evidence to support the assertion
that this is indeed the case. There is also some
evidence that oral dosing with anthocyanins reduces
elastase activity in diabetics and of improved wound
and ulcer healing in rats (cited in Ref 56).

REGULATORY POSITION AND SAFETY

Anthocyanins (E163) have been approved for use in

foods based on very limited toxicological data.
JECFA83 reviewed the limited toxicological data,
including data on mutagenicity, reproductive toxicity
and teratogenicity, and concluded that anthocyanincontaining extracts are of a very low order of toxicity.
The only negative effects observed were reduced organ
weights (liver, adrenal and thyroid) and reduced body
weights, associated with reduced energy intakes at the
highest dose (grape skin extract, containing 3%
anthocyanins, incorporated at 15% of diet), and
probably reecting reduced palatability of the diet.
In 1982, an ADI of 02.5 mg kg1 bw (for anthocyanins present in a grape skin preparation at a level of
3%) was calculated. This translates into 150175 mg
per day maximum for a 6070 kg adult. The derivation
of this ADI83 is cryptic and I am grateful to Professor R
Walker (occasional Chairman of JECFA) for the
clarication in Table 2. Interestingly it is appreciably
lower than some estimates of average daily consumption.

FUTURE RESEARCH REQUIREMENTS

It is necessary to determine:
1. whether or not anthocyanins are absorbed by
humans and, if so, the associated pharmacokinetics, speciation and tissue distribution;
2. whether or not the sodium-glucose cotransporter
(SGLT1) is involved in absorption;
3. the identity of the gut ora metabolites;
4. the dietary burden and variations within and
between populations, and the contribution of
anthocyanin transformation products to the total
burden.

Table 2. Derivation of the ADI for a Grape-skin Extract

1. The no-observed-effect-level (NOEL) for a grape-skin extract containing 3% anthocyanin pigments was 7.5% of the diet of rats
in a two-generation reproduction study (observation period up to 18 weeks).
2. 7.5% of the diet of a young rat equates to 7500 mg kg1 bw per day.
3. The preparation tested contained 3% anthocyanins.
4. NOEL for anthocyanins = 3  7500/100 = 225 mg kg1 bw.
5. ADI = NOEL/100 = 2.25 mg kg1 bw per day, which was rounded to 2.5 mg kg1 bw per day.
J Sci Food Agric 80:10631072 (2000)

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MN Clifford

Such studies will require the synthesis of labelled

anthocyanins in which the label is stable during pH
equilibration.
This information decit, compounded by the lack of
information on the burden of such compounds,
variations in burden with populations, etc, is the major
impediment to evaluating properly the role of dietary
phenols in disease prevention.

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