Natural Pigments Carotenoids Anthocyanins and Beta

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Natural Pigments: Carotenoids, Anthocyanins, and Betalains — Characteristics,

Biosynthesis, Processing, and Stability
Article in Critical Reviews in Food Science and Nutrition · June 2000

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Critical Reviews in Food Science and Nutrition, 40(3):173–289 (2000)
Natural Pigments: Carotenoids, Anthocyanins, and

Betalains — Characteristics, Biosynthesis,
Processing, and Stability
F. Delgado-Vargas,1 A. R. Jiménez,2 and O. Paredes-López3*
1Fac. de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sin.; 2Centro de Productos
Bióticos del Instituto Politécnico Nacional, Yautepec, Mor.; 3Centro de Investigación y de Estudios Avanzados
del Instituto Politécnico Nacional, Unidad Irapuato, Apdo. Postal 629, Irapuato, Gto. 36500, México
Dr. F. J. Francis, Dept. Food Science, Chenoweth Lab, University of Massachusetts, Amherst, MA 01003
I. INTRODUCTION ...............................................................................................................................176
Downloaded By: [Cinvestav del Ipn] At: 15:20 24 November 2009
II. PIGMENTS IN GENERAL ..............................................................................................................176

A. Distribution ................................................................................................................................ 176
B. Classification ..............................................................................................................................176
1. By Their Origin .....................................................................................................................176
2. By the Chemcial Structure of the Chromophore ...................................................................176
3. By the Structural Characteristics of The Natural Pigments .................................................176
4. As Food Additives .................................................................................................................177
III. NATURAL PIGMENTS ................................................................................................................177

A. Distribution ..............................................................................................................................177
1. Tetrapyrrole Derivatives .......................................................................................................177
2. Isoprenoid Derivatives .........................................................................................................177
3. N-Heterocyclic Compounds Different from Tetrapyrroles ..................................................178
a. Purines .....................................................................................................................178
b. Pterins .....................................................................................................................178
c. Flavins ......................................................................................................................178
d. Phenazins .................................................................................................................180
e. Phenoxazines ...........................................................................................................180
f. Betalains ..................................................................................................................180
4. Benzopyran Derivatives .......................................................................................................180
5. Quinones ..............................................................................................................................182
6. Melanins ...............................................................................................................................182
B. Functions ..................................................................................................................................182
1. Tetrapyrrole Derivatives .......................................................................................................182
2. N-Heterocyclic Compounds Different from Tetrapyrroles ..................................................185
3. Benzopyran Derivatives .........................................................................................................185
a. Antioxidant ..............................................................................................................185
b. Color and Sexual Processes in Plants .....................................................................186
c. Photoprotection ..........................................................................................................186
*Corresponding author.
1040-8398/00/$.50
© 2000 by CRC Press LLC
173
d. Defense Mechanism in plants ...................................................................................187
e. Other Ecological Functions .....................................................................................188
f. Pharmacological Effects .............................................................................................189
4. Quinones ................................................................................................................................192
5. Iridoids ...................................................................................................................................193
6. Melanins ..............................................................................................................................193
C. Importance As Food Colorants ................................................................................................193
1. Reasons to Use Color Additives .........................................................................................193
2. Importance of Natural Colorants .........................................................................................194
D. Some Regulatory Aspects About Color Additives ..................................................................195
IV. SOME IMPORTANT PLANT PIGMENTS: CAROTENOIDS,

ANTHOCYANINS, AND BETALAINS ..................................................................................................197
A. Carotenoids ...............................................................................................................................197
1. Definition ...............................................................................................................................197
2. Classification ..........................................................................................................................197
3. Distribution ...........................................................................................................................197
a. Higher Plants .............................................................................................................197

b. Algae .........................................................................................................................200
c. Bacteria .....................................................................................................................200
d. Fungi ........................................................................................................................200
4. Biosynthesis: Biochemistry and Molecular Biology ............................................................200
a. Biochemistry .............................................................................................................200
b. Biosynthesis Regulation .............................................................................................205
c. Molecular Biology of Carotenogenesis .........................................................................207
5. Functions ................................................................................................................................214
a. Color ........................................................................................................................214
b. Photosynthesis ...........................................................................................................214
c. Xanthophyll Cycle ...................................................................................................214
d. Antioxidant ..............................................................................................................214
e. Pharmacological Effects .........................................................................................217
6. Methodological Aspects ......................................................................................................220
a. Extraction ................................................................................................................221
b. Saponification .........................................................................................................222
c. Separation ...............................................................................................................222
d. Charaterization ........................................................................................................224
7. Importance as Food Colors — Stability, Processing, and
Production ...............................................................................................................................................225
a. Stability in Model Systems ......................................................................................225
b. Processing and Stability in Foods ............................................................................227
c. Production of Carotenoids in Bioreactors ................................................................230
B. Anthocyanins .............................................................................................................................231
1. Definition ...............................................................................................................................231
2. Classification .........................................................................................................................231
3. Distribution ...........................................................................................................................231
4. Biosynthesis: Biochemistry and Molecular Biology ...........................................................231
a. Biochemistry ............................................................................................................231
b. Biosynthesis Regulation ............................................................................................236
c. Molecular Biology of Anthocyanin Biosynthesis ....................................................237
174
5. Functions ...............................................................................................................................240
a. Color and Ecological Functioins ............................................................................240
b. Marker for Good Manufacturing Practices in Food ................................................240
c. Pharmacological Effects ..........................................................................................240
6. Methodological Aspects ........................................................................................................241
a. Extraction .................................................................................................................241
b. Separation ................................................................................................................242
c. Characterization .......................................................................................................242
7. Importance as Food Colors — Stability, Processing, and Production ................................244
a. Stability in Model Systems .......................................................................................244
b. Processing and Stability in Foods ..........................................................................247
c. Production of Anthocyanins by Plant Tissue Culture...............................................249
C. Betalains ..................................................................................................................................251
1. Definition ................................................................................................................................251
2. Classification .........................................................................................................................253
3. Distribution ...........................................................................................................................253
4. Biosynthesis: Biochemistry and Molecular Biology ............................................................254
a. Biochemistry .............................................................................................................254
b. Biosynthesis Regulation ...........................................................................................259
c. Molecular Biology of Betalain Biosynthesis .................................................................259
5. Functions ...............................................................................................................................260
a. Taxonomic Markers................... .............................................................................260
b. Ecological and Physiological Aspects ...................................................................260
c. Pharmacological Effects ..........................................................................................261
6. Methodological Aspects .......................................................................................................261
a. Extraction .................................................................................................................261
b. Separation .................................................................................................................262
c. Characterization ........................................................................................................263
7. Importance as Food Colors — Stability, Processing, and Production ...............................266
a. Stability in Model Systems .....................................................................................266
b. Processing and Stability in Foods ..........................................................................267
c. Production of Betalains by Plant Tissue Culture ...................................................269
V. FUTURE TRENDS .........................................................................................................................270
ACKNOWLEDGMENTS ......................................................................................................................271
REFERENCES .......................................................................................................................................271
ABSTRACT: Pigments are present in all living matter and provide attractive colors and play basic roles in the
development of organisms. Human beings, like most animals, come in contact with their surroundings through
color, and things can or cannot be acceptable based on their color characteristics. This review presents the basic
information about pigments focusing attention on the natural ones; it emphasizes the principal plant pigments:
carotenoids, anthocyanins, and betalains. Special considerations are given to their salient characteristics; to their
biosynthesis, taking into account the biochemical and molecular biology information generated in their elucida-
tion; and to the processing and stability properties of these compounds as food colorants.
KEY WORDS: color, carotene, xanthophyll, betacyanin, betaxanthin, food.
175
I. INTRODUCTION for each pigment group. Consequently, it is not our
major goal to present a thorough review of all pig-
Pigments produce the colors that we observe at ment groups. This would be impossible in a single
each step of our lives, because pigments are present document, and although we present here a global
in each one of the organisms in the world, and plants overview about natural pigments our work focuses on
are the principal producers. They are in leaves, fruits, the more representative pigments in the main natural
vegetables, and flowers; also, they are present in skin, producers, namely, plants, which in addition are the
eyes, and other animal structures; and in bacteria and more highly consumed as food products. In the fol-
fungi. Natural and synthetic pigments are used in lowing pages, we emphasize their salient characteris-
medicines, foods, clothes, furniture, cosmetics, and in tics; their biosynthesis, taking into account the bio-
other products. However, natural pigments have im- chemical and molecular biology information generated
portant functions other than the imparted beauty, such for their elucidation, and the processing and stability
as the following: we could not have photosynthesis or properties of these pigments as food colorants.
probably life all over the world without chlorophylls
and carotenoids. In animals how could oxygen and
carbon dioxide be transported without hemoglobin or II. PIGMENTS IN GENERAL
myoglobin? Under stress conditions plants show the
A. Definition
synthesis of flavonoids; the quinones are very impor-

tant in the conversion of light into chemical energy.
The melanins act as a protective screen in humans and Pigments are chemical compounds that ab-
other vertebrates, and in some fungi melanins are sorb light in the wavelength range of the visible
essential for their vital cycle; last but not least, a lot of region. Produced color is due to a molecule-spe-
pigments have a well-known pharmacological activ- cific structure (chromophore); this structure cap-
ity in sickness such as cancer and cardiovascular tures the energy and the excitation of an electron
diseases, and this has stressed pigment importance for from an external orbital to a higher orbital is
human beings.162,196,259,327 produced; the nonabsorbed energy is reflected
Additionally, since time immemorial human and/or refracted to be captured by the eye, and
beings have associated product qualities with their generated neural impulses are transmitted to the
colors, this is especially true for meals. Historically, brain where they could be interpreted as a color.196
at the beginning of the food industry consumers did
not take care about the kind of pigments used in food
coloring (natural or synthetic), but recently people B. Classification
have shown their phobia to synthetic pigments when
the concepts “synthetic pigments” and “illness” were 1. By Their Origin
associated, and when the attributed pharmacological
benefits of natural pigments came into consider- Pigments can be classified by their origin as
ation. However, the natural pigments that are per- natural, synthetic, or inorganic. Natural pigments are
mitted for human foods are very limited, and the produced by living organisms such as plants, animals,
approval of new sources is difficult because the U.S. fungi, and microorganisms. Synthetic pigments are
Food and Drug Administration (FDA) considers the obtained from laboratories. Natural and synthetic pig-
pigments as additives, and consequently pigments ments are organic compounds. Inorganic pigments
are under strict regulations.80,144,507,508 can be found in nature or reproduced by synthesis.29
Thus, an adequate understanding of the actual
sources of pigments will contribute to their better use.
In this review we present the basic information about 2. By the Chemical Structure of the
pigments focusing our attention on the natural pig- Chromophore
ments. At this time, it must be emphasized the ubiq-
uity of pigments in living organisms (plants, fungi, Also, pigments can be classified by taking
bacteria, among others), the variety of chemical struc- into account the chromophore chemical structure
tures, and the large quantity of information generated as:510
176
Chromophores with conjugated systems: caro- basic structure (lineal array). In the cyclic com-
tenoids, anthocyanins, betalains, caramel, syn- pounds, we can mention the heme group (the
thetic pigments, and lakes. porphyrin ring is bonded to an iron atom); this
Metal-coordinated porphyrins: myoglobin, group is present in hemoglobin and myoglobin,
chlorophyll, and their derivatives. present in animals, and also in cytochromes,
peroxidases, catalases, and vitamin B12 as a pros-
thetic group, all of them with a wide distribu-
3. By the Structural Characteristics of tion. However, chlorophylls constitute the most
the Natural Pigments important subgroup of pigments within the tet-
rapyrrole derivatives. Chlorophyll is mainly
Moreover, natural pigments can be classified present in the chloroplasts of higher plants and
by their structural characteristics as:29,196 most algae. The number of known chlorophyll
Tetrapyrrole derivatives: chlorophylls and heme structures have shown increments through the
colors. years: up until 1960 only three structures had
Isoprenoid derivatives: carotenoids and iridoids. been described, 20 up until 1970, and more than
N-heterocyclic compounds different from tetrapy- 100 up until 1980. Higher plants, ferns, mosses,
rroles: purines, pterins, flavins, phenazines, green algae, and the prokaryotic organism
phenoxazines, and betalains. prochloron present only two chlorophylls (“a”

Benzopyran derivatives (oxygenated heterocyclic and “b”), and the rest of them have been found
compounds): anthocyanins and other flavonoid in other groups such as algae and bacte-
pigments. ria.60,87,196,279,404
Quinones: benzoquinone, naphthoquinone, an-
thraquinone.
Melanins. 2. Isoprenoid Derivatives
Isoprenoids, also called terpenoids, repre-

4. As Food Additives sent a big family of natural compounds; they are
found in all kingdoms where they carry out mul-
By considering the pigments as food addi- tiple functions (hormones, pigments, phytoalex-
tives, their classification by the FDA is150,162,510 ins). Over 23,000 individual isoprenoid com-
Certifiable. These are manmade and subdi- pounds have been identified and many new
vided as synthetic pigments and lakes. structures are reported each year. By their abun-
Exempt from certification. This group in- dance and structure, two subgroups of compounds
cludes pigments derived from natural sources such are considered pigments: quinones and caro-
as vegetables, minerals, or animals, and man- tenoids. However, in addition, and only recently,
made counterparts of natural derivatives. iridoids is a third group of plant isoprenoid com-
pounds that have acquired some relevance.
Quinones are considered another group because
III. NATURAL PIGMENTS not all of them are produced by this biosynthetic
pathway. On the other hand, carotenoids as a
A. Distribution major point of our review are described below.
In relation to iridoids, these are found in
1. Tetrapyrrole Derivatives about 70 families (Capriofilaceae, Rubiaceae,
Cornaceae, among others) grouped in some 13
These compounds have a structure with pyr- orders. Saffron (Crocus sativus L.) and cape
role rings in linear or cyclic arrays. Figure 1 jasmine fruit (Gardenia jasminoids Ellis) are the
shows some common structures of tetrapyrrole best-known iridoid-containing plants, but their
derivatives. Phytochrome is very common in al- colors are more importantly influenced by caro-
gae (Rhodophyta, Cryptophyta), and bilin is this tenoids.406
177
FIGURE 1. Basic structure of porphyrinic pigments (pyrrol) and of some porphyrinic pigments with biological
importance.
3. N-Heterocyclic Compounds Different b. Pterins

from Tetrapyrroles
The pteridin ring system is probably present in
Figure 2 shows some structures for compounds every form of life. Most of the natural pteridins have
of this group. an amino group at C-2 and an hydroxyl group at C-
4. Also, 2,4-dihydroxipteridins have been described
as important components in the flavin biosynthesis.
a. Purines Pterins are responsible for color in some insects, in
vertebrate eyes, human urine, and bacteria (Lacto-
As most of the nucleotides, purines are found bacillus casei and Streptomyces faecalis R.).152,196
in two macromolecules: deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA). These mol-
ecules are an essential component of life, thus c. Flavins
they are present in each living organism. Free
purines have been found in animals (golden and In these compounds a pteridin and a ben-
silvery fish).196 zene ring are condensed. Riboflavin is the main
178
FIGURE 2. N-heterocyclic compounds.
179
compound of this group, and it is synthesized in also contribute to the yellow color of flowers,
all live cells of microorganisms and plants. Ri- where they are present with carotenoids or
boflavin is found in milk. Other sources are a alone in 15% of the plant species. 259
wide range of leafy vegetables, meat, and The aurones are present in flowers (Bidens
fish.87,152,196 sp., Cosmos sp., Dahlia sp.), wood (Rhus
sp., Schinopsis sp.), bryophytes (Funaria
hygrometrica), and in Cypreceae plants. They
d. Phenazines are more common in inflorescences, seeds, and
leaves. The chalcones are common in mixtures
They are found in bacteria.196 with aurones to generate the anthclor pigment in
the Compositae). In nature, it is uncommon to
find them without substitutions, but hydroxy-
e. Phenoxazines lated have been described in wood and peels
of trees in the genus Acacia, Rhus, Macherium,
They are found in fungi and insects.196 and Adenanthera. Flavonols are ubiquitous in
woody angiosperms, common in shrubby an-
giosperms, and exceptionally in inferior plants.
f. Betalains
Flavones and flavanones are found free

or glycosylated in leaves of angiosperms.
As a main component of our review, this Flavanones are especially common in
subgroup of compounds are discussed below. Rosaceae, Rutaceae, legumes and Compositae.
Dihydrochalcones are found mainly hydroxy-
lated in apple and in some species of Rosaceae,
4. Benzopyran Derivatives Ericaceae, Fagaceae, legumes, and Salicaceae.
Leucoanthocyanidins (flavan 3,4-diols) are
The most studied secondary metabolites are widely distributed in plants, and they have been
the flavonoids. These are phenolic compounds isolated from wood and peel of trees (particu-
with two aromatic rings bonded by a C3 unit larly Acacia) and methylated, and C-alkylated
(central pyran ring) and divided in 13 classes leucoanthocyanidins have been identified in a
based on the oxidation state of the pyran ring variety of sources (e.g., Neuroautanenia
and on the characteristic color: anthocyanins, amboensis, Marshallia sp.). Catechins flavan 3-
aurons, chalcones, yellow flavonols, ols) and flavans are found mainly in leaves. Cat-
flavones, uncolored flavonols, flavanones, echins and epicatechins are among the common-
dihydroflavonols, dihydrochalcones, leuco- est flavonoids known, sharing a distribution
anthocyanidins, catechins, flavans, and almost as widespread as quercetin in the
isoflavonoids. Figure 3 shows some flavonoid Dicotyledoneae. The 3,4,5-trihydroxy B-ring
structures. Each type of flavonoid can be modi- flavan 3-ols gallocatechin and epigallocatechin
fied by hydroxylation, methylation, acylation, have also a wide distribution (paralleling
and glycosylation to obtain a great natural diver- myricetin), especially in more primitive plants
sity of compounds. Flavonoids are water soluble (the Coniferae being outstanding). Many flavans
and show a wide distribution in vascular plants. are lipid soluble and appear to be leaf-surface
They are present in each part of the plant. More constituents. Isoflavonoids have the B ring in C-
than 5000 flavonoids have been chemically char- 3 instead of C-2, and many natural products are
acterized, and new structures are described con- in this group: isoflavones, rotenoids, pterocarps,
tinuously.194,259 and coumestans. Isoflavones are the most com-
In the flavonoids, the anthocyanins are the mon in legumes, especially in Lotoideae, al-
most important pigments; they produce colors though they have also been reported in
from orange to blue in petals, fruits, leaves, Amaranthaceae, Iridicaceae, Miristicaceae, and
and roots and are discussed below. Flavonoids Rosaceae.29,157,193,259
180
FIGURE 3. Benzopyran derivatives.
181
5. Quinones color phenomena is conspicuous for the survival
of animals and plants; (2) the relation between
Quinones have a great number of coloring color and evolution theories; and (3) their impor-
compounds (Figure 4). This group is the biggest tance in comparative physiology. Thus, the stud-
one in number and structural variation. Also, they ies on pigments were greatly impulsed by their
are more widely distributed than other natural multiple functions.396
pigments (with the exception of carotenoids and In agreement with the distribution and abun-
melanins). The basic structure consists of a dance, the most important pigments in higher
desaturated cyclic ketone that is derived from an plants were chlorophylls, carotenoids, and antho-
aromatic monocyclic or polycyclic compound. cyanins when considering their capacity to impart
Quinones can be divided by their structure as colors. By the same criteria, algae phycobilins are
benzoquinones, naphthoquinones, anthraquinones, considered important pigments. Other pigments
and miscellaneous quinones; moreover, have less importance as colorant substances be-
dibenzoquinones, dianthraquinones, and cause they are present in low quantities.465 In the
dinaphthoquinones have been reported. The vari- following paragraphs we describe other functions
ability in the kind and structure of substituents of this colorant substance.
conduce to large number of quinones. Quinones
are found in plants: plastoquinones are found in

chloroplasts of higher plants and algae; ubiquino- 1. Tetrapyrrole Derivatives
nes are ubiquitous in living organisms;
menaquinones are found in bacteria; The heme groups show chemical combina-
naphthoquinones in animals; and anthraquinones tion with metals (Fe, V, Cu, Mg) to form metal
in fungi, lichens, flowering plants, and insects. porphyrins. The metal atom is bonded to the ni-
Many quinones are byproducts of the metabolic trogen atoms of the heme group by a coordination
pathways and a few organisms (fungi) produce bond. The most important metal porphyrins are
large quantities. In general, quinones produce formed with Fe. In these compounds, nature has
yellow, red, or brown colorations, but quinone exploited the change of the valence states of iron
salts show purple, blue, or green colors.196,464 from ferric (Fe+3) to ferrous (Fe+2) and vice versa
to establish a system for electron transport. This
system gives the connection between the intracel-
6. Melanins lular dehydrogenases with atmospheric oxygen
and in consequence the support for the transport
Melanins are nitrogenous polymeric compounds and storage of oxygen, an essential component
whose monomer is the indole ring (Figure 5). In for life. Additionally, the bonding of metal to
general, melanins are not homopolymers but present cytochrome permitted joining the air oxygen with
a mixture of macromolecules. Melanins are respon- the metabolic substrates in the cell, contributing
sible of many of the black, gray, and brown colora- with electron transportation, and consequently
tions of animals, plants, and microorganisms. providing the energy for vital processes.396 Cata-
Eumelanins are widely distributed in vertebrate and lase and peroxidase are enzymes with an heme
invertebrate animals. Phaemelanins are macromol- cofactor and have a participation in reduction-
ecules of mammals and birds. Allomelanins have oxidation (redox) reactions of living organisms.
been described in seeds, spores, and fungi, and Vitamin B12 with its heme group participates in
esclorotins in arthropods.64,196,465 chemical-biological reactions that involve rear-
rangements.196
The most important function of chlorophylls
B. Functions is in the photosynthetic process. Chlorophylls
are porphyrins and have a phytyl group confer-
Up to 1898 the increased interest on color in ring on them hydrophobic characteristics. The
organisms was due to three main reasons: (1) the metal bonded in chlorophylls is magnesium in-
182
FIGURE 4. Basic structures of Quinones (A, B, and C) and some of their most known compounds.
183
FIGURE 5. Melanin-related structures and some properites. (A) Basic structure (indolic ring). (B) Resonance
structures that are probaly involved in the process of color. (C) Some of the suggested forms of indolic polymerization
to form melanins. The arrows show the points and sense of polymerization.
184
stead of the iron in the heme. These compounds all the information for the survival of organisms
absorb light in the visible region, red (peak at is described. Some pterins are growth factors:
670 to 680 nm) and blue (peak at 435 to 455 rhizopterin (Streptomyces faecalis), pteroyl-
nm). The reflection and/or transmittance of the glutamic acid (Lactobacillus casei and chickens),
nonabsorbed green light (intermediate wave- other growth factors are teropterin, vitamin Bc,
length) gives the characteristic green color of folinic acid, and probably the most important pterin
plants and chlorophyll solutions. Light absorp- is folic acid, which is an essential vitamin in-
tion is carried out by the porphyrin ring and in volved in the transference of methyl groups and
the absorption process a π-electron is excited to in the insertion of two carbon atoms in the pyri-
the higher energy π*-orbital. Part of the lumi- midine ring.152,196 Flavins are in the molecule of
nosity energy is absorbed by the chlorophyll flavinadenin mono- (FMN) and di-nucleotide
molecules, where it is transformed into biochemi- (FAD), important molecules in the redox reac-
cal energy. Normally, in this process an electron tions of organisms, where they are found. FMN
is lost and transferred to an acceptor molecule. and FAD are coenzymes of many enzymes (e.g.,
This process needs highly specialized chloro- nitrate reductase, pyruvate decarboxylase). Ribo-
phyll molecules that are associated with mem- flavin is an essential vitamin in animals.152 Phena-
branes (reaction centers P680 and P700). A small zines are in bacteria and have bacteriostatic prop-
portion of chlorophyll molecules shows this func- erties. Phenoxazines are in fungi and insects; for
tion, but the reaction centers are the starting example, Streptomyces sp. produces actinomycin
point of the chain of electron transportation that (antibiotic).196
will finally produce the energy in the form of
ATP and NADPH2, and these molecules support
all the plant metabolism. Photosynthesis as 3. Benzopyran Derivatives
known nowadays cannot be possible without
chlorophyll. Thus, chlorophyll is a very impor- a. Antioxidant
tant molecule for life all over the world.404
Among the linear tetrapyrroles, bilins pigment Several reports have shown the antioxidant
the integuments of many invertebrates (worms and activity of flavonoids, which is important in the
insects). In algae, the function of phycobilin is simi- development of their functions such as scavenging
lar to chlorophyll as accessory pigments in light of radicals and disease treatment and prevention.
harvesting, transmitting this energy to chlorophyll Rutin inhibited malonaldehyde formation from ethyl
molecules in the photosynthetic membrane. It has arachidonate by 70% at the level of 0.125 µmol.
been described that pigment content in cyanobacteria With ethyl linoleate, naringin, galangin, and rutin
and some algae is regulated by light.196 exhibited dose-related activities and showed inhi-
Phytochrome is a blue-green photochromic bitions of 30% at 0.5 µmol. It has been pointed out
pigment and controls a wide variety of metabolic that the major mode of flavonoids as antioxidants
and developmental processes in green plants. The is in their ability to scavenge free radicals, and that
chromophore of phytochrome is a lineal tetrapyr- hydroxylation of the B-ring is an important con-
role and has been shown that the activity of many tributor to such activity; in particular, hydroxyl
enzymes is regulated by phytochrome at biochemi- groups at the 3′- and 4′- positions of the B-ring
cal and transcriptional level (mRNA species). Phy- exhibited the highest antioxidant activity, but it
tochrome is involved in the germination, flower- was lower than the observed with BHT.484 It
ing, ripeness, anthocyanin, and protein synthesis.196 was shown that luteolin has good antioxidant
activity evaluated by the β-carotene
bleaching method, and it was suggested that C2-C3
2. N-Heterocyclic Compounds Different double bond and C4 keto group seem to be essen-
from Tetrapyrroles tial for high antioxidant activity (quercetin > (+)-
catechin). Also, it was shown that aglycone fla-
The main function of purines is to serve as vonoids are more potent antioxidants. However,
structural components in DNA and RNA, where luteolin did not exhibit quality antioxidant activity
185
with the α,α-diphenyl-β-picrylhydrazyl (DPPH) established that flavonols are required to induce
method, and quercetin and other flavonoids were pollen germination in vivo and in vitro. Some of
better, and a similar trend was obtained the structural requirements of compounds to de-
with the Rancimat method. 484 Another velop this function are a double bond between
study used 18 different intestinal bacteria to me- carbons two and three, a keto group at carbon four,
tabolize eriocitrin (eriodictyol 7-rutinoside), and a hydroxyl group at carbon three of the hetero-
which is an antioxidant in lemon fruit. It was shown cycle, and only the flavonol class has all these
that in a first step eriocitrin is transformed to features. It has been established that phenylalanine
eriodictyol by different bacteria except Clostridium ammonia-lyase (PAL) might play an essential role
spp., but from eriodictyol Clostridium spp. pro- in the development of microspores to mature grains.
duced 3,4-dihydroxyhydrocinnamic acid and phlo- The introduction of the sweet potato PAL into
roglucinol. Interestingly, eriodictyol showed higher tobacco generated transgenic plants with reduced
activity than eriocitrin or α-tocopherol.324 The an- pollen fertility (10 to 45%). It was suggested that
tioxidant activity of wines has also been evaluated, pollen reduction in PAL activity could reduce the
and it has been reported that almost 96% of the levels of flavonols and a reduction of levels of
activity can be explained by the content of cat- sporollenin of the pollen cell walls, and conse-
echin, m-coumaric acid, epicatechin, cis-polydatin, quently affect the development, germination, and
and vanillic acid, showing higher correlations with growth of pollen tubes in tobacco. Thus, PAL ac-
several flavonoids (catechin, myricetin, quercetin, tivity in the anther tapetum is a significant factor in
rutin, epicatechin, cyanidin, and malvidin 3-gluco- the development of pollen.301 p100 cDNA of
side). Wine flavonoids also show a good Solanum tuberosum is up-regulated in pistils after
peroxynitrite scavenging activity; this was demon- pollen tube growth and, much more transiently, by
strated by removing the activity with the elimina- touching the stigma. This cDNA clone shows ho-
tion of these compounds, an interesting aspect of mology with various isoflavone reductase-like se-
the antioxidant profile of wine.356 quences that have been involved in the response to
pathogen attack and stress conditions. In potato
pistils was shown that not only pollination but also
b. Color and Sexual Processes in Plants the physical presence of the pollen tubes in the
pistil is a constant stimulus for expression. An
Most of the pigments in flowers are flavonoids, antioxidant protective activity has been proposed
and they impart colors in the range red or purple for p100 because oxygen radicals are natural
associated with anthocyanins to yellow associated byproducts of metabolism, and a high metabolism
with aurones and chalcones. Also, flowers contain is required for the fast-growing pollen tubes.483
flavonols and flavanones that by themselves do not
have colors, but modify the coloration by complex-
ation with anthocyanins and/or metallic ions (co- c. Photoprotection
pigmentation). The coloration patterns of flowers
attract insects to anthers (masculine organ) and Severe illumination produces and oxidative stress
pistils (feminine organs) and contribute to the pol- in plant tissues and flavonoids protect them from
linating process, and as a matter of fact it is well damage. One of the responses of UV-illuminated
known that many species accumulate flavonoids in seedlings is the transcriptional activation of flavonoid
these organs. The most common flavonoids in biosynthetic genes. Also, it must be considered that
anthers are anthocyanins, flavonols, and chalcones. UV-B radiation itself modify the pigment composi-
Also, flavonoids have been involved in the sexual tion and consequently induces a reduction of the
process: in the cross- pollination of two varieties of radiation levels over plant tissues. Moreover, it has
forsythia, rutin, and quercetin are required. An- been established that better UV-B photoprotectors are
other important aspect: animals are attracted by the noncolored flavonoids (flavones, flavonols, and
fruit color, and after the consumption the seeds are isoflavonoids). Additionally, it is known that the pro-
dispersed through the excrement.193,259,455 It has been duction of pyrimidine dimers and 6,4-photoproducts
186
is higher in plants without flavonoids.196,259,327 were studied. It was determined that the flavonoid
Middleton and Teramura314 showed that flavonoids compounds were induced following infection by
protect soybean but without an effect on the photosys- P. viticola in both the resistant and in the interme-
tems, and they suggested that carotenoids develop diate resistant species. However, the kind of fla-
this function. In 1992, it was shown that quercetin and vonoids was different. In the resistant species,
its glycoside (rutin), acting as antioxidants, reduced necrosis of the stomatal tissues occurred after
the rate of formation of malondialdehyde and lipid infection, resulting in the cessation of fungal
peroxidation in chloroplast thylakoids of wheat under growth of the tissue. This response was also ob-
severe illumination; it was suggested that flavonoids served in the species with intermediate resistance,
acted by trapping the singlet oxygen produced in the but response time was longer in the last one.
biological reactions.96 Thus, it is clear that flavonoids play a key role in
the high resistance of V. rotundifolia and that
precocity in the resistance of Vitis sp. to P. viticola
d. Defense Mechanism in Plants is very important.111 Flavonoids of sour cherries
were extracted and assayed against the ascomycete
When attacked by pathogens, some plants shoot Leucostoma persoonii that causes the perennial
their flavonoid biosynthesis (phytoalexins, as those canker on the bark of peach, plum, and sweet
from Broussonetia papyrifera and Narcissus cherry. In general, all tested flavonoids slowed
pseudonarcissus); interestingly, these flavonoids are down the mycelial growth. It was shown to have
different to those induced under severe illumination. significant differences in the toxicity of aglycones
Sorghum (Sorghum bicolor) synthesizes the and glucosides, showing higher toxicity the agly-
phytoalexins apigeninidin and luteolinidin cones; therefore, suggesting glucoside hydrolysis
(deoxyanthocyanidin flavonoids) as a response to as a defense reaction. Naringenin and chrysin
attempted infection by the fungus Colletotrichum showed the highest fungistatic effects.167 Erwinia
graminicola that causes the anthracnose disease. It carotovora causes blackleg and soft-rot of pota-
was reported that susceptible plants lose the ability to toes by macerating activity in the host tissue. The
respond rapidly to fungal infection. It has been ob- main enzymes of this bacterium are pectate lyases
served that after synthesis of phytoalexins in inclu- (PL), and the transformation of potato with PL
sions within the cell under attack, the pigments re- was used as a protection mechanism. It was deter-
leased were observed to accumulate in the fungus.443 mined that transgene was expressed in different
The major rice pathogens are Xanthomonas tissues and tuber disks of transformants inhibited
oryzae pv. Oryzae (the bacterial blight pathogen), the Erwinia growth. Interestingly, a strong induc-
Pyricularia oryzae (the fungal blast pathogen), tion of PAL was observed in the transformant
and Rhizoctonia solani (the fungal sheath blight with the high inhibitory effect that was a marker
pathogen); several flavonoids with variable activ- for the activation of defense-related genes. Thus,
ity have been reported. Naringenin inhibited the the resistance might be based on the liberation of
growth of X. Oryzae, naringenin, and kaempferol, phenolic compounds and phenol oxidases.501
the spore germination of P. oryzae. It was estab- Isolated in Arabidopsis thaliana was a
lished that nonpolar flavonoids showed higher sulfotransferase cDNA (RaR047) that regulation
inhibition than their polar counterpart, but none is developmentally regulated and associated with
of the tested compounds showed inhibition to stages of active plant growth. RaR047 was in-
Rhizoctonia. Also, cyanidin and peonidin glyco- duced under stress or pathogen attack. RaR047
sides (anthocyanins) showed growth inhibition of showed a maximal expression in the incompatible
Xanthomonas. 354 Downy mildew caused by interaction between 3 and 7 days after inoculation
Plasmopara viticola (Berk et Curt.) is an impor- for Xanthomonas campestris pv. campestris and
tant disease threatening the world’s viticultural between 7 and 24 h after inoculation with
areas. Three Vitis sp.(V. vinifera cv. Grenache, Pseudomonas syringae pv. maculicola. It was sug-
susceptible; V. rupestiris cv. du Lot, intermediate gested that sulfotransferase could be involved in
resistant; and V. rotundifolia cv. Carlos, resistant) the molecular communication between the inter-
187
acting organisms; in particular, it was proposed cipally influence, the shoot growth of seedling,
that the RaR047 product participates in the syn- but germination and radical length can be af-
thesis of toxic compounds directed against the fected by chalcones. Tambulin did not show
pathogen during the resistance reaction.268 any effect against germination and radical length
Several studies have been carried out to induce of tomato and barley but inhibited shoot growth
virus resistance, and flavonoids have shown antivi- of tomato (approximately 25%) and barley
ral activity against tomato ringspot virus (22%). Kukulkanin B and heliannone A differ
(TomRSV). The most effective compounds were in the presence of an additional methyl group in
quercetin, quercetin 7,4′-dimethyl ether, quercetin heliannone A; however, their activities are quite
3,7,4′-trimethyl ether, and fisetin 4′-methyl ether, different. Heliannone A inhibited germination
causing a 67 to 76% inhibition of TomRSV infec- of tomato (20%) and barley (35%) and slightly
tivity when applied at 10 µg ml-1. Quercetin did not affected barley shoot growth length. On the
affect viral replication. When quercetin was used other hand, kukulkanin B only showed inhibi-
in combination with RNA, an increment in the tory activity on the shoot growth of tomato.290
infectivity was observed. Thus, it was concluded
that inhibitory effect of quercetin is dependent on
the presence of the coat protein for antiviral activ- e. Other Ecological Functions
ity, possibly by the inhibition of uncoating. Fla-

vonoids are proposed as a class of compounds with Studying the loblolly pine (Pinus taeda) needles,
potent antiviral activity that can be used to reduce it was shown that increments in catechin and
or eliminate viruses from plant material.294 On the proanthocyanidin concentration are generated by the
other hand, leucoanthocyanins are accumulated in effect of O3 exposure. It was suggested that these
some seeds and make them less attractive for the increments are an adaptive response by acting as an
consumption by animals or the attack of other antioxidant, and it was proposed that elevated cat-
pests. Additionally, these leucoanthocyanins pro- echin levels might also be a suitable biochemical
vide a seed protection against digestive enzymes, indicator of forest damage.51
permitting that after the excretion the seed can Flavonoids are important compounds used as
germinate. The flavan from Lycoris radiata bulbs ecological indicators.169 It has been reported con-
was found to be antifeedant for the larvae of the siderable quantitative and qualitative differences
yellow butterfly Euroma hecabe mandarina.193,259,455 of flavonoids in propolis and has been concluded
Studying the root bark and stem wood of Erythrina that such differences were dependent mainly on
sigmoidea, the methylene chloride extract was found plant ecology and the variety of the bee.261 More-
to be active against the Gram-positive bacteria over, it has been established that bee pollen can
Staphylococcus aureus. After successive purifica- be used to identify their plant origin, for example,
tions, it was isolated with the bioactive flavonoid the major compound in almond bee pollen is
neobavaisoflavone, which showed a good inhibi- 8-methoxykaempferol 3-glycoside, while jara
tory action compared with streptomycin sulfate.346 bee pollen contained mainly quercetin
Also, it has been established that proanthocyanidins and isorhamnetin 3-glycosides. Thus, flavonoids
are the main components of tannin. In particular, can be used as a markers of pollens.470 The fla-
bark procyanidins are dominant (Monterrey pine vonoid profile has been used to evaluate the evo-
50%, Scots pine 75%, and birch 65%), being lution in the Phaseolinae: it was determined that
prodelphinidins in a higher proportion. It has been kaempferol and quercetin glycosides are the main
suggested that proanthocyanidins contribute to pro- flavonoids, whereas the glycosidic pattern was
tecting internal plant tissues (phloem, cambium, 3,7-O-diglycoside and 3-O-glycoside. These fea-
and xylem) against invasion by pathogens because tures represent intermediate to advanced evolu-
they are particularly abundant in the more exposed tionary characters among the Phaseolinae fla-
outer bark.302 vonoids. The flavone frequency was higher than
It was proposed that flavonoids isolated in other subtribes, thus indicating a highly ad-
from Helianthus annuus possibly play roles in vanced position in the subtribe Phaseolinae within
the allelopathic activity of sunflower. They prin- the subfamily Papilionoideae, which was sup-
188
TABLE 1
Pharmacological Activities of Some Flavonoids
Flavonoid Activity
Rutin, silibin Against disorders of the respiratory system

Naringenin Antimicrobial and antifungic on skin
Quercetin, morin, procyanidin, pelargonidin Antiviral
(+)-Catechin, 3-O-Metil-(+)-Catechin, Against ulcers
naringenin
Butrin, isobutrin Hepatic disorders and viral hepatitis
Chisin, floretin, apigenin, quercetin, Antiinflammatory
kaempferol, baicaelin
Epicatechin Against diabetes
Genistein, kaempferol, sophoricoside Antifertility
Galangin Activity against Staphylococcus epidermis
Proanthocyanidins Astringent for digestive system, diuretic, cardiac
tonic, in the treatment of high blood pressure
Adapted from Refs. 10, 198, 509.

ported by the C-glycosylation and C-acylation rieties, and is especially used as indicator of grape-
profiles, and it was concluded that flavonoids of fruit addition to orange juice.398
these Phaseolinae species correspond to an inter-
mediate evolutionary level.522
On the other hand, leaf waxes of 32 Aeonium f. Pharmacological Effects
species were obtained, and 32 flavonoids were
identified as methyl ethers of kaempferol, 6- It has been clearly established that flavonoids
hydroxykempferol, quercetin, myricetin, and in natural products (e.g., grape, soybean, peanut,
scutellarein (6-hydroxyapigenin). It was suggested wine, tea) have a good antioxidant activity. In
that waxes of different plants of the same species some instances a better activity than in the com-
contained the same principal flavonols, with in- mercial antioxidants has been determined.159 Ad-
traspecific variation limited to the minor fla- ditionally, flavonoids contribute to the protection
vonoids. It was determined that in the Aeonium and/or regeneration of antioxidants. It has been
genus the occurrence of myricetin methyl ethers suggested that the flavonoid action is by trapping
and 6-hydroxykaempferol methyl ethers is taxo- free radicals and that flavonoids are antimutagenic
nomically most significant.450 In addition, fla- that can reduce the atherogenesis and the risk for
vonoids have been used to characterize wines, for strokes, and that are modulators of arachidonic
example, in Pinot Noir wine, the highest concen- acid metabolism. This last point involves fla-
trations of catechin and epicatechin were found, vonoids in many metabolic processes.159,198,338
while Cabernet Sauvignon shows quercetin (typi- Interestingly, flavonoids are ubiquitous of plants
cal of red wines).444 Citrus has been studied by and a person can consume up to 1 g of them. This
considering their flavonoid composition, which quantity implies that flavonoids can reach phar-
are quantitative fingerprints of the juices: the pre- macological concentrations in the organism. Thus,
dominant flavonoids in lemon were hesperidin it is interesting to point out that many flavonoids
and eriocitrin, which were also present in addition have assigned pharmacological activities (Table
to neoponcirin in lime, while pummelo contains 1), and that many medicinal plants are known and
almost exclusively naringin. In grapefruit, the used by their flavonoid content. Moreover, it has
profile was more complex with neohesperidoside been determined that flavonoid structure has the
as the main component. Based on this informa- responsibility of a large part of the activity. Also,
tion, naringin is used as a chemotaxonomic marker it has been shown that different substitutes give a
to distinguish sweet orange from other citrus va- molecule with an specific polarity that permits
189
the presence of privileged bonds in the active act at the level of the union of topoisomerase
sites.10 This characteristic could have a relation enzymes with DNA. Flavones can either stabilize
with the antioxidant activity, because it is known the catalytic topoisomerase I-DNA intermediate
that variations in glycosylation and/or acylation or inhibit the DNA binding of the free enzyme.
induce differences in the activity and stability of Thus, in view of the relatively low toxicity of
flavonoids.233 From an acetone extract of the root quercetin and related antitumor activity, its use as
bark of Ormosia monosperma several flavonoids an anti-cancer drug seems feasible.43
were isolated, and it was observed that It is well known that iron could induce the
2,3-dihydroauriculatin showed a moderate activ- generation of harmful reactive species and that
ity against oral microbial organisms such as Strep- special proteins are required to capture iron (trans-
tococcus mutants, Porphyromonas gingivalis, and ferrin, ferritin, heme proteins) and to prevent its
Actinobacillus actinomycetemcomitans, each at reaction with oxygen species. Released iron in-
6.3 µg ml-1. 234 From Ononis spinosa subsp. duces the peroxidation of membrane lipids and
Leeiosperma, two flavonoids with biological ac- hemolysis, and it has been established that quer-
tivity were isolated: spinonin showed a moderate cetin inhibits the harmful effect of free iron prob-
activity against P. aeruginosa and higher activity ably by forming a complex with it (in assays with
was obtained with ononin against β-hemolytic erythrocytes). Interestingly, it has been suggested
Streptococcus (minimal inhibitory concentration that the release of iron in a redox-active form
or MIC = 25 µg/ mL).256 It was shown that some correlated with the generation of senescent anti-
flavonoids (isorhamnetin, rhamnetin, and querce- gen (the antigen appearing on aged erythrocytes),
tin) diminished the total serum cholesterol levels and quercetin induces the chelation of iron at
when rats were fed with these compounds; athero- intracellular level and delays the formation of
genic index, bile acids, serum triacylglycerols, senescent antigen, prolonging the storage time of
and thiobarbituric acid-reactive substances blood in blood stores.145 Also, it was shown that
(TBARS) tended to low as well. Flavonoids in- an hydroxyl group at C-3 and carbonyl at C-4 of
hibited the generation of superoxide anion (higher the C-ring are necessary to bind iron. From ethyl
with rhamnetin) and the oxidation of linoleic acid acetate extracts of peppermint, sage, and thyme,
(up to 84% with quercetin). The activities of su- luteolin was identified as the main active com-
peroxide dismutase and xanthine oxidase did not pound, showing desmutagenicity against
vary, thus the flavonoid abilities could be ascrib- Trp-P-2. It was suggested that mutagenicity gen-
able in part to the scavenging of free radicals and erated from consumption of 1 g of broiled meat
in particular to their antioxidative activities.198 could be mitigated with very small amounts of
Studies on oregano extracts showed that these herbs: 2.8 mg of peppermint, 13 mg of sage,
galangin and quercetin are active desmutagens or 0.9 mg of thyme. A correlation was identified
against the mutagenicity of Trp-P-2 (3-amino-1- between a desmutagenic mechanism of flavonoids
methyl-5H-pyrido[4,3-b]indole), a liver-specific and their antioxidant potency.413 Also, it was sug-
carcinogen, and other mutagens and it was suggested that the choleretic, antirheumatic, and di-
gested that flavonoid desmutagenicity plays an uretic activities of dandelions could be related
important role in cancer prevention; however, it is with the content of luteolin glycosides (7-gluco-
convenient to mention that under certain condi- side and 7-diglucosides).506 Also, luteolin glyco-
tions these flavonoids did not inhibit mutagenic- sides have been isolated from Vitex agnus-castus
ity, that is, in the presence of a buffered mixture (e.g., luteolin 6-C-[4′′-methyl-6′′-O-trans-
of polychlorinated biphenyls, essential cofactors, caffeoylglucoside]; luteolin 6-C-[6′′-O-trans-
NADP, and glucose-6-phosphate to form the S9 caffeoylglucoside]) and these compounds showed
mix.249 It has been reported that quercetin or its cytotoxic activity against P388 lymphocytic leu-
analogs could be interesting substitutes of kemia cells IC50 (values were 0.1 for 4′ to
camptothecin, which is used in the treatment of 5-dihydroxy-3,3′,6,7-tetramethoxy-flavone and
cancer and produces undesirable secondary reac- 0.31 for luteolin).214 Trypsin and leucine ami-
tions. It was proposed that all of these substances nopeptidase have been implicated in a variety of
190
pathological process such as acute pancretitis, in- vone) showed activity against the human immuno-
flammatory process, various cancers, etc. Inter- deficiency virus (HIV) reverse transcriptase (HIV-
estingly, most flavonoids showed inhibitory prop- 1 RT). It was indicated that biflavones with two
erties against trypsin aminopeptidase (strongest apigenin units linked either with C-C or C-O-C
inhibition with quercetin and myricetin), but only bonds (robustaflavone, hinokiflavone) exhibited sig-
3′,4′-dihydroxyflavone and quercetin inhibited nificant inhibitory activity. However, in a whole
leucine aminopeptidase. From the studies with cell assay (human PBM cells infected with HIV-1
the trypsin aminopeptidase inhibition, it was es- strain (LAV-1) morelloflavone exhibited potent
tablished that hydroxyl groups at C-5 and C-7 in inhibitory activity (promising anti-HIV activity),
ring A are essential, while a double bond at posi- while it possessed moderate activity in the HIV-1
tion C-2, C-3 in ring C and hydroxylation at both RT, emphasizing the importance of cellular mecha-
the C-3′ and C-4′ enhance the inhibitory activity. nisms in flavonoid action. It was concluded that the
On the other hand, inhibition of leucine ami- presence of an unsaturated double bond at C-2, C-
nopeptidase by flavonoids requires of a double 3 and three hydroxyl groups at C-5, C-6, and C-7
bond at C-2, C-3.358 L868276 is a synthetic ana- positions were prerequisites for inhibition of RT.284
log of flavopiridol that is obtained from Dysoxylum From the ethanolic extract of the stem bark of
binectariferum. These substances are used to in- Mitrella hentii, it was isolated linderatin. This com-
hibit the growth of breast and lung carcinoma cell pound was tested in vitro on a non-small-cell bron-
lines by its effect on the cyclin-dependent kinase chopulmonary lung carcinoma type of cancer that
activities. Interestingly, it has been shown that represents 80% of all human bronchopulmonary
flavopiridol do not inhibit other protein kinases.113 cancers that is highly chemoresistant to medical
Brysonima crassifolia (known in México as treatment. It was reported that only (-)-linderatin
nanche) extracts have been used in traditional exhibited a significant activity toward this type of
medicine by Mixe Indians in the treatment of gas- carcinoma (IC50 = 3.8 µg mL-1).34 It is interesting
trointestinal disorders and skin infections, and in to mention that quercetin or its structural analog
vitro assays have shown that ethanolic extracts fisetin) enhanced the estradiol-induced tumorigen-
inhibited the nematode multiplication (IC50 = 175 esis in hamsters. The effect of quercetin was medi-
ppm). In this extract the main components were ated by increased levels of S-adenosyl-L-homocys-
proanthocyanidins.168 In Brazil, decoctions or infu- teine (SAH) which inhibited the methylation of
sions of Stryphnodendron adstringens are used tra- 2- and 4-hydroxyestradiol by catechol
ditionally in the treatment of leukorrhoea, diar- O-methyltransferase. Also, it was shown that low
rhoea, and as antiinflammatory. An acetone-H2O renal pools of S-adenosyl-L-methionine contrib-
extract was analyzed and prodelphinidins with uted to this phenotype.527 The estrogenic activity of
bioactive pyrogallol units (epigallocatechin deriva- isoflavonoids has been reported, and
tives) were identified and proposed as the pharma- 7-isopropoxyisoflavone is now sold on the market
cological active compounds.122 In Guazuma (‘Osten’ from Takeda Chemical Industries, Ltd.)
ulmifolia, which is used in the treatment of diar- as a therapeutic drug for osteoporosis. Also, it has
rhoea by the Mixe Indians were identified been reported that daidzein increased the cell num-
procyanidins (epicatechins). These compounds in- ber of mouse osteoclasts at a very low concentra-
activate the cholera toxin, and toxin-binding activ- tions.466 One of the main flavonoids in teas is
ity increased with their molecular weight.219 Also, scutellarein, which has been used as a diuretic,
flavonoids have been involved in the inhibition of antiinflammatory, and antiasthmatic drug.218 The
several individual steps related to the thrombosis wide spectrum of resistance of cancer cells to natu-
process, and one of the most active compounds ral agents has been assigned in part to the
was the biflavonoid hinokiflavone. The activity of overexpression of proteins that belongs to the ATP
this flavonoid has been suggested that is mediated binding cassette transporter proteins (ABC).
by the inhibition of induction of tissue factor Multidrug resistance protein (MRP), pertains to
expression by interleukin-1.270 Other biflavonoids ABC proteins, has been cloned from a drug-resis-
(e.g., amentoflavone, agathisflavone, robusta- fla- tant small cell lung cancer line. Several flavonoids
191
(genistein, kaempferol and flavopiridol) have shown quinone reactions only one is common to all of
inhibition of MRP-mediated transport of anti-can- them, a reversible reduction. By this reaction,
cer drugs by a direct interaction of flavonoids on quinones participate in the redox reactions of the
the active site of MRP. Additionally, it has been organisms where they are present. Ubiquinone
suggested that a glucose moiety in the flavonoid and plastoquinone are essential components of
structure plays an important role for its activity.218 the electron transport in mitochondrial membranes
Flavonoids have also been proposed in the (ubiquinone) and chloroplasts (plastoquinone).
treatment of obesity. Extracts of Cassia nomame Ubiquinone is an hydrophobic compound of low
have lipase-inhibitory activity, and it was deter- molecular weight, and it can be found in its re-
mined that (2S)-3′,4′,7-trihydroxyflavan-(4α→ 8)- duced or oxidized state: ubiquinone is lipid soluble
catechin has a significant inhibition of the porcine and can diffuse through the membranes to act as
pancreatic lipase (IC50 = 5.5 µM). However, inter- an electron carrier between the components of the
estingly, an oligomeric fraction showed the most respiratory chain. Plastoquinone has similar char-
potent inhibitory effect (IC50 = 0.20 µM). This acteristics to ubiquinone, and it is involved in the
oligomer was mainly composed of 3′,4′,7- electron transport between PSI and PSII in the
trihydroxyflavan and catechin.199 photosynthetic process.196,464
Moreover, it has been reported that several
enzymes have a quinone as cofactor. Methanol

4. Quinones dehydrogenase has a pyrroloquinoline quinone
(PQQ) as cofactor, and also other bacterial dehy-
As colorant substances, quinones are not very drogenases are quinone dependent (quinoproteins).
important. They only provide coloration in some These enzymes are involved in the oxidation of
higher organisms and microorganisms: in mem- substrates such as alcohols, amines, and sugars.
bers of the Echinodermata family, quinones con- Additionally, it has been found that some copper-
tribute to the pigmentation of spines, shell, ova- dependent amino oxidases (AO) have an alternate
ries, and eggs. In microorganisms, Polyporus cofactor called topaquinone (TPQ). Several me-
rutilans is a fungus and accumulates up to 23% thyl-amine dehydrogenases have the tryptophan
dry weight (d.w.) of polyporic acid, a terpenyl tryptoquinone (TTQ) as a cofactor. TPQ is a cofac-
quinone of bronze color. Helminthosporium tor ubiquitous in bacteria, yeasts, plants, and mam-
gramineum reaches up to 20% d.w. of quinones mals. Bacterial and yeast TPQ enzymes permit
(islandicin, crisophanol, emodin). Some bacteria them to grow in different amines as a nitrogen
produce good quantities of quinones; Streptomy- source, and in some instances as source of carbon.
ces coelicor accumulates up to 15% d.w.464 In plants, the main function of the AO is in the
On the other hand, some quinones have been production of hydrogen peroxide to heal wounds
used as food colorants at the industrial level. Rubia by the induction of new cell wall. Moreover, it has
tinctoreum Linn. is obtained from an extract com- been suggested that plant AO participate in the
posed of quinones (alizarin, xanthopurpurin, growth process by the regulation of polyamine
rubiadin, purpurin, etc.). The peel of Coprosoma levels. The AO functions in animals are more elu-
acerosa A. contained 3-hydroxi-2-methyl-an- sive and diverse. The well-defined enzyme is the
thraquinone, methyl-ether of rubiadin, and lucidin, mammal lysyl-oxidase that catalyzes the connec-
etc. From the root of Rumex chinensis is obtained tive tissue maturation by the crossover of elastin
a denticulate, which in China is used as a substi- and collagen. On the other hand, some evidence
tute of rhubarb. However, the most important indicates that free PQQ has other functions in eu-
quinones at the industrial level are the anthraquino- karyotes: in the production of superoxides in
nes: carminic acid is obtained from the Mexican neutrophiles (neuroprotector); prevents cataracts
species of Dactylopuis coccus; carmesic acid is by exposure to cortisone; edemas induced by car-
obtained from Laccifer lacca Kerr.464 rageenans; liver injury by hepatotoxins (carbon
Quinones are very reactive compounds due tetrachloride). In plants, it has been shown that
their structural characteristics. Between all the PQQ stimulates 5 to 10 times the conversion of
192
acetyl-CoA to hydroxymethylglutaryl-CoA (HMG- By considering the pharmacological effect, it
CoA).258 However, new methodologies for the as- has been found that allomelanins (free of pro-
say of PQQ (bioassay and GC-MS) have shown teins) from plants, black bean, soybean, and
their presence in Gram-negative bacterium, and it sesame suppress the growth of tumorigenic cells
has been reported in only one Gram-positive bac- of animals and mammals. Also, it has been men-
teria (Amycolatopsis methanolica). However, as tioned that melanin inhibits the in vitro infection
such, it was concluded that PQQ is not present in of the lymphocyte cell line by the HIV-1 virus.
materials obtained from plants and animals, conse- However, the free radicals in melanin molecules
quently, previous reports about their presence in could produce a toxic effect in cells or the growth
these organisms must come from contamination.323 inhibition by melanin association with minor el-
Defense mechanisms. Streptomyces sp. pro- ements such as iron and copper. It has been re-
duces the antibiotic tetracycline. Many insects ported that soluble melanins and their pre-
(Dyctiopters, Diplopodes, and Opiplions) produce cursors (catecholamines, 3-hydroxyquinurenine,
alkylbenzenequinones as a defense mechanism. The 3-hydroxyanthranilic acid, catechol) can induce
African tree Mansonia altissima excretes mansonons degenerative processes in connective tissues, blood
that protect it from fungi and insect attack.465 In vessels, heart valves, liver, and other tissues, simi-
addition to the pharmacological contributions men- larly to that observed in the illness called alcapto-
tioned above, it has been reported that several natu- nuria. Parkinson’s disease and the normal aging
ral laxatives rhubarb, senna) have mixtures of an- process are characterized by the loss of pigmented
thraquinones and anthrones of the emodine type, neurons in nigra substance, which correlates with
and vitamin K functions as a cofactor of the the accumulation of neuromelanin and with the
prothrombines (coagulation factor). At the indus- oxidative conditions in that brain region, and it
trial level, skikonin is the most important quinone has been hypothesized that neuromelanin acts as
with pharmacological activity, and it is produced by a protector under oxidative stress in normal indi-
tissue cultures of Lithospermum erythrorhizon.196,464 viduals, but in those with Parkinson’s this mol-
ecule has the potential to exacerbate the oxidative
conditions through the generation of hydrogen
5. Iridoids peroxide or through the liberation of active met-
als for the redox process. This information em-
These compounds are not particularly impor- phasizes the importance of protection by antioxi-
tant as colorants and their relevance, until a few dants in the normal human metabolism, in diseases,
years ago, had been restricted to be taxonomic or in the aging process.32,248
markers. Iridoid glucosides are only found in more
advanced dicotyledonous plants. Remarkably, seco-
iridoids are biosynthetic precursors of alkaloids.406 C. Importance as Food Colorants
1. Reasons to Use Color Additives

6. Melanins
Color is associated with many aspects of our
In general, melanins are not essential for the life. For example, the main factors to evaluate
growth and development but induce an increment food quality are color, flavor, and texture, but
in the possibilities of organism survival by acting color can be considered the most important of
as a defense mechanism. Melanins have been also them, because if it is not appealing consumers
associated with the immune response. It has been will not enjoy the flavor and texture of any given
observed that rice resistance to Piricularia oryze food. Color may as well give the key to cataloge
is associated with the induction of phenol oxi- a food as safe, with good aesthetic and sensorial
dase/polyphenol oxidase and the synthesis of characteristics: the undesirable colors in meat,
melanin. In fungi, melanins provide resistance to fruits, and vegetables warn us about a potential
stress conditions.32,64,456,465 danger or at least of the presence of undesirable
193
flavors, among other reactions. Many studies have problems. Lead chromate and copper sulfate were
emphasized the relation of color with the flavor used to pigment candies and sauerkraut, but in
detection threshold, with the sweetness or salinity the pigmentation process arsenic and other ven-
sensations, with the susceptibility and preference omous impurities were added frequently. Also,
for products.80,162,339 Nowadays, people know that in that epoch began the use of tar colorants and
foods have their own natural colors, but most other petroleum derivatives in the processing of
people live in big cities, far from the places of foods, medicines, and cosmetics.144 Synthetic
food production, and processing and transporta- colorants have been used for many years, in
tion of foods are required. During such events, 1938 it was recognized the use of approximately
foods may lose some of their properties and some 200, in 1970 to 1965, and nowadays only seven
additives must be added to permit such products can be used in food pigmentation.144,150 How-
having desirable appearance and are safe for con- ever, in the last 30 years synthetic additives have
sumption. Thus, additives are used to emulate been severely criticized, and consumers show a
colors that are found in natural products such as phobia toward these products and consequently
pepper, red beet, grapes, saffron, meat, and people prefer the natural colorants.157,161 In the
shrimp.58,144 Moreover, it is possible to enumerate period 1960 to 1970 in the U.S., the environ-
the reasons to use food colorants:150 mental activist movements attacked the food
additives, and this attitude was converted in a

1. To restore the original food appearance world-wide phenomenon. In general, this move-
because of the occurrence of changes ment was against the technologies that have a
during processing and storage. harmful effect on the environment, and they used
2. To assure the color uniformity to avoid a very particular association between food and
variations in tone color by seasonal varia nutrition. The criticism of these groups was fo-
tions. cused on the fast food and was difficult for them
3. To intensify colors that are normally found to attack these products as a group. However,
in food and the consumer will associate colorants were an easy target; they indicated that
this improved color with food quality. pigments only have a cosmetic value, and, on the
4. To protect the flavor and light susceptible other hand, they could cause damage. The activ-
vitamins. ist consumers reviewed the concepts health and
5. To give to food an attractive appearance, found it harmful to many foods and diets, and
whereas without the additive food will companies began with the aim of producing
not be an appetizing item. healthier foods. They started using the nutri-
6. To preserve the identity or character by tional characteristics as a sale tool, a strategy
which food is recognized. that has previously failed, but after the social
7. To help in the visual assignation of the movement was converted into a total success;
food quality. thus, a world-wide tendency to use natural
colorants was generated. At the present time,
most people interpret the content of chemical
2. Importance of Natural Colorants products as a contaminant and the tendency has
been reinforced, and all seems to indicate that it
Since the early civilizations, natural prod- will continue to in the future.158 To emphasize
ucts were used to give an attractive presentation this situation, it should be pointed out that up to
to man-made products. Saffron and other spe- 1986 356 patents on natural colorants have been
cies were used frequently to provide yellow color registered and for the synthetics only 71.156 Nowa-
in a variety of foods and evidence exists that days, the number of advantages of natural over
butter was pigmented with these products. The synthetic colorants have been increased because
first reports regarding the use of derived colors of the pharmacological properties of the natural
of minerals dated from the ninteenth century; pigments. In 1994, the market of natural colorants
however, some of them caused serious health had an estimated value of $250 million U.S.
194
dollars, and 65% of this corresponded to color gate the safety of these products before issuing any
additives for food, with an annual growth rate of regulation. With the 1906 act, the control on the
5 to 10% in relation to 3 to 5% for the synthetic color additives was greater than on the other addi-
pigments. However, it is necessary to note that tives. In 1938, the Food, Drug & Cosmetic Act
synthetic colorants have well-known advantages (FD&C) was established and published a list of tar
over the natural ones based on the higher pig- colorants that could be used as food additives; also,
menting power, stability, storage, facility in the it was decreed that the FDA had the authority to
processing, and they are cheaper and available certify the color lots. With this, color additives
in unlimited quantities.507 On the other hand, were the first substances that must be revised be-
some products have a good market value only if fore their commercialization. In the first years,
they are colored with natural products: in the synthetic colors were prohibited as health-harmful
manufacture of Cheddar cheese, only the annato products. The next important change came in 1958;
pigment is used;161 in the pigmentation of poul- additives were redefined and a new classification
try products, synthetic colorants are not ad- with three categories appeared: (1) substances ap-
equate.297 With the information mentioned above proved by the FDA or the USDA (United States
and by considering the large number of papers Department of Agriculture) during 1938 to 1958
about natural colorants, one tends to believe that (prior-sanctioned substances); (2) substances that
the area of these food color additives is very are Generally Recognized as Safe (GRAS sub-
active, but this is a mistake because most of stances), which did not require of a previous FDA
them are not applicable at the industrial level evaluation to be commercialized; and (3) all the
because they have not covered the economical, remaining added substances in the food supply
legal, and safety requirements that most govern- food additives), which must be evaluated by the
ments have established for food additives. Nowa- FDA before commercialization.
days, the number of approved colorants for food All new and color additives were included in
industry is very limited, and this phenomenon is the last category. Moreover, in 1958 included was
observed practically all over the world (Table the Delaney clause “no additive shall be deemed to
2), probably with the exception of Japan. be safe if it is found to induce cancer when ingested
Colorants produced by the Monascus fungi are by man or animal, or if it is found after tests which
well-known pigments. Traditionally, Monascus are appropriated, for the evaluation of the safety of
pigments are obtained from fermented products food additives, to induce cancer in man or animal”.
of rice and bread and have been used as food In 1960 and with the FD&C amendment, Congress
colorants and medicinal agents. Interestingly, established that all color additives required the
iridoids and Monascus pigments have remark- prior FDA approbation for their commercializa-
able properties, such as a good range of colors tion. It may appear that it did not difference existed
and stability, that can be used as food colorants; between this and the regulation before 1958. How-
however, none of these groups are approved in ever, the new regulation also included prior-sanc-
neither the U.S. nor in the European Union.58 tioned colors (before 1958). When the 1900 act
was published in 1912, 80 tar-derived colors had
been used in food industry, the number was re-
D. Some Regulatory Aspects About duced to 12 after the FD&C act, and nowadays
Color Additives only 7 are approved. Additionally, color additives
have another disadvantage, that is, all must be
Color additive regulation has an older history considered as additives and not as GRAS sub-
than any other additive regulation, with the excep- stances, thus the regulations for color additives are
tion of that of preservatives. These two kinds of stronger than for other additives, and certainly up
additives were the subject of a special legislation to date there exists no scientific basis to establish
by the U.S. Congress in the act of 1900 on colors differences between color and other additives.507,508
and preservatives. In this act it was pointed out that On November 9 1990, an act was approved in
the government had the responsibility to investi- which the producers are obliged to label all products
195
TABLE 2
Approved Colors for Food Industry in the European Union and in the Food and Drug
Administration (FDA) of USA
Color CEE FDA
Certifiable
Red allure AC No Yes (red #40)
Brilliant blue No Yes (blue #1)
Carmosine Yes (E122) No
Eritrosin Yes (E127) Yes (red # 3)
Fast green FCF No Yes (green # 3)
Indigotine Yes (E132) Yes (blue # 2)
Ponceau 4R Yes (E124) No
Sunset yellow FCF Yes (E110) Yes (yellow # 6)
Tartrazine Yes (E110) Yes (yellow # 5)
Polymeric colors No No
Citric red # 2 No No
Exempt of certification
Annato extract Yes Yes

Dehydrated red beet Yes Yes
Ultramarine blue Yes
Canthaxanthin No Yes
Caramel Yes Yes
β-apo-8'-carotenal No Yes
β-carotene Yes Yes
Dactylopuis coccus extract Yes Yes
Meal of cotton seeds Yes Yes
Iron glutamate No Yes
Skin grape extract Yes Yes
Iron oxide No Yes
Fruit juices Yes Yes
Vegetable juices Yes Yes
Algae meals No No
Tagetes and extracts Yes? No
Carrot oil Yes Yes
Oil of corn endosperm Yes Yes
Paprika Yes Yes
Paprika oleoresin Yes Yes
Riboflavin Yes Yes
Saffron Yes Yes
Titanium dioxide No Yes
Turmeric Yes Yes
Turmeric oleoresin Yes Yes
Chlorophyll Yes No
Xanthophylls, flavoxanthins, rubiaxanthin, Not all Not all
zeaxanthin and other natural products with
some of these carotenoids
under certain regulation. With respect to color, the suspicious about synthetics. On the other hand, the
act established that all certified colors used in foods exemption of certification additives are from com-
must be indicated in the label, but the exempt of mon natural products: red beet, carrot, fruits, pepper,
certification (this includes the accepted natural pig- among others, and regulation could be expected to
ments) must be generically grouped as colorants. be less severe for these products; however, it is clear
This act gave a clear advantage to the use of natural that FDA policies do not share this idea, and the
over synthetic colorants, because consumers are research for natural colorant sources is uncertain
196
because of the difficulty in getting FDA approval. In 2. Classification
this respect, the history of color additive amendment
is disastrous; red #40 is a colorant that was devel- Carotenoids are classified by their chemical struc-
oped in the 1960s and approved by FDA in 1974, ture as: (1) carotenes that are constituted by carbon and
the only synthetic colorant approved in the last 75 hydrogen; (2) oxycarotenoids or xanthophylls that have
years that it is still used). In general, the approbation carbon, hydrogen, and, additionally, oxygen.
process is expensive, a synthetic colorant requires Also, carotenoids have been classified as pri-
$2.5 millions dollars only for the FDA evaluations, mary or secondary. Primary carotenoids group
and in the European Union about $1 million dollars. those compounds required by plants in photosyn-
Nevertheless, the regulations for the GRAS sub- thesis (β-carotene, violaxanthin, and neoxanthin),
stances are very successful: olestra lipid substitute) whereas secondary carotenoids are localized in
was invented in 1968, this product was cataloged as fruits and flowers (α-carotene, β-cryptoxanthin,
an additive and it was approved by the end of 1996, zeaxanthin, antheraxanthin, capsanthin,
28 years after its invention; on the other hand, the capsorubin).279 Some carotenoid structures are
inventors of the high fructose corn syrup (HFCS) presented in Figure 7.
decided that their product was a GRAS substance,
thus HFCS was immediately commercial-
ized.144,229,310,507,508 Additionally, FDA uses the term 3. Distribution
indirect additives to group those additives used in

the pigmentation of animal meals, and finally ani- Carotenoids are the widest distributed group
mals will be used as human food. Something that it of pigments. They have been identified in photo-
is not easy to understand.161 synthetic and nonphotosynthetic organisms: in
Undoubtedly, it is technologically feasible to higher plants, algae, fungi, bacteria, and at least in
obtain new colorants from plants and microorgan- one species of each form of animal life. Caro-
isms, but the greater obstacles for the introduction tenoids are responsible for many of the brilliant
of new colorants are (1) the current legislation; (2) red, orange, and yellow colors of fruits, vegetables,
cost of manufacture, which includes that of the fungi, flowers, and also of birds, insects, crusta-
biotechnological culture or production; and (3) the ceans, and trout.179,180,182,196,510 Only microorgan-
acceptance of unknown materials by consumers.507 isms and plants can synthesize carotenoids de
novo; carotenoids in animals come from these
two sources, although they can be modified dur-
IV. SOME IMPORTANT PLANT ing their metabolism to be accumulated in tis-
PIGMENTS: CAROTENOIDS, sues.179 More than 300 carotenoids have been
ANTHOCYANINS, AND BETALAINS identified up to 1972, and around 600 up to 1992.
Actually, this number has been exceeded by con-
A. Carotenoids sidering that many carotenoids have been isolated
from marine organisms.477 Total production of
carotenoids in nature has been estimated at 108
ton/year, most of which is concentrated in four
1. Definition carotenoids: fucoxanthin, in marine algae; and
lutein, violaxanthin, and neoxanthin, in green
In general, carotenoids are compounds comprised leaves.200
of eight isoprenoid units (ip) whose order is inverted
at the molecule center (Figure 6). All carotenoids can
be considered as lycopene (C40H56) derivatives by a. Higher Plants
reactions involving: (1) hydrogenation, (2) dehydro-
genation, (3) cyclization, (4) oxygen insertion, (5) Carotenoids are accumulated in chloroplasts
double bond migration, (6) methyl migration, (7) of all green plants as a mixture of α- and
chain elongation, (8) chain shortening.179 β-carotene, β-cryptoxanthin, lutein, zeaxanthin,
197
FIGURE 6. Carotenoid structure. (Adapted from Ref. 179.)
violaxanthin, and neoxanthin. These pigments are ated carotenoids, frequently 5,8-epoxydes; (2) prin-
found as complexes formed by a noncovalent bond- cipally β-carotenes; and (3) carotenoids that are
ing with proteins. In green leaves, carotenoids are species specific (e.g., eschscholzxanthin in pop-
free, nonesterified, and the composition depends pies). Fruits are yet more prodigious in their syn-
on the plant and developmental conditions. Some thetic ability than flowers. More than 70 character-
leaves of gymnosperms accumulate not very com- istic carotenoids have been described and have
mon carotenoids in oily droplets, which are been classified as those with minimal quantities,
extraplastidial: rhodoxanthin in some members of higher quantities, and specific carotenoids, for ex-
the families Cupressaceae and Taxaceae, and semi- ample, capsanthin and capsorubin in pepper
β-carotenone in young leaves of cycads. In repro- fruits.180,181,279 Interestingly, carotenoids have been
ductive tissues the following have been found: identified in wood: samples of oak (Quercus robur
liliaxanthin in white lily and crocetin in Crocus sp. L., Quercus petrae Liebl., and Quercus alba L.),
stigmas; in flowers more than 40 pigments exclu- chestnut (Castanea sativa Mill.), and beech (Fagus
sive of petals have been identified. Flowers have silvatica L.) were studied at different ages and
been identified that synthesize: (1) highly oxygen- sections. Lutein and β-carotene were identified in
198
FIGURE 7. Structures of common carotenoids.
199
oak wood and also in others deciduous species. harvesting complexes or reaction centers in mem-
These carotenoids could be the origin of β-ionone branal systems of bacterial cells; and (5) usually
and more than 30 other norisoprenoid substances structural elements are not found, that is, allenic
identified in oak wood. Considering that carotenoids or acetylenic bonds, epoxydes, furanoxides C45 or
are hydrophobic and not soluble in sap, it is sug- C50 carotenoids. In vivo, one of the main groups of
gested the in situ formation of carotenoids in the carotenoids are the sulfates of eritoxanthin sulfate
living cells is in the sapwood. It was reported that and of caloxanthin sulfates. The sulfates of caro-
sapwood was richer in β-carotene than lutein, and tenoids are not associated with pigment-protein
the ratio was reversed in the heartwood. Also, it complexes, for example, they are neither part of
was found that lutein could be used as a marker to the light harvesting complexes nor of the reaction
distinguish between the wood samples.299 centers. In non-photosynthetic bacteria, caro-
tenoids appear sporadically and when present,
they have unique characteristics, for example,
b. Algae some Staphylococcus accumulate C30 carotenoids,
flavobacteria C45 and C50, while some mycobacte-
Carotenoids are in chloroplasts as com- ria accumulate C40 carotenoid glycosides.180
plex mixtures that are characteristic of each
class; the exceptions are Chlorophyta caro-

tenoids, which have a tendency to accumulate d. Fungi
the pigments characteristics of higher plants.
The red algae Rhodophyta have α- and Carotenoid distribution in fungi, non- pho-
β-carotene and their hydroxylated derivatives. tosynthetic organisms, are apparently capricious, but
In the Pyrrophyta, the main pigments are they usually accumulate carotenes, mono-
peridinin, dinoxanthin and fucoxanthin. and bi-cyclic carotenoids, and without
Chrysophyta accumulates epoxy-, allenic-, and carotenoids with ε-rings. For example,
acetylenic-carotenoids, and between them fu- plectaniaxanthin in Ascomycetes and canth-axanthin
coxanthin and diadinoxanthin. Eutreptielanone in Cantharellus cinnabarinus has been found.180
has been found in Euglenophyta. The princi-
pal carotenoids in Chloromonadophyta
are diadinoxanthin, heteroxanthin, and 4. Biosynthesis: Biochemistry and
vaucheriaxanthin. Chryptophyta is character- Molecular Biology
ized by their acetylenic carotenoids, for ex-
ample, alloxanthin, monadoxanthin, and
crocoxanthin. While the Phaeophyta is char-
acterized by its main pigment, fucoxan- a. Biochemistry
thin.180,181
Carotenoids as terpenoids are synthesized by
the isoprenoid pathway (Figure 8).185 Most of the
c. Bacteria pathways have been elucidated, but more infor-
mation is required about the involved en-
Approximately 80 different carotenoids are zymes.179,180,181 Isopentenyl pyrophosphate (IPP)
synthesized by photosynthetic bacteria. Usually, is the common precursor of many of the iso-
the characteristics of the accumulated carotenoids prenoid compounds (Figure 8), thus sophisticated
are (1) most of carotenoids are aliphatic, but in mechanisms of control must exist to assure the
Chlorobiaceae and Chloroflexaceae some caro- production of the appropriate levels of these com-
tenoids have aromatic or β-rings; (2) aldehydes pounds in the context of the metabolic pathway,
with crossover conjugations and tertiary methoxy developmental stage, and environmental condi-
groups; (3) various classes of carotenoids in each tions.305 Initial steps of the pathway involve the
species; (4) all carotenoids are bound to the light- fusion of three molecules of acetyl-CoA to pro-
200
FIGURE 8. (A) Pathway of biosynthesis of isoprenoid compounds. (B) Proposed stages for the carotenogenesis
pathway. (Adapted from Ref. 185.)
201
duce 3-hydroxi-3-methylglutaryl-coenzyme A which is rearranged to IPP. Thus, they suggested
(HMG-CoA). In animals and yeasts these reac- the existence of an alternative pathway for iso-
tions are catalyzed by two enzymes, the acetyl- prenoid synthesis in higher plants.
CoA transferase and the HMG-CoA synthase. Isopentenyl pyrophosphate is the basic unit
However, these reactions have not been studied for constructing terpenoids of longer chains.
extensively in plants, but apparently the reaction IPP itself is not reactive enough to start the
must be similar, a cabocationic mechanism. Evi- condensation reactions. Thus, the first step is
dence exists that only one enzyme catalyzes both its isomerization to dimethyl-allyl pyrophos-
reactions with Fe+2 and quinone as cofactors.305 phate (DMAPP), the reaction is catalyzed by
The conversion of HMG-CoA to mevalonate the IPP isomerase with a divalent metallic ion
is catalyzed by the HMG-CoA reductase. This as cofactor. The next step is the condensation
enzyme is very important in animals, limiting the of IPP and DMAPP to form geranyl pyrophos-
stage of the reaction, thus this enzyme is highly phate (GPP). After this, two molecules of
regulated. The HMG-CoA reductase activity has IPP are condensed to GPP to obtain the
been detected in endoplasmic reticulum, mito- geranylgeranyl pyrophosphate (GGPP) by ca-
chondria, and plastids, and catalyzes two steps of talysis with GGPP synthase. The GGPP syn-
reduction and uses NADPH as a cofactor.305 thase has been purified to homogeneity, and the
IPP synthesis from mevalonate is well charac- activity has been corroborated by in vitro reac-
terized in plants (Figure 9A). Mevalonate tions.123 The enzyme has requirements for two
is phosphorylated sequentially by the en- ions, Mg+2 or Mn+2, per catalytic site. The ion is
zymes mevalonate kinase and after by bonded to the pyrophosphate group of GPP for
mevalonate-5-pyrophosphate kinase to form a better salient group. The ionization of the
5-pyrophosphomevalonate. This compound suffers allylic pyrophosphate leads to the formation of
a decarboxylating elimination catalyzed by an allylic carbocation with a stabilized charge.
pyrophosphomevalonate decarboxylase that re- The electrophilic addition of the IPP molecule
quires ATP and a divalent cation.185,305 These enzy- generates the intermediate carbocation of 15
matic activities have been characterized carbons that is attacked by another IPP mol-
in plant extracts or in vitro. It seems that mevalonate ecule to obtain the GGPP (Figure 9B). Because
kinase is feedback regulated by phosphomevalonate GGPP is a precursor of many other compounds
and other allylic-diphosphate intermediates. It has (phytyls, phylloquinones, plastoquinones, taxol,
been shown in potato that mevalonate diphosphate tocopherol, gibberellic acid, diterpenoid phy-
decarboxylase is induced by pathogen attack or by toalexins, and diterpenes), it is possible that the
treatment with chemical products. It could be pos- transformation of GGPP, in this branching of
sible that the main controversy is in the subcellular the pathway, is highly regulated by complex
localization of these enzymes.185 mechanisms and is possible to be compartmen-
In 1997 Lichtenhaler et al.280 proposed an talized. These assumptions are supported by
alternative pathway for the IPP synthesis in the the existence of multiple genes for the GGPP
chloroplasts of higher plants. These authors used synthase of Arabidopsis.24,93,185,266,305
labeled glucose (1-13C-glucose) to grow L. gibba, The first specific step for the synthesis of caro-
D. carota, and H. vulgare. They then analyzed the tenoids is the condensation of two molecules of
labeling in sterols produced in cytoplasm and GGPP to cis-phytoene (C40). The reaction has the
found that they were synthesized from acetate prephytoene pyrophosphate PPPP) as an interme-
through the mevalonate pathway as it had been diate (Figure 10A).185 In tomato and pepper it has
described previously. However, the labeling pat- been shown that one enzyme phytoene synthase)
tern of carotenoids, phytyl, and plastoquinone were catalyzes both steps.172 Complexes with IPP
different (produced in plastids). The pattern sug- isomerase, GGPP synthase, and phytoene synthase
gested an alternative pathway that uses pyruvate activities have been isolated.70 In the mechanism,
and 3-phosphoglycerate (from the glycolysis path- two GGPP molecules are condensed, initially hy-
way) to obtain D-1-deoxy-xylulose-5-phosphate, drogen and one of the pyrophosphate groups are
202
FIGURE 9. Initial stages in the carotenogenesis pathway. (A) Isopentenyl pyrophosphate biosynthesis. (B) Isopentenyl pyrophosphate polymerization to geranylgeranyl
pyrophosphate. The involved enzymes are (1) Mevalonate kinase; (2) Mevalontate-5-pyrophosphate isomerase; (3) Mevalonate-5-pyrophosphate decarboxylase;
(4) Isopentenyl pyrophosphate isomerase; (5) Farnesyl pyrophosphate synthase; (5) Geranylgeranyl pyrophosphate synthase. (Adapted from Refs. 185, 422.)
203
FIGURE 10. (A) Phytoene biosynthesis and (B) desaturation reactions to form lycopene. (Adapted from Refs.
179,185.)
204
produced to obtain PPPP. Afterward, the PPPP are believed to happen in the last stages of the
molecule is rearranged to form phytoene. Most of biosynthetic pathway (Figure 11C).68,72 The first
the organisms (especially higher plants, algae, and xanthophylls are formed from cyclic carotenoids
fungi) synthesize 15,15′-cis-phytoene. The 15,15′- (e.g., β-carotene) by hydroxylation in the 3 and 3′
cis-phytoene has the basic structure of the caro- positions of the ionone ring, followed by the
tenoids, and subsequent reactions involve chemi- epoxydation in the 5,6 and 5′,6′ positions. By
cal transformations of this structure. However, most considering the high diversity of xanthophylls in
of natural carotenoids have a trans configuration, nature, nowadays a lot of information is required
and it seems that the cis-trans transformation does to completely define this route (enzymes, reac-
not require any specific isomerase.14,24,93 tion mechanisms). In pepper, the capsanthin
Later, phytoene suffers four desaturation re- capsorubin synthase that catalyzed the conver-
actions to give phytofluene, ζ-carotene, sion of antheraxanthin and violaxanthin to the
neurosporene, and finally lycopene (Figure ketocarotenoids capsanthin and capsorubin, re-
10B).24,179 It has been established that iron could spectively, has been purified to homogeneity.14,24,69
play an important role in the desaturation elec- In 1996, a protein of 43 kDa was purified from
tron-transport chain involved in phytoene lettuce thylakoids that corresponded to a
desaturation. This conclusion was reached by violaxanthin deepoxydase. This was the first re-
growing sycamore (Acer pseudoplatanus L.) cells ported enzyme that catalyzes the conversion of
in an iron-deprived medium, producing a large violaxanthin and antheraxanthin into zeaxanthin.
accumulation of phytoene and corresponding dimi- Also, it was established that acidic conditions and
nution of carotenes and xanthophylls.362 Addi- ascorbate in lumen are required for the reaction.399
tionally, studies on chromoplasts of Narcissus Afterward, the violaxanthin deepoxydase from
pseudonarcissus have been established spinach was isolated and shown that it is a mono-
that quinone compounds (plastoquinone/ mer of 46 to 50 kDa molecular weight.201
plastohydroquinone) are involved in the redox By using Arabidopsis thaliana mutants, it was
reactions in membranes. Membrane oxidation shown that plants with deficiencies in carotenoid
leads to a loss of desaturase activity and the main synthesis also had low levels of abscisic acid
redox active components in chromoplast mem- (ABA), and it was suggested that ABA was formed
branes, quinones and tocopherols, become oxi- by the oxidative breakdown of epoxyxanthophylls,
dized. Then, NADPH is able to reactivate specifically it was related with violaxanthin and
desaturase reactions by the reduction of these antheraxanthin.24,293 This was supported by the
components. By considering that chromoplasts very recent isolation of genes of the biosynthetic
showed a pronounced NADPH-dependent respi- pathway of ABA from Nicotiana plumbaginifolia,
ratory activity, it is thus considered that phytoene Arabidopsis, and maize.383 The fast conversion of
desaturation and chromorespiration are overlap- cis-xanthoxin (obtained from epoxyxanthophylls)
ping biochemical phenomena by using in the re- to cis-ABA was reported in tomato plants, and it
dox process the same quinone pool.2,341 was observed that cis-xanthoxin levels were sig-
Lycopene is converted to β- or ε-carotene by nificantly induced by water stress in older plants.515
lycopene cyclases (Figures 11A and 11B); these
enzymes accept other acyclic substrates, such as
neurosporene. In Capsicum annuum the direct con- b. Biosynthesis Regulation
version of lycopene to β-carotene catalyzed by
lycopene cyclase has been detected.71 This reaction Regulation of the carotenoid biosynthesis is
requires FAD as a cofactor, and the proposed complex, as been observed for most of the terpe-
mechanism is shown in Figure 11A. However, this noids because it seems that regulation must occur
reaction is not yet perfectly defined.14,24,179 at several levels. Evidence shows that carotenoid
The hydroxylation reactions occur in an early biosynthesis is restricted to specific tissues, where
stage, but different structural and functional modi- they are used. Carotenoids are produced in fruit
fications (epoxy, furanoxy, and oxy derivatives) chromoplasts during the maturation process (or-
205
FIGURE 11. (A) Proposed mechanism for ring formation of carotenoids. Examples of (B) cyclization reactions of
lycopene, and (C) hydroxylation and other modifications. Some of the involved enzymes are (1) β-lycopene cyclase;
(2) ε-lycopene cyclase; (3) β-carotene hydroxylase; (4) zeaxanthhin epoxidase; (5) violaxanthin de-epoxidase.
(Adapted from Ref. 90, 179.)
206
gan-specific regulation). Also, it has been deter- c. Molecular Biology of Carotenogenesis
mined that GGPP synthase activity is in chro-
moplast stroma (tissue-specific regulation).24 En- Starting models. Biochemical studies have
zymes of carotenoid biosynthesis, and other related outlined the carotenoid biosynthetic pathway;
proteins, are functional in chloroplast/chromoplast however, the biochemical approach for studying
but are codified by nuclear genes; then, the corre- the involved enzymes have shown to be ineffi-
spondent precursors are posttranscriptionally im- cient, because most of them are in membranes
ported toward plastids. This could suggest the and are difficult to isolate. On the other hand,
existence of transcriptional or posttranscriptional carotenoids are very important for human beings,
pathway control. However, although some ge- and scientists work to control the carotenogenesis
netic mutants in fruit ripening and carotenoid ac- pathway (overproduce or change). It is clear that
cumulation have been identified (from maize, to- pathway control requires a well-defined pathway
mato, and Arabidopsis), none of the regulatory and that molecular biology has permitted advances
genes have been isolated.24,93 in this area.
Studies on carotenogenesis provided signifi- The current knowledge on the molecular bi-
cant advances thanks to use of mutants of oxy- ology of the carotenogenesis pathway is derived
genic photosynthetic organisms. However, the from studies of specific organisms. Complete
identification of mutants block in the initial steps sequences of clusters of genes for carotenogenesis
of the biosynthesis pathway has proven to be have been reached in the photosynthetic prokary-
difficult because of cells with chlorophyll that are otes Rhodobacter capsulatus and Rhodobacter
simultaneously exposed to illumination and oxy- sphaeroides. Much data on the carotenogenesis
gen, in the absence of carotenoids, suffer photo- pathway have been reported for Synechococcus,
oxidative damage and then die. Additionally, mu- and all the genes of some nonphotosynthetic
tations in the final stages are masked by bacteria, for example, Myxococcus xanthus, My-
endogenous chlorophylls; thus, few mutants in cobacterium aurum, Thermus thermophilus, and
carotenoid synthesis have been isolated. In plants, some bacteria Erwinia sp. Also, the pathway is
the situation is not so discouraging: potential le- well defined in the fungi Neurospora and
thal mutants affecting green photosynthetic Phycomyces.13,25,160,221,321 It was isolated and char-
tissues can survive in heterozygosis, and acterized the Neurospora wc-2 gene that codes
carotenogenic mutants in flower and fruits do not for a second central regulator of blue light re-
necessarily affect the viability. In particular, a sponses. The coded proteins represent the first
collection of mutants affected in carotenoid bio- two putative GATA transcription factors that
synthesis in leaves and endosperm of maize have have been characterized in any organism being
been used.14 involved in light-activated gene regulation rather
Interestingly, exogenous regulatory agents have than in a general transcriptional regulation.285
been used to direct the carotenoid synthesis or block Gene clusters have been observed in some
catabolism; they are attractive approaches for produc- microorganisms but not in eukaryotes
ing fruits or vegetables with improved characteristics. or cyanobacteria; however, generated informa-
2-(4-chlorophenylthio)-triethylamine hydrochloride tion from microorganisms and from
(CPTA) and N,N-diethyl nonylamine cause an accu- the carotenogenesis genetics of higher plants
mulation of the red carotenoid lycopene. 2-(4-ethyl- helped to identify carotenogenic genes
phenoxy)-triethylamine hydrochloride has been used of cyanobacteria and of higher plants (Zea
to improve orange color by postharvest application mays, Lycopersicon esculentum, Narcissus
induce the accumulation of xanthophylls in endocarp). pseudornarcissus, and Capsicum annuum).14,23,420
Esters of 2-diethylaminoethanol induce the accumula- Altogether, these models provided the essential
tion of β-carotene, while p-bromobenzylfurfurylamine tools to obtain the plant carotenogenesis genes
applied on grapefruit produces poly-cis lycopene reported up to date (Table 3).422 In general,
prolycopene), which has a characteristic orange color carotenogenesis eukaryotic genes seem to be in
contrasting with the red color of lycopene.293 only one copy with the exemption of phytoene
207
TABLE 3
Some Genes (genomic or cDNA Clones) Isolated from Plants That Are Directly Related to the
Carotenogenesis Pathwaya
Enzyme that is coded by the geneb
Plant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Arabidopsis thaliana ● ● ● ● ● ● ● ● ● ● ● ● ●
Capsicum annuum ● ● ● ● ● ● ●
Lycopersicon ● ● ● ● ●
esculentum
Zea mays ● ●
Narcissus ● ● ●
pseudonarcissus
Nicotiana ● ● ●
tabacum
Glycine max ● ●
Cucumis melo ●
Oryza sativa ● ● ●
Brassica ● ●
campestris
Catharanthus ●
roseus
Lupinus albus ●
Nicotiana ●
benthamiana
Sinapsis alba ● ● ●
Haematococcus ●
pluvialis
Clarkia breweri ●
Clarkia xantiana ●
Lactuca sativa ●
a Adapted from Refs. 422, 498.

b (1) 3-hydroxy-3-methyl glutaryl coenzyme A reductase; (2) mevalonate kinase; (3) mevalonate 5-pyrophosphate
decarboxylase; (4) isopentenil pyrophosphate synthase; (5) isopentenyl pyrophosphate isomerase; (6)
geranylgeranyl pyrophosphate synthase; (7) phytoene synthase; (8) phytoene desaturase; (9) ζ-carotene
desaturase; (10) β-lycopene cyclase; (11) ε-lycopene cyclase; (12) capsanthin capsorubin synthase; (13)
β-carotene hydroxylase; (14) β-carotene ketolase; (15) violaxanthin de-epoxidase; (16) zeaxanthin epoxidase.
synthase PSY) and GGPP synthase of pep- rate-limiting step. However, plants obtain more
per.90,278,423 products from the mevalonate pathway than ani-
Initial stages (Figure 9). Mevalonate syn- mals, thus some tissues and organelles have mul-
thesis is catalyzed by HMG-CoA reductase tiple HMGR genes (Arabidopsis, Hevea, and
(HMGR). In animal systems isoprenoid biosyn- Solanum). These genes are differentially regu-
thesis is strongly regulated at HMGR level, and it lated: different genes are expressed in different
was suggested that in plants mevalonate-synthe- parts and developmental stages.272 On the other
sis catalyzed by HMGR is also the first major hand, one cDNA clone of mevalonate kinase has
208
been isolated from Arabidopsis and IPP isomerase ported two enzyme forms: one soluble and inactive
from some plants and yeasts. All the information in stroma and another active, which are membrane
indicates that reactions catalyzed by HMGR, bound. The carotenoid content was increased dur-
mevalonate kinase, and IPP isomerase are not ing flower development; however, the mRNA level
limiting steps of carotenoid biosynthesis.93,185,305 was constant during the process. Also, there was an
The number of plant prenyltransferase genes increment of the PSY protein level that was not
isolated is very limited, for example, GGPP syn- related with activity, because the soluble PSY pro-
thase cDNAs from C. annuum 266 and from tein was only increased. It was reported that mem-
Arabidopsis.421 The pepper cDNA was obtained by brane galactolipids are necessary for PSY activity,
using antibodies against the purified GGPP syn- in particular an aldopyranose is necessary to acti-
thase in a cDNA expression library;266 this cDNA vate and lipids are necessary to membrane enzyme
was used as a probe to obtain the Arabidopsis bounding. Additionally, sequence analysis showed
gene.421 However, most of the information about that PSY is membrane integral protein, and it was
prenyltransferases come from animal and micro- suggested the existence of postranscriptional regu-
bial systems. Prenyltransferases have high homol- lation mechanisms that involve the membrane re-
ogy between different kingdoms. They are struc- dox state.423 In 1996,65 phytoene synthase y1 gene
turally and functionally conserved, suggesting the was identified from maize and evidence of mul-
presence of a common ancestor for prokaryotes tiple copies was presented. Interestingly, y1 is re-
and eukaryotes.13 However, GGPP synthase of sponsible of leave and endosperm carotenogenesis.
pepper chromoplasts segregates with eubacterial When tobacco plants were transformed with
prenyltransferases, thus this gene could be trans- PSY1 cDNA, an increment was observed in caro-
ferred from a bacterial symbiont. Also, a consensus tenoid content but with a reduction in gibberellic
region has been observed, DDXX(XX)D, which acid and chlorophyll levels. This information sug-
must have an important role in substrate bond- gested the existence of a common GGPP pool for
ing.305 Recently, a new GGPP synthase (GGPS6) these compounds. On the other hand, the cDNAs
gene from Arabidopsis thaliana was isolated. GGPP clones for GGPS, PSY, and PDS were isolated
synthase showed a strong amino acid homology from white mustard (Sinapis alba). It was estab-
with other GGPP synthases. This is the first GGPP lished that PSY transcript is up-regulated by light
synthase localized in mitochondria; thus, it was during etioplast/chloroplast conversion, but nei-
suggested that this isozyme produces the precur- ther GGPS nor PDS. Also, it was shown that this
sors of the side chain of isoprenoid quinones, and, response is mediated by phytochrome in mutants
importantly, that GGPP synthase could be pro- the PSY mRNA level was not induced). Thus,
duced in different organelles rather than organelle- enhanced carotenoid biosynthesis should occur
specific production and posterior distribution.528 only after the light-dependent development of
Phytoene synthesis and desaturation reactions structured chloroplast membranes.498
(Figure 10). Tomato phytoene synthase was the Phytoene desaturases (PDS) of anoxygenic pho-
first cDNA clone obtained for a carotenogenic tosynthetic microbes catalyzes the conversion of
enzyme that was isolated by using the correspon- phytoene to lycopene or neurosporene; in contrast,
dent cyanobacterial cDNA.389 Later on, the exist- PDS cDNAs of Arabidopsis, tomato, and soybean
ence of two phytoene synthase genes was discov- catalyzes the transformation phytoene to ζ-carotene.420
ered: one leaf-specific (PSY1) and another fruit In both models, a dinucleotide binding site are iden-
specific(PSY2). In contrast to tomato, pepper PSY tified in their coding sequence.26 Norris et al.343 used
expression is not induced by fruit ripening.400 It Arabidopsis mutants that accumulate phytoene to map
was suggested that differences between these ma- three loci (Figure 12). Two of these loci do not
terials could be assigned to differences between correspond to the PDS gene locus. Thus, at least three
climacteric and non-climacteric fruits.24 Schedz et genes are required in the phytoene desaturation; it
al.423 isolated the PSY cDNA clone from daffodil was showen that mutants showed deficiencies in plas-
by using an heterologous tomato PSY1 probe; the toquinone and α-tocopherol accumulation. Then a
gene was in one copy (Southern blot). They re- complementation assay with quinone biosynthesis
209
intermediates (homogenistic acid or plastoquinone) the isolation by a complementation analysis of
was used and carotenoid production was recovered. It the gene of the cyanobacteria Synechococcus.91
was concluded that plastoquinones and/or tocopherols By using this sequence, β-lycopene cyclase
are essential components for phytoene the desaturation cDNAs were obtained from tobacco and tomato.364
reaction in higher plants. It was proposed that plasto- Protein sequences are conserved between plants
quinone is an important component in electron trans- and cyanobacteria but different in bacteria. The
portation and ubiquinone was proposed as the trans- enzyme showed activity in the cyclization of
porter of anoxygenic photosynthetic organisms, PDS neurosporene (neu) to β-zeacarotene and of lyco-
expressed in E. coli is active, where plastoquinone is pene to β-carotene, suggesting that the enzyme
absent. In general, it was concluded that quinones are could perform two cyclizations. However, β-ly-
universal adapter molecules in the carotenoid copene cyclase was not capable of cyclizing ζ-
desaturation reactions.343 carotene, and it was proposed that the presence of
Corona et al.86 studied carotenogenesis regula- a double bond in the 7 to 8 or 7′ to 8′ is required
tion in tomato fruits. During tomato development, for the cyclization process. Sequence analysis
carotenoid content was increased (approximately showed FAD or NAD dependence. Also, the en-
10 times), which corresponded to PDS mRNA zyme was expressed in an active form in E. coli,
increment. A 2-kb region was identified upstream showing that membrane special elements are not
required. β-lycopene cyclase mRNA decreased
of the PDS gene that shows a cis-activation of the

chromoplast-specific PDS expression. Also, it was during fruit ripening, contrary to the observation
reported that chromoplast differentiation and PDS with PSY and PDS, and a transcriptional regula-
expression are coordinately regulated in petals and tion mechanism was proposed. Up to date, infor-
tomato fruits, and it suggested the presence of a mation of regulatory mechanisms of fruit
“chromoplast factor” that activates the PDS pro- carotenogenesis does not exist, but it is clear that
moter. This relation was not found in tobacco, regulation must be different in relation to leaves.
which does not have chromoplasts in its leaf struc- Also, a locus B that seems to be a β-lycopene
ture but accumulates good carotenoid levels. It was cyclase regulatory element was identified, al-
proposed that PDS could be regulated by the end though its mode of action is unknown.364
product (probably β-carotene, xanthophylls, or ab- As could be expected from the evidence men-
scisic acid) that the effect of specific inhibitors tioned above, β- and ε-lycopene cyclases cDNAs
activates the PDS promoter, and that etioplasts also were obtained from Arabidopsis thaliana (Figure
accumulate carotenoids.86 Recently, maize PDS 13).90 The importance of 7 to 8 or 7′ to 8′ insaturation
cDNA clone was isolated. This enzyme catalyzes for the cyclization process was reiterated. It was
the transformation of phytoene to ζ-carotene, an found that ε-lycopene cyclase is a monocyclase that
heterologous complementation assay was used in cannot introduce a second ε-ring where previously
the demonstration. With these results the require- another exists. It was concluded that lycopene cy-
ment of two enzymes for the desaturation of clization is a key process in plant and algae caro-
phytoene to lycopene in higher plants were con- tenoid biosynthesis, because the relation between
firmed and observed both in mono- and dicotyle- these two enzymes gives the balance between cyclic
donous plants. Southern blot analysis showed only carotenoids with or without an ε-ring. Also, it was
one gene copy. Three possible regulatory locus reported that these genes are in one copy.90
vp2, and especially vp9, were reported because Hydroxylation and other modifications
recessive alleles accumulate ζ-carotene. Northern (Figure 11C). A. thaliana β-carotene hydroxy-
analysis showed that mRNA level was constant lase cDNA was isolated. Hydroxylation activity
despite the carotenoid content increments. It was of the ε-ring was low, and by considering the
pointed out that maize PDS regulation contrasts differences in chirality of β- and ε-rings, the pres-
with that observed in the temporal control of PSY ence of an ε-carotene hydroxylase was sug-
and PDS of tomato fruits. gested.376 In addition, the Zeaxanthin epoxydase
Cyclization (Figures 11A and 11B). The ini- gene was identified, and it was shown that
tial step to clone a plant β-lycopene cyclase was epoxyxanthophylls are the precursors of abscisic
210
FIGURE 12. (A) Pathway for α-tocopherol and (B) model for the participation of plastoquinone in carotenoid
desaturations. Involved enzymes are (1) OHPP dioxygenase; (2) Phytyl/Prenyl transferase; (3) methyl transferase;
(4) phytoene desaturase; (5) ζ-carotene desaturase. Arabidopis plants with mutations on the steps signaled with
stars were analyzed. (Adaped from Ref. 343.)
211
FIGURE 13. Carotenoid cyclization reactions in Arabidopsis. the involved enzymes are (e) ε-lycopene cyclase; (b)
β-lycopene cyclase. Numbered double bonds are required for the cyclase activities. Observed activities are
represented by solid arrows. Broken arrows are proposed activities. Parallel lines over the arrow indicate that the
enzymatic activity was observed but no product was detected. (Adapted from Refs. 90, 364.)
212
FIGURE 14. Hydroxylation and other modifications in the carotenoid production by pepper fruits. Enzymes involved
in the latter steps are (1) zeaxanthin epoxidase and (2) capsanthin capsorubin synthase. (Adapted from Ref. 54.)
acid (ABA). The enzyme catalyzes the conver- similar to pepper fibrillin was isolated.490 This
sion of zeaxanthin to antheraxanthin and protein does not contain cysteines and has several
violaxanthin.295 aspartic and/or glutamic residues arranged in tan-
Carotenogenesis of pepper (Capsicum dem; it is peripheral in thylakoid membranes; the
annuum) fruit is one of the better studied models, mRNA level have a temporal and tissue specific
and molecular biology has permitted the elucida- regulation and shows increments during flower
tion at the enzymatic level the last biosynthetic development up to anthesis, then decreases; and
steps for its major carotenoids, capsanthin, and mRNA level is higher in corolla. Similar results
capsorubin (Figure 14).24 were observed in melon and watermelon (fibrilar
In the elucidation of carotenogenesis regula- chloroplasts), tomato (crystalline chromoplasts),
tory mechanisms, some interesting genes have and orange (globulous chromoplasts). It was con-
been isolated. A cDNA that codes for a protein cluded that some apoproteins act as chromoplast
213
sequestrate agents of carotenoids, and altogether tioned that these results were attractive because
gene expression of these proteins and those of the goal was 2 µg β-carotene/g dry seed weight to
PSY1 and PDS genes are strongly regulated at the cover the minimal requirements of young chil-
transcriptional level. Something similar has been dren (100 µg retinol equivalents). Thus, the intro-
observed with chloroplastidial proteins. Interest- duction of the next carotenogenesis enzymes (ζ-
ingly, corolla-specific carotenoid-associated pro- carotene desaturase and lycopene cyclase) could
tein (CHRC) is highly unstable and shows a fast generate rice transformants with good levels of β-
disappearance when it is not bounded to caro- carotene.13,66
tenoids.490 It was established that the CHRC ex-
pression is up-regulated in cucumber (Cucumis
sativus) corollas by GA3. Moreover, carotenoid- 5. Functions
associated genes were not affected, and it was
suggested that modulation of carotenogenesis by a. Color
GA3 is at the level of carotenoid sequestration.
Thus, CHRC up-regulation is proposed to be cru- Carotenoids provide colors to flowers, seeds,
cial for enhanced carotenoid accumulation while fruit, and to some fungi, and color has an impor-
preserving the structural organization of the orga- tant role in reproduction: coloration attracts ani-
nization of chromoplast.491
mals that disperse pollen, seeds, or spores. In

Nowadays, molecular biology has prospected Phycomyces blakesleanus it was observed that
new horizons to produce materials with a high intracellular accumulation of excess carotenoids
content of biological active carotenoids (e.g., with disturb the mating recognition system, which ap-
vitamin A activity) or to introduce new caro- pears to be involved in the later stages of mating
tenoids.13 The crtI gene of Erwinia uredovora by inhibiting the cell-to-cell recognition systems.351
codes for phytoene desaturase. Nicotiana tabacum
was transformed with crtI via Agrobacterium
tumefaciens. Transformed plants were herbicide b. Photosynthesis
resistant: norfluorazon, fluridone, diflufenican,
flurtamone, and fluorochloridone were inhibitors Main pigments involved in photosynthesis
of phytoene desaturase, and KM143-958, J852, are chlorophylls and carotenoids. Carotenoids
and LS80707 interfere with ζ-carotene desaturase. have two well-known functions in photosynthe-
Interestingly, higher levels of β-carotene and its sis: (1) accessory pigments in light harvesting,
xanthophyll derivatives (e.g., violaxanthin) and and (2) as photoprotectors against oxidative dam-
lower levels of lutein were observed in trans- ages. It has been proposed that carotenoids as
formed tobacco, while total carotenoid content light harvesting compounds evolved from anaero-
was conserved between transformed and control bic organisms, then generalized to all of the
plants. The meaning of the change in the xantho- aerobic photosynthetic organisms. One of the
phyll pattern have not yet been explained.13 An- carotenoid structural characteristics is their abil-
other important goal of biotechnology is the pro- ity to absorb visible light: p delocalized elec-
duction of important crops of high human trons suffer a photoinduced transformation in
consumption with enhanced carotenoid content. which a singlet state (s2) is produced, then en-
Rice endosperm does not contain carotenoids, and ergy is efficiently transferred to chlorophyll (chl)
to obtain plants that produce carotenoids it was to form singlet chl with a slightly higher energy.
necessary to introduce carotenogenesis genes. Rice The physical structure of chloroplasts facilitate
plants were transformed via biolistic with the transference of energy absorbed by caro-
the phytoene desaturase gene of Narcissus tenoids to chl. In thylakoidal membranes, caro-
pseudonarcissus; this rice formed phytoene, the tenoids are bound to chls and proteins to form
precursor of colored carotenoids. The high specific complexes called photosystem I (PSI)
phytoene production with transformants was and photosystem II (PSII). It is known that PSI
0.74 µg phytoene/g dry seed weight. It was men- is a pigment protein complex functioning as a
214
plastocyanin: ferredoxin oxidoreductase. This to chl a.84 The photosynthetic efficiency, O2 evo-
process is highly endergonic (∆G° = 77 kJ mole- lution, and nonphotochemical quenching was
1) utilizing light energy to drive the reaction. PSI similar between mutants and wild type. How-
complex is composed of 11 to 17 different ever, under strong light stress and long exposi-
polypeptide subunits, about 90 chl “a” molecules, tion time, the ABA mutants showed an acceler-
10 to 15 β-carotene molecules, two phylloquinone ated photoinhibition compared with wild-type
molecules, and three [4Fe-4S] clusters.424 PSII leaves.139
functions as a water-plastoquinone oxidoreduc- Carotenoid functions are greatly determined
tase. It is a membranal complex that comprises by their associated proteins. These proteins are
more than 25 different proteins, and the heart of mainly membranal, usually hydrophobic, which
the complex is the reaction center (RC) consist- bound carotenoids by noncovalent bonds.183
ing of the D1 and D2 proteins. In all the redox Also, it has been established that protein cor-
process, quinones have an important participa- rect folding in the photosynthetic complexes
tion. It has been suggested that all pigments requires chl and carotenoids.186 A cDNA of pro-
involved in photosynthesis are bound to proteins tein that bound fucoxanthin-chl was isolated
of the individual thylakoid membranes, and cer- from Heterosigma carterae. This protein
tainly in bacteria it has been established that showed a high similarity with proteins that bind
bacteriochlorophyll molecules are bound to His chl a/b. With this evidence, divergent evolution
residues of α and β apoproteins. Even less is was suggested, while the energy-harvesting
known about how carotenoids are bound to the complexes of algae evolved their ability to bind
photosynthetic apparatus, but it has been pro- accessory pigments (chl’s and carotenoids) in-
posed that carotenoids span the membrane, with dependently to increase their absorption spec-
the groups interacting with polar amino acids tra and consequently to have a more efficient
near the membrane surface, and the middle parts energy utilization.131
making van der Walls contacts with hydropho- On the other hand, Phaffia rhodozyma exposed
bic side groups of the protein.21 Main carotenoid to singlet oxygen increased the astaxanthin content
in PSI is β-carotene and lutein in PSII. In PSII, as a protection mechanism.428 Phytoene desaturase
all β-carotene is located in the complex nucleus, mutants of Chlamydomonas reinhardtii showed se-
very near of the reaction center, while xantho- vere defects during PSII assembly, PSII repair dur-
phylls are bound to the remaining chl “α” and ing photoinhibition, degradation of D1 protein, and
“β” molecules in the energy-harvesting an- in the binding of D1 with PSII. Cells without β-
tenna. 14,61,81,179,314,349 Wild-type Arabidopsis carotene did not show PSII activity, and they did not
thaliana and ABA-3 mutant, which lacks of have D1 protein. It was concluded that, in addition
epoxydase activity (contains only lutein and ze- to its role in photosynthesis and photoprotection β-
axanthin), were used to study the function of carotene stabilizes PSII or permits the correct as-
carotenoids in the light harvesting complex II semble of D1 and PSII, protecting D1 of a fast
(LHCII). It was found the same chl a/b ratio in degradation. Also, it was proposed that phytoene
both wild-type and ABA-3 mutant, but differ- desaturase is plastoquinone dependent; plastoquinone
ences were detected in the carotenoid levels. redox state could limit β-carotene biosynthesis, which
The wild-type ratio was 7/5/2/1 chl a/chl b/lutein/ limits the PSII assemble, and this regulates the plas-
neoxanthin, whereas in ABA-3 mutant it was toquinone redox state. Consequently, PSII could be
7/5/1.4/0.6 chl a/chl b/lutein/zeaxanthin. This adjusted to illumination conditions by a fast turn-
study showed that the predominant sequential over of β-carotene, D1 protein, and its correspond-
energy transfer is from carotenoids (predomi- ing assemble into PSII.471 In addition, it has been
nantly lutein) to chl “b” and then to chl “a” in shown that synthesis of D1 protein increases during
wild-type Arabidopsis. In contrast, mutants the transference of Chlamydomonas cells from low
showed substantial carotenoid to chl “a” energy light to high light conditions, and it was suggested
transfer. It was suggested that zeaxanthin is the that it tends to favor the mitigation of photoinhibition
component in ABA mutant that transfers energy and to ensure the function of photosystem II.436
215
c. Xanthophyll Cycle antioxidant activity or with modulation of other
cellular antioxidants. Also, it has been established
When leaves are exposed to high illumina- that carotenoid structure has a great influence in
tion, epoxy xanthophyll groups are removed of its antioxidant activity; for example, canthaxanthin
violaxanthin to initially form antheraxanthin and and astaxanthin show better antioxidant activity
then zeaxanthin. This is one of the plant protec- than β-carotene or zeaxanthin.281,300,355 Chloro-
tion mechanisms against light damage. The num- phyll-sensitized photooxidation (4000 lx) of
ber of carotenoid molecules is higher in sun-ex- soybean oil was studied in the presence of
posed leaves than darkness maintained leaves. carotenoids (lutein, zeaxanthin, lycopene,
Also, xanthophyll cycle carotenoids (violaxanthin, isozeaxanthin, or astaxanthin). It was found that
antheraxanthin, and zeaxanthin) are increased in higher carotenoid concentrations decreased the
sun-exposed leaves. This phenomenon is very peroxide value of soybean oil by quenching sin-
important, sun exposed leaves in a fast-growing glet oxygen, and it was shown that longer chro-
stage use not more than 50% of absorbed energy mophores favor this reaction. Thus, astaxanthin
during the stage of maximum radiation (midday), with 13 conjugated double bonds was the best (in
and in some species only 10% is used. Thus, 50 methylene chloride).274 Packer352 evaluated the
to 90% of absorbed light is in excess and must be antioxidant activity of different carotenoids by in
eliminated in order to avoid cellular damage. Xan- vitro assays. It was reported that antioxidant ac-
thophyll cycle is a process that makes the energy tivity depends on the used system.352 Other stud-
dissipation easy and protects the photosynthetic ies indicated an isomer-specific activity of caro-
apparatus. It has been established that energy trans- tenoids: using in vitro assays, it was determined
ference from chl to zeaxanthin is theoretically that cis-lycopene isomers showed better absorp-
possible, and this gives support to the observed tion than β-carotene and were more efficient in
zeaxanthin increments under high illumination. singlet oxygen quenching.447
Moreover, xanthophyll cycle carotenoids are as- The antioxidant activity of lutein, lycopene,
sociated with the energy harvesting complexes annato, β-carotene, and γ-tocopherol was evalu-
PSI and PSII.14,349 ated on triglycerides by the effect of air and light.
Xanthophyll cycle involving violaxanthin, It was reported that lutein, lycopene, and β-caro-
antheraxanthin, and zeaxanthin is ubiquitous of tene act as prooxidants, favoring the formation of
higher plants and green and brown algae. In hydroperoxides; however, if a small quantity of
Dunaliella salina Teod. and Dunaliella bardawil γ-tocopherol is added to these pigments, the phe-
the accumulation of β-carotene has been observed nomenon is reverted and they act as antioxidants
in response to a combination of high light, hyper- with a higher activity than γ-tocopherol. Annato
salinity, and nutrient stress. Also, substantial showed antioxidant activity, however, this could
amounts of zeaxanthin and a continued operation be assigned to unknown fluorescent compounds.189
of the xanthophyll cycle have been observed. In- β-Carotene also showed prooxidant activity in
terestingly, during diurnal illumination variations, oil-in-water emulsions evaluated by the forma-
stressed (nutrient and high salt concentration) cells tion of lipid hydroperoxides, hexanal or 2-heptenal;
did not show high xanthophyll levels at midday in the activity was reverted with α- and γ-tocopherol.
contrast with nonstressed cells. It was explained With this evidence, it was suggested that toco-
that in stressed cells high levels of xanthophylls pherols effectively protect β-carotene against radi-
are maintained, which work as an adaptative func- cal autoxidation. 204 Antioxidant activity of
tion to protect the photosynthetic apparatus.369 capsanthin and lutein was evaluated using chloro-
phyll as photosensitizer. Capsanthin was a better
antioxidant, and it was concluded that the antioxi-
d. Antioxidant dant activity depended on the number of double
bonds, keto groups, and that cyclopentane rings
In vivo and in vitro studies have shown that in the carotenoid structure enhanced their activ-
carotenoid photoprotective role is related to its ity. It was suggested that carotenoids can be used
216
in foods to prevent degradation of other compo- droxy-β-carotene, and the synthetic retro-dehydro-
nents.100 Reactivity of singlet and triplet states β-carotene show an efficient induction of the gap
was evaluated in carotenoid photodegradation; it junctional communication (GJC) in murine fibro-
was found that canthaxanthin is more stable than blasts. The GJC stimulation was more than five
β-carotene. β-Carotene could be degraded in oxy- times, depending on the carotenoid. β-Carotene
gen absence (in toluene), but canthaxanthin and retrohydro-β-carotene were the most efficient
showed good stability. It was established that inducers of GJC, indicating that the presence of a
carbonyl groups facilitate energy transference by six-member ring is important in the GJC induc-
giving greater stability because they have less tion; carotenoids with five-member rings showed
diradical character, thus transformation between little activity. Interestingly, the induction of GJC
excited states is easier. Also, it was suggested the did not show correlation with the quenching of
use of ketocarotenoids as food antioxidant. Inter- singlet oxygen. Thus, it was suggested that GJC
estingly, research has shown than astaxanthin is a and antioxidant activities in cancer prevention
better agent to destroy free radicals than other operate independently of each other.448 The im-
carotenoids.340 Miller et al.315 evaluated carotenoid portance of carotenoids with rings of six mem-
antioxidant activity against radicals and estab- bers was emphasized by the isolation of a caro-
lished the following order of activities decreas- tenoid-binding protein (CCBP) of 67 kDa from
ing): lycopene > β-cryptoxanthin > lutein = zeax- ferret liver. Carotenoids with substituted β-ionone
anthin > α-carotene > echineone > canthaxanthin rings, without β-rings, or with an intact β-ring
= astaxanthin. Lycopene showed three times more with a shorter lineal chain were not bonded by
activity than γ-tocopherol, and it was concluded CCBP. Consequently, it was showed that CCBP
that antioxidant activities are influenced by po- binds β-carotene mole per mole with high effi-
larities that are increased with the presence of ciency and specificity. Thus, CCBP may play a
functional groups in terminal rings.315 major role in the storage, transport, and targeting
of β-carotene in mammalian systems. Addition-
ally, it was suggested that the high affinity be-
e. Pharmacological Effects tween CCBP and β-carotene may protect the caro-
tenoid from degradation, and its antioxidant
Many diseases, such as cancer and strokes, activity must be better.388 There exists evidence of
involve oxidative processes mediated by free radi- the effectiveness of β-carotene in the treatment of
cals. Carotenoids, by their antioxidant effect, can certain kinds of cancer, for example, smoking-
show benefits in such diseases; however, this func- related cervical intraepithelial neoplasia and cer-
tion is not completely demonstrated in vivo. Caro- vical and stomach cancer.94 Siefer et al.438 showed
tenoids are an integral part of membranes. that β-carotene affects the immune response in
Carotenes are immersed in membranes, but xan- rats, and by this means tumor growth is inhib-
thophylls showed a variable membranal position, ited.438 In contrast, it was reported that retinoic
polar groups in xanthophylls affect their position acid and citral act as suppressors of this system.
and mobility. Consequently, carotenes are able to However, it was demonstrated that retinoic acid
react efficiently only with radicals generated in- regulates the γ-interferon (IFN-γ) gene, which has
side the membrane. While zeaxanthin, with their an important role in practically all the stages of
polar groups aqueous exposed, is able to react the immune and inflammatory responses.78,217
with radicals produced in that zone. Additionally, More than 600 carotenoids are known, and 50
it has been suggested that carotenoids influence of them are consumed in meals to be transformed
the strength and fluidity of membranes, thus af- into the essential nutrient vitamin A. After their
fecting its permeability to oxygen and other mol- absorption, these carotenoids are metabolized by
ecules. Also, it has been determined that caro- an oxidative rupture to retinal, retinoic acid, and
tenoids have a remarkable effect in the immune small quantities of breakdown products. Caro-
response and in intercellular communica- tenoids are transported by plasma lipoproteins.
tion.61,79,94,217 β-carotene, canthaxanthin, 4-hy- Carotenes are mainly associated with low-density
217
lipoproteins, while xanthophylls show a uniform that stage, alcohol dehydrogenase class IV was
distribution between the low- and high-density identified, and it was proposed that negative ef-
lipoproteins.360 Vitamin A is required in the vi- fects of alcohol consumption during fetus devel-
sion process, epithelial maintenance, mucose se- opment could be caused by the inhibition in
cretion, and reproduction.348,385,460 In the process retinoic acid synthesis, catalyzed by alcohol de-
of senile macular degeneration, retinol is related hydrogenase, which conduces to a failure in func-
to the induction of a gene cascade permitting the tion of retinoic acid receptor (necessary for nor-
phagocytosis of damaged cell in retina; this pro- mal development).9
cess is critical for the photoreceptor survival.138 Retinoic acid (RA) has also been related to
Also, it has been established that retinoids affect the aging process. RA affects the gene expression
many biological process, such as cellular prolif- levels through its interaction with triiodothyro-
eration, differentiation, and morphogenesis. More- nine receptor (TR) and RAR. Interestingly, when
over, retinoids have been used in treatments of the TR and RAR mRNA levels were analyzed in
certain kinds of cancers and some dermatological the brain of young, adult, or aged rats, lower
activities. Additionally, it is mentioned that a diet values were found. Additionally, it was showed
with deficiencies in vitamin A or supplemented that RA supplementation increased the TR and
with an excess of retinoic acid could induce ter- RAR mRNA levels. Also, the transglutaminase
atogenesis.306
activity was reduced in aged rats and recovered

Differentiation is a complex process, during after RA treatment. Transglutaminase has involved
development of the monoblastic cell line U-937, the memory process.385
the activation of both the retinoic acid receptor Prostaglandins are substances that have been
(RAR) and the 9-cis-RA receptor (RXR) are re- involved in several physiological process, and in
quired. Thus, it was suggested that different com- particular prostaglandin D2 has been involved in
binations of retinoids produced different pharma- the endogenous sleep promotion, modulation of
cological responses in the specificity and potency several central actions (regulation of body tem-
in cancer therapy.53,133,187 Sun et al.453 found that perature, release of the luteinizing hormone), etc.
retinoic acid induces the response of proteins as- Recently, it was shown that prostaglandin D (PGD)
sociated with damage by ultraviolet light in F9 synthase binds retinoic acid and retinol. In addi-
and NIH3T3 cells. However, this induction does tion, it was demonstrated that all-trans-retinoic
not show a correlation with the repairer of DNA acid inhibits its enzymatic action but not retinol.
damage.453 Another research showed that retinoic With the generated information, it was suggested
acid conduces to differentiation of F9 cells by an that retinoids may regulate the synthesis of PGD2,
increment of cellular communication. It was sug- and that PGD synthase may be a transporter of
gested that this process is mediated by the protein retinoids to the place where they are required.
connexin 43 that induces the expression of impor- Thus, it was suggested that PGD synthase plays a
tant molecules for cellular adhesion.79,94,133 Cur- critical role in regulating the development of neu-
rently, the effect of retinoic acid in arthritis treat- rons by the regulation of the transfer of all-trans-
ment is controversial.267 In these studies, synthetic or 9-cis-retinoic acid to RAR or RXR in the im-
retinoic acid (Am-80) was used, and a suppres- mature nerve cell.457 Additionally, it has been
sion in the incidence of arthritis, back-feet swell- mentioned that the overexpression of the
ing, and bone destruction in collagen-treated rats cyclooxygenase gene (a key enzyme in the forma-
was observed. However, 13-cis-retinoic acid did tion of prostaglandins) is an early and central
not show the above-mentioned effects, and this event in colon carcinogenesis.217
suggest that Am-80 could pertain to a family of In contrast with that discussed above, it was
antiinflammatory compounds but shows some sec- shown in transgenic mice that inhibition of the
ondary toxic effects similar to those observed transcription activator protein-1 (AP1) has an im-
with other retinoids.267 Ang et al.9 studied mice portant antitumor promotion activity, but the ac-
embryogenesis and found that retinoic acid has an tivation of the retinoic acid-responsive element
important role during the gastrulation process. In has not. AP1 regulates the expression of genes
218
with the recognition sequence designed as 12-O- Phaeophyta) diminished the risks of being af-
tertadecanoylphorbol-13-acetate (TPA)-respon- fected by certain types of cancer. Also, it was
sive element (TRE) in their promoter region, and established that the main antitumor agent is not
many TPA-responsive genes include several β-carotene, but other components (carotenoids)
protooncogenes (e.g., c-fos, c-jun). Thus, the de- that are present in algae. In this sense, mixtures of
sign of specific inhibitors of the transcription of carotenoids (α-carotene, fucoxanthin, and
AP1may be an interesting strategy in the treat- halocintiaxanthin) have shown a higher inhibi-
ment of cancer.227 tory activity than β-carotene in proliferation of
Carpenter et al.75 observed that mixtures of human neuroblastoma cells. In addition, α-caro-
canthaxanthin with low-density lipoproteins (LDL) tene showed higher antitumorigenic activity than
inhibited macrophage formation from human β-carotene in rat cancer induced by glycerol, and
monocytes. However, if canthaxanthin and LDL it was mentioned that carotenoids with an ε-ring
were added simultaneously (but without previous (absent in β-carotene) have higher inhibitory ac-
mixture) to cellular medium, it was not observed tivity.94,337
to any effect. The same was noted with β-caro- The antimutagenicity of carotenoids in Mexi-
tene, but with zeaxanthin the opposite was found. can green peppers Capsicum annuum) were stud-
It was explained that all evaluated carotenoids ied. The experiment was carried out by determin-
show good antioxidant activity, and observed dif- ing the number of revertants in the plate
ferences are caused because they act at different assay with Salmonella typhimurium. The
levels: zeaxanthin can quench radicals in the aque- antimutagenicity inhibition by nitroarenes was
ous phase, while β-carotene inhibits lipid higher than 90%. Pepper carotenoids were more
peroxidation; on the other hand canthaxanthin efficient antimutagens than pure β-carotene, sug-
was the most potent agent in the inhibition of gesting that other carotenoids (e.g., lutein, zeax-
methyl linoleate, and it was concluded that anti- anthin) in the pepper extracts showed a synergis-
oxidant activity depends on the particular system: tic effect with β-carotene. Also, it was mentioned
radical, carotenoid, microenvironment, etc. Thus, that the antimutagen activity might be from block-
diets with carotenoid mixtures are recommended ing the entrance of toxic compounds into the cell
instead of having just one particular carotenoid, or by their antioxidant activity.384 Also, the
because in vivo a great variability of radicals and antimutagenicity activity of carotenoids of Aztec
microenvironments take place. marigold (Tagetes erecta) was evaluated. It was
Carotenoids have been considered that pro- concluded that lutein was the compound with a
vide benefits in age-related diseases, against some higher activity on marigold extracts, but similar
forms of cancer (in especial lung cancer), strokes, to the observation mentioned for the pepper ex-
macular degeneration, and cataracts. However, tracts, the mixture of carotenoids in the marigold
most studies relate dietary components with sick- extract had higher antimutagenicity activity. In
ness incidence or symptoms; thus, these studies addition, it was suggested that lutein and
cannot establish a direct cause-effect relationship. 1-nitropyrene (mutagen) formed an extracellular
On the other hand, it is clear that carotenoids in complex that limits the bioavailability of
association with other components of fruits and 1-nitropyrene and consequently its mutagenicity.177
vegetables seem to have a protective effect against Böhm et al.44 indicated that carotenoids,
some chronic diseases and precancerous condi- α-tocopherol radicals, and ascorbic acid develop
tions. Additionally, in some studies β-carotene their function by diminishing the content of harm-
was supplied to smokers, and it was found that ful nitrogenous compounds. Also, a synergistic
cancer mortality indexes were higher in smokers effect between β-carotene and vitamins E and C
than in their respective controls.14,349,460,529 It has was observed in cellular protection. It was ex-
been signaled that a combined supply of β-caro- plained that β-carotene not only destroys
tene, α-tocopherol, and selenium reduces stom- oxyradicals but repairs tocopherol radicals pro-
ach cancer mortality, and it was pointed out that duced when α-tocopherol destroys oxyradicals.
the consumption of marine algae (especially Additionally, it was suggested that low antioxi-
219
dant levels (e.g., ascorbic acid) in smokers, in Many methodologies used in the analyses of
contrast with non-smokers, could be related with organic compounds are inadequate for carotenoid
an apparent failure in the recycling of α-toco- analyses. Their study is complicated by the great
pherol by β-carotene.44 variability in nature, the existence of many isomers,
Carotenoids protect lab animals of UV-in- great structural similarity, instability, presence of
duced inflammation and certain type of cancers. substances that interfere the determination, and the
Historically, carotenoid supplementation has been absence of an exact quantitation method.252,480
used in the treatment of diseases produced by The stages to be considered for carotenoid
light sensitivity, which are usually hereditary: 84% studies are extraction, saponification, separation,
of patients with erythropoietic protoporphyria, identification, and quantitation, and the develop-
consuming diets supplemented with β-carotene, ment of a good work depends on our knowledge
increased by a factor of 3 their ability to resist of carotenoid properties:61,112
sunlight exposition without presenting symptoms.
Also, carotenoids have been used in other photo- 1. Carotenoids are lipids and thus soluble in
sensitivity diseases: congenital porphyria, other lipids or in nonpolar solvents.
sideroblastic anemia, and have shown only a lim- 2. Carotenoid color is imparted by conjugated
ited success in treatment of polymorphic light double bonds that are mainly in trans con-
eruption, solar urticaria Hydroa vacciforme, Por- figuration (in their natural form), in ex-
phyria variegata, Porphyria cutanea tarda, or tended form; hence, they are lineal and
actinic reticuloid.300,529 rigid molecules. The properties of cis-caro-
Lutein and zeaxanthin have been considered tenoids show substantial differences with
as protective agents against aging macular degen- the trans ones; they are more easily solubi-
eration and senile cataracts.460 Also, it has been lized, absorbed, and transported. Factors
suggested that β-carotene suppress the increment generating isomerization are free halogens,
of hormones related to stress syndrome.197 acids, excessive light, and high tempera-
Certainly, up to date studies on anti-carcino- tures.
genic activity have produced unexpected results. 3. Conjugated double bonds in carotenoids are
Massive studies the α-tocopherol, β-carotene highly delocalized, and consequently caro-
[ATBC] cancer prevention study, the β-carotene tenoids have a low-energy excited state.
and retinol efficacy trial [CARET], and the phy- Hence, the energy of transition is in the
sicians health study) gave no results or do not visible region (400 to 500 nm); thus,they are
inclusively give a higher incidence of cancer. intensely colored yellow, orange, or red).
Nowadays it is thus premature to enunciate final Also, because of this property carotenoids
conclusions regarding the potential role of caro- are involved in photosynthesis and
tenoids in the therapeutics of degenerative dis- photoprotection.
eases. However, the consumption of fruits, veg- 4. The polyene structure of carotenoids makes
etables, and fortified foods with antioxidants is themselves highly reactive molecules. The
encouraged.94 structure is rich in electrons and suscep-
tible to be attacked by electrophilic reagents
responsible for carotenoid instability against
6. Methodological Aspects oxidation and give them their radical char-
acteristics. Carotenoids in the crystalline
It is evident that new methodologies usually state are susceptible to oxidation after their
give better results and confidence when identify- isolation; their degradation is very quick if
ing and quantitating the carotenoid components, stored in the presence of oxygen traces.
an important aspect if we consider that biological Electrons are delocalized but not with a
activity of different carotenoids is different, for uniform distribution, electronic density is
example, not only β-carotene is source of vitamin higher at molecule ends, and these are their
A, and not all carotenoids show this activity. more reactive part: deepoxydation reaction
220
preferentially occurs in carbons 7 and 8, high temperatures in order to minimize degra-
diminishing the reactivity by the presence dation.60
of keto groups (astaxanthin and Currently, industrial extraction consists of
canthaxanthin). pressing and solvent extraction of materials:
material is milled and pelleted, mixed with
hexane, and heated in a special recipient cov-
ered with a vapor jacket; hexane is eliminated
a. Extraction in a film evaporator and afterward by vacuum
distillation, and the main problems to be solved
With solvents. Carotenoids are soluble in are to diminish pigment degradation, increase
lipids or in nonpolar solvents, except when they extraction performance, and solve safety and
form complexes with proteins and sugars. environmental problems. More than 50% of the
Hence, they are extracted with nonpolar sol- pigments is lost during this extraction process;
vents. If the tissue is previously dried, then oil extraction industries emitted into the envi-
water-immiscible solvents are used such as ronment 210 to 430 million liters of hexane that
petroleum or ethyl ether; with the fresh materi- together with other organic compounds can
als acetone or ethanol are used, which have two produce nitrogen oxides and other pollutants.
functions, extracting and dehydrating solvents. These products in the last stage generate ozone
Solvents used in extraction must be pure (with- and other highly dangerous photochemical oxi-
out oxygen, acids, halogens) to avoid degrada- dants.403 With these perspectives, several re-
tion. Up to now, no solvent is optimal for the searchers have proposed alternative extraction
extraction of all carotenoids: carbon disulfide solvents. Heptane has been used, and the fol-
is the best solvent, but volatility, flammability, lowing characteristics have been mentioned:
toxicity, and degradation limit its use. Chloride lower volatility than hexane, similar extraction
solvents are good, but they show high toxicity; efficiency, and good quality product (oil).83
free peroxide ether, despite its efficiency, is not When mixtures of heptane-isopropanol or only
used because of its flammability and volatility; isopropanol are used, more antioxidants are ex-
other solvents such as hexane, heptane, and tracted and oils with enhanced stability are ob-
isooctane are not so good for extraction, but tained, and considering that isopropanol has
their other characteristics are favorable. On the lower flammability than hexane, this solvent
other hand, it must be considered which com- could be a good alternative in oil extraction.247,381
pounds will be extracted: polar solvents (such Enzymatic and/or aqueous extraction. Food
as acetone, methanol, ethanol) are good with industries have used enzymatic methods to obtain
xanthophylls but not with carotenes. As a gen- a diversity of products: maize starch, gluten and
eral rule, the extraction process consists of the starch of wheat, gelatin, deboned meat, among
removal of hydrophobic carotenoids from an others. The main advantages of these procedures
hydrophilic medium. The use of nonpolar sol- are specificity, moderated temperature and pH,
vents is not recommended because of penetra- treatments are mild, secondary products are scarce,
tion through the hydrophilic mass that surrounds and the final product is almost not affected. In
pigments is limited, while slightly polar sol- enzymatic processing, enzymes with mixed ac-
vents dissolve poorly carotene in dried samples tivities are used because of cell wall complex-
and solubility diminish in fresh samples. Thus, ity.106,119,132,298,353 Successful application of en-
it was postulated that complete extraction can zymes have been reported in oil extractions, and
be reached by using samples with low mois- extraction performance increments in the range
ture, and slightly polar plus nonpolar solvents.112 50 to 90% have been reported, and byproducts
Literature describes different solvent systems have shown higher digestibility than control.124,403
used in carotenoid extraction. In general, ex- Aqueous extraction has been proposed since
traction must be carried out very quickly, avoid- 1950 as an alternative to organic solvent. This
ing contact with light, oxygen exposure, and technology was implemented because of safety
221
and the cheapness of the process, which is based ization was observed with this extraction pro-
on oil-water insolubility and phases are separated cess.445 SFE and traditional extraction processes
by differences in density. Up to now, many re- were compared in the extraction of carrot caro-
ports have been published on extraction by simul- tenoids. The highest efficiency was obtained by
taneous enzymatic and aqueous processes: in oil using ethanol (10%) as a co-solvent, and it was
extraction of avocado, olive, coconut, rapeseed, reported that the traditional method extracts more
maize, cocoa, among others.52,97,106 However, re- α-carotene than SFE. Interestingly, vitamin A
ports on the application of this technology in equivalents obtained by SFE were higher than
pigment extraction are scarce. Pommer 377 proposed by traditional methods and extraction time was
an extraction method of vegetable oleoresins, and diminished from 6 to 1 h.22 Despite the above
between them pigments where plant material is mentioned, no commercial process of pigment
mixed with water and an water-immiscible or- SFE is in use nowadays.
ganic acid. Organic acid was selected with a lin-
ear chain of 6 to 12 carbons. Optionally, enzymes
are used in the extraction process, and pigment- b. Saponification
extraction efficiency was enhanced for pigments
of marigold (Tagetes erecta) flowers. On the other As mentioned above, xanthophylls are usu-
hand, Delgado-Vargas and Paredes-López120 re- ally esterified,279 which produces additional analy-
ported a procedure in which enzymatic treatment ses complications, for example, a pigment with
and aqueous extraction were carried out in order two hydroxyl groups can be without one or two
to obtain marigold flower meals with a higher positions esterified, which requires both separa-
carotenoid content. tion and identification. Thus, saponification ob-
Supercritical fluid extraction (SFE). The tains less complex mixtures when only
above-described method appears to have the re- nonesterified pigments appear. Another advan-
quirements to be considered a good extraction tage of saponification is chlorophyll destruction
process: fast, simple, and cheap. However, it gen- in the saponified samples.112
erally implies that considerable time is needed for Most carotenoids are stable under alkaline
it to be carried out, and a concentration stage may treatments; thus, the use of methanolic solutions
be necessary as well. Consequently, SFE has been of potassium hydroxide is a common method of
considered to perform a better extraction process. saponification, sometimes at environmental tem-
Up to 1986, only two works using SFE had perature or by heating.11,59,179,252 When carotenoids
been reported, but in the period 1986-mid-1989 are sensitive (e.g., astaxanthin and fucoxanthin),
26 works were published; this large increment alternatively lipases have been used.59 Candida
could be explained because this technique shows cylindracea lipase has been used in the saponifi-
the following advantages: rapidity, solvent cation (darkness, under nitrogen atmosphere, and
strength can be controlled, and the solvents used 4 h) of red palm oil and neither loss nor isomer-
are gases friendly to the environment and with ization of β-carotene was observed.282 In caro-
low toxicity. Interestingly, with this method the tenoid extraction from paprika, it was reported
concentration stage is avoided because solvents that carotene is sensitive to alkaline saponifica-
are eliminated immediately under environmen- tion, thus mild conditions were evaluated and
tal conditions.104,203,387 With these characteris- samples were saponified with potassium
tics, SFE has been used commercially to obtain methoxide.237
caffeine from coffee and hop oil.387 Lutein and
carotene were extracted from protein leaf con-
centrate by using CO2 as a solvent in SFE, and c. Separation
it was shown that conditions can be manipulated
to make a selective extraction.143 β-Carotene SFE Separation methods can be classified as
of sweet potato was three to five times more nonchromatographic and chromatographic.
efficient than traditional methods, but isomer- Nonchromatographic method uses mainly phase
222
partition, for example, by using petroleum ether reverse phase TLC chromatography to reach the
and aqueous methanol (90%). Carotenoids are separation.235
dissolved and nonpolar compounds recovered in High-performance liquid chromatography
epiphase, petroleum ether.59 Also, this method (HPLC) is the preferred column chromatography
has been used in lutein purification by using to carry out the qualitative and quantitative analy-
hexane:acetone:toluene:absolute ethanol (10:7:7:6 ses of carotenoids.60 This technique is ideal be-
v/v) and then hexane. 478 Another example: cause carotenoids can be monitored easily with
xanthophyll esters are separated by Craig the UV-visible detector, and methodology has
countercurrent distribution using dimethyl- converted it into a powerful technique with the
formamide:dichloromethane:hexane (8:2:10 v/v) introduction of the diode array detector (DAD),
as solvent.166 which permits detection at several wavelengths
In chromatographic methods adsorbents have and simultaneous tentative identification by UV-
been used such as sucrose, cellulose, starch, spectral analyses. In addition, information about
CaCO3, Ca3(PO4)2, Ca(OH)2, CaO, MgCO3, MgO, purity of compounds is obtained with a DAD
ZnCO3, Al2O3, silicic acid, silica gel, kieselguhr, detector.57,427,480 In general, HPLC shows the fol-
Microcel C, and mixtures. A general strategy for lowing advantages over the traditional methods:
obtaining a pure carotenoid uses open column greater sensitivity, resolution, reproducibility, and
chromatography in alumina followed by thin layer speed of analysis; additionally, it is possible to

chromatography (TLC) in silica, MgO-kieselguhr use inert conditions.36,181,415,480
G TLC, and silica TLC again, with different sol- It has been reported that HPLC has reproduc-
vent systems. The criteria for choosing a solid ibility advantages inclusive in relation to
support depends on the carotenoid to be purified; supercritical fluid chromatography.36 Additionally,
for example, alumina must not be used to separate separation of carotenoid geometric isomers has
astaxanthin because of oxidation problems; addi- been tried with reverse phase C18 column but with
tionally, alumina can produce isomerization of limited success.427 However, a successful separa-
other carotenoids. Magnesium oxide is not rec- tion was reached by using a column with poly-
ommendable when acetone is used as solvent; a meric surface C30. The resolution of isomers was
possibility of solvent polymerization reaction higher for polar and nonpolar carotenoids than
exists. Alumina and silica separations are based C18 resolution.415 This column has been evaluated
on polarity; on the other hand, magnesium oxide in the separation of isomers of lutein, zeaxanthin,
or calcium hydroxide permits separation by num- β-cryptoxanthin, α- and β-carotene and lycopene,
ber and type of double bonds in the structure; which were produced by iodine isomerization.
calcium hydroxide or zinc (II) carbonate sepa- The main isomers identified were 9-, 9′-, 13-,
rates cis-trans isomers. It is usually recommended 13′-, 15-cis and all-trans carotenoids. It has been
to use each one of these three supports (alumina, commented that a good separation is a prerequi-
magnesium oxide, and calcium hydroxide) to reach site to make more exact determinations of provi-
the separation, and, generally, preceded by silica tamin A content, isomeric profiles in biological
or alumina open-column chromatography.59,60 tissues, comparative physiological roles between
Nowadays, open column chromatography is used the cis and trans configurations, purity determi-
as a prepurification stage to separate groups of nation, and purification of carotenoids.136 This
carotenoids with similar characteristics in special column has been used in the evaluation of bio-
flash open column chromatography.264,308 logical samples with excellent results.117,118 When
In chromatography, TLC is still used because a C34 column was evaluated, better resolution was
new advantages have been incorporated into this obtained for only some isomers, and, in general,
technique in addition to its main characteristics: the results were comparable to those obtained
low cost and simplicity. Also, it must be pointed with the C30 column.33 On the other hand, with
out that this is the starting technique used in Ca(OH)2 columns, a good resolution has been
whatever purification, and that is the wider used obtained in the separation of carotene geometric
technique.59,60,378 In addition, it is possible to use isomers with 5, 7, and 11 double bonds.425
223
All the information indicates that HPLC analy- electrospray detector that is 100 times more sen-
ses are required when analyzed carotenoids are sitive than conventional techniques (picomolar).480
minority compounds in a given sample.1 Also, it Another ionization technique is Atmospheric Pres-
is necessary to establish the content of bioactive sure Chemical Ionization (APCI), which produces
compounds, such as provitamin A precursors. The a better fragmentation pattern than FAB or
analysis of leafy vegetables used in the Indone- electrospray and shows a good compatibility with
sian food table (spinach, cassava, papaya, mango, HPLC equipment, and, recently, matrix-assisted
sweet shoot, and jointfir spinach) showed that laser desorption ionization was introduced and its
with HPLC were obtained lower values for provi- detection limit is in a femtomolar-attomolar
tamin A than previously reported values.228 range.251,480 These detectors have been used in
several studies to identify carotenoids from bio-
logical samples, and it has been established that
d. Characterization carotenoid identification must cover at a mini-
mum the following criteria: co-chromatography
Spectroscopy. The most important technique with authentic samples, UV visible, and mass
in carotenoid analyses is UV-visible spectroscopy, spectra.
which gives information about the presence of RAMAN spectroscopy and circular dichro-
rings, carbonyl groups, and isomeric ef- ism have permitted the study of carotenoids in
fects. 59,92,112,179,181 In this analysis, absorption biological systems.200,435 Kull and Pfander264 stud-
maxima, form, and fine structure of spectra are ied canola carotenoids and used UV visible, MS,
characteristic of the molecule’s chromophore. NMR, and circular dichroism to identify several
Nowadays, the introduction of DAD permits to isomers of luteoxanthin and violaxanthin. Also, it
make in situ identifications immediately after was mentioned that in nature both trans and cis
HPLC separation.60 isomers exist.
Infrared spectroscopy gives information on Photoacoustic spectroscopy (PA) was used to
the kind of bonds and atoms in the analyzed com- evaluate pigment composition of paprika. It was
pound, while nuclear magnetic resonance (NMR) possible to identify peaks or shoulders in the vis-
of proton and carbon permits the assigning of ible spectrum region that corresponds to caro-
these atoms to a certain structure. However, the tenoids or chlorophylls. With the generated infor-
technique used most in carotenoid analysis is mass mation, it was possible to distinguish between
spectroscopy (MS), mainly because of the sample different samples of paprika, and, when PA was
quantity required for analysis is very small. Mass used in the near infrared region (800 to 1000 nm),
spectroscopy provides information on carotenoid semiquantitative information on total pigment
MW, and fragmentation pattern helps us to deter- composition was obtained.489
mine the carotenoid structure.179 However, it is Chemical tests. By considering carotenoid
very important to choose an adequate MS equip- chemical structure, several simple tests are used
ment because of carotenoid instability, and one of to corroborate the presence of chemical groups:
the initial and most successful MS ionization tech- 5,6-epoxydes react with HCl to form 5,8-epoxyde
niques is fast atom bombardment (FAB).200,426 isomers; this chemical modification is accompa-
The main advantage of this methodology is that nied by a hypsochromic shift of 7 to 22 nm for
MW is determined unambiguously; the molecular monoepoxydes and 40 nm for diepoxydes. Allyl
ion usually appears, and one of its disadvantages alcohols treated with HCl are dehydrated, and
is a poor fragmentation pattern, thus structural another double bond is formed with a consequent
elucidation limited and serial detectors must be change in their UV visible spectra. Another test to
used to differentiate between geometric isomers. evaluate allyl alcohols is the reaction with HCl in
Nowadays, new techniques have been introduced: methanol to obtain methyl esters. To determine
HPLC coupled with an MS detector (LC/MS), a the presence of aldehyde groups, aldol condensa-
very convenient technique by considering intrin- tion was carried out by treatment with acetone in
sic carotenoid instability, and MS with an a basic system producing an extension in the con-
224
jugated system, then spectra showed a change antioxidants, have led to their wide application in
toward higher wavelengths. In carotenoids with the food industry; preparations to apply them in
carbonyl groups, these can be identified by reac- oily or aqueous media have been produced, in-
tion with hydrides in ethanol or tetrahydrofuran; cluding emulsions, colloidal suspensions, and
thus, a hypsochromic change of 20 to 30 nm and complexes with proteins. These preparations have
finest spectra are observed. Another simple test is found applications to pigment margarine, butter,
iodine isomerization, which produces a mixture fruit juices and beverages, canned soups, dairy
of equilibrium isomers (cis-trans); if the starting and related products, deserts and mixes, preserves
pigment is all-trans, a hypsochromic shift is ob- and syrups, sugar and flour confectionery, salad
served (3 to 4 nm), while if it is cis, hyperchromic dressings, meat, pasta and egg products, among
(1 to 3 nm).112,179 others,257 and, interestingly, other important areas
Silver nitrate is used to discriminate between of application of carotenoids have been emerged.
β and ε carotenoid rings. Carotenoids are sepa- It has been reported that corn carotenoids inhibit
rated by TLC (preferable in silica gel) and then the synthesis of aflatoxin by Aspergillus flavus
developed with a methanolic solution of silver (90%) and by most of the A. parasiticus (30%)
nitrate. Carotenoids with β-rings produce a strains. Interestingly, carotenoids with an α-ionone
bathochromic shift; the shift depends on the num- ring showed higher inhibition (25%) than the
ber of β-rings, for example, after development β-ionone ring. Thus, lutein and α-carotene showed
zeaxanthin spot presents red tones, while lutein higher activities than β-carotene or zeaxanthin,
spot has yellow tones.236 and it was determined that several corn inbred
Evaluation of pigmentation efficiency. Color lines have high enough levels of carotenoids to
is a complex process and color evaluation also, affect aflatoxin formation in the endosperm (0.09
because we need to evaluate eye perception and to 72 µg/g). It was shown that the antioxidant
brain interpretation, and pigment deposition and activity of carotenoids are not responsible of inhi-
color perception do not show a direct relation- bition of aflatoxin synthesis, and it was suggested
ship. All attempts are only approaches to reality. that they may affect cell membranes enough to
The most common used methods are (1) sample indirectly modify polyketide synthase activity
carotenoid extraction followed by UV-visible (cytosolic enzyme) or by direct interaction with
spectroscopy analysis,11,313 and (2) the use of pho- the synthase or other enzymes of aflatoxin syn-
toelectric instruments that measure reflectance. thesis.344
Hunter-Lab is the most common equipment; it is
based on tristimulus effect.31,297 Several studies
have shown that reflectance colorimetry is a good a. Stability in Model Systems
descriptor of the pigment content from different
samples (pepper, red beet, marigold). This re- In practice, it is very important to take into
search has given high correlation coefficients account the carotenoid instability, and the estab-
(> 0.9), and it has been concluded that the assess- lishment of processing conditions must consider
ment of pigment content by color determination this aspect.297 However, most studies have been
is highly feasible at industrial level.121,230,394 carried out with β-carotene. It has been reported
that some pepper components facilitate β-caro-
tene oxidation. β-Carotene oxidation was acceler-
7. Importance As Food Colors — ated by increased levels of linolenic acid. The
Stability, Processing, and Production ascorbic acid effect depends on its concentration
and if copper ions are present. It was mentioned
Carotenoids have been used as food colors that high aqueous activity favored prooxidant
for centuries: saffron, pepper, leaves, and red palm activity. Additionally, high ascorbic concentra-
oil have carotenoids as their main color compo- tions (100 µmol/g of cellulose) in the presence of
nents. Color of carotenoids, together with benefi- copper ions induce a greater antioxidant effect,
cial properties such as vitamin A precursors and and under these conditions prooxidant activity of
225
peroxidase was inhibited. Moreover, it was con- The models were in matrixes of gelatin/sugar and
cluded that ascorbic acid concentration needed acacia gum/sugar. In first-order kinetics in the
for food protection depends on the content and degradation and isomerization reaction was ob-
identity of lipids present.250 served. Also, it was shown that in illuminated
At incubation conditions (37°C, 5% CO2, dark- aqueous β-carotene, the main isomers formed were
ness), β-carotene was slightly stable during 48 h 9-cis-, 13-cis-, and all-trans-β-carotene. In dark-
and showed fast degradation with the effect of ness, 13-cis-isomer was favored, while 9-cis was
UV and visible light in the presence of oxygen; under light conditions, showing a lower stability
the degradation rate was increased with incre- of 13-cis-β-carotene.
ments in O2 turnover. β-Carotene was stabilized Another study showed that all-trans-β-caro-
with antioxidants (e.g., BHT); it was thus con- tene isomerization depends on which solvents were
cluded that degradation is by the effect of free dissolved. Faster reactions were observed in non-
radicals.419 polar solvents, identifying the higher rate of for-
Another study evaluated the effect of tem- mation for the 13-cis-β-carotene.368 However,
perature (100°C by different times) on aqueous other studies showed contrasting results: β-caro-
suspensions of β-carotene and lycopene. It was tene, diesterified capsanthin, and capsanthin dis-
considered that this treatment is representative of solved in anhydrous (cyclohexane) or aqueous
those commonly applied on vegetables. Volatile ethanol, water) solvents were illuminated
compounds were trapped in column or in organic (1000 l×). Degradation in anhydrous medium fol-
solvents then analyzed by gas chromatography lowed a zero-order kinetics and of first-order in
(flame ionization detector) and mass spectrom- aqueous medium. In addition, it was shown that
etry. The main degradation products of β-caro- deesterified pigments were more stable in aque-
tene were identified as 2,6,6-trimethyl-cyclohex- ous than in nonpolar medium, while at intermedi-
anone; β-cyclo-citral; 5,6-epoxy-β-ionone and ate polarity both forms esterified and deesterified)
dihydroactinidiolide. The products of lycopene showed similar stability. β-Carotene was more
degradation were 2-methyl-2-hepten-6-one; labile than capsanthin, and as in other studies
pseudo-ionone; 6-methyl-3,5-hepadien-2-one; degradation increased with temperature.319
geranial, and neral, and the following nonvolatile Crystals of α- and β-carotene were isomer-
compounds of β-carotene degradation were iden- ized by temperature, light, and iodine. The main
tified: aurochrome; mutatochrome; 5,6,5′,6′- components in isomerized samples were
diepoxy-β-carotene and 5,6-epoxy-β-carotene.89 all-trans-, 15-cis-, 9-cis-, and 13-cis-β-carotene
An oxidation study of β-carotene by peroxide and all-trans-, 15-cis-, 13-cis-, and 9-cis-α-caro-
radicals was carried out at 37°C in benzene, and tene (in decreasing order). Minimal isomerization
alkylperoxyl radicals were generated by was observed in the range 50 to 100°C/30 min,
2,2′-azobis (2,4-dimethylvaleronitril). The analy- but it was considerable at 150°C/30 min. The
sis of oxidation products suggest that β-carotene photoisomerization reactions were of first-order:
is destroyed by a free-radical-addition mecha- reaction constant was higher for the conversion
nism. Products were analyzed by HPLC, UV- toward all-trans-β-carotene, and with α-carotene
visible spectroscopy, MS, and NMR (proton and the 13-cis-isomer was favored. With iodine cata-
carbon-13).519 lyzed reaction, β-carotene degradation followed a
On the other hand, many studies have been first-order kinetics, while isomerization produced
carried out to evaluate the kinetics of carotenoid first- and second-order kinetics.98
degradation and/or biosynthesis. Degradation Photostability (sunlight 8 h/day) of marigold
mechanisms based on free radicals have been pigments was studied in aqueous emulsions with
proposed by using solid supports (carboxymeth- Arabic gum and/or mesquite gum. Stability in
ylcellulose, Al2O3 or MgO).174 Also, it has been emulsions with mesquite gum was better 3.83
established that low aw (around 0.3) diminish the times) than with Arabic gum. Also the synergistic
carotenoid degradation.174,192 Photodegradation effect between the two gums was observed. High-
2690 lx) of aqueous β-carotene has been studied. est stability was reached at pH 5.0. Kinetic deg-
226
radation was of zero-order. It was concluded that was observed, and the most affected carotenoid
polyelectrolytic nature and MW of gums are im- was β-carotene followed by β-cryptoxanthin and
portant in color protection, and dark emulsion hue zeaxanthin, while the most stable were capsanthin
could act as a sun filter.486 and capsorubin. Also, it was observed that drying
process produces a net carotenoid biosynthesis
that is enhanced by illumination: drying under
b. Processing and Stability in Foods darkness conditions increased the carotenoid con-
tent in 15%, while under illumination it was 47%.
Processing effects were analyzed in tomato Higher increments were observed in β-carotene,
and green vegetables (broccoli, spinach, and green cryptoxanthin, zeaxanthin, and capsanthin, while
beans). Moderate processing did not produce caro- violaxanthin and capsorubin were destroyed. The
tenoid modifications, but a prolonged heating biosynthesis occurred in a first stage (35 to 40%
(1 h boiling) conduces to total destruction of of moisture), then degradation was observed.318,320
epoxycarotenoids; it was shown that under this The effect of mango processing on carotenoid
condition only the most sensitive carotenoids are content was evaluated. Mango was sliced and
highly destroyed. Boiled tomato showed an iden- stored at vacuum or frozen (-40°C) conditions
tical carotenoid profile with fresh tomato, and during 6 months; other samples were commer-
differences in content were only detected.253 A
cially canned. Fresh and frozen fruits showed

similar result was obtained with vegetables used similar characteristics. Canned mango showed
in Spain, and it was mentioned that carotenoid great changes in color and carotenoid profile. The
saponification results in losses that are more criti- most stable carotenoid was β-carotene.73
cal in xanthophyll than in carotenes.184 All-trans-β-carotene isomerization sensitized
The effect of freezing and canning of kiwi by chlorophyll was studied. Main formed isomers
was analyzed by TLC, HPLC, UV-visible spec- were 9-, 13-, and 15-cis-β-carotene, with a higher
troscopy and chemical tests. The principal com- proportion of the 9-cis-isomer. In control without
ponents of fresh and frozen kiwi were 9′-cis- chlorophyll, isomerization was imperceptible dur-
neoxanthin, trans- and cis-violaxanthin, ing the first 2, then a quick increment was ob-
auroxanthin and lutein. In frozen kiwi stored for served during the first day and then remained
6 months (-18°C), the carotenoid profile was simi- constant. The main isomers under these condi-
lar to fresh fruit, but in addition antheraxanthin tions were 9- and 13-cis-β-carotene. The chloro-
was detected. On the hand, canned kiwi showed a phyll sensitizer effect supports the idea that cis-
complicated carotenoid profile because of ther- isomers in photosynthetic tissues are not
mal treatment. Neoxanthin and violaxanthin were artifactual.350
some of the degraded xanthophylls.74 Chen et al.99 studied the effect of juice carrot
Processing of fresh pepper to obtain paprika processing on its carotenoid content. Pasteuriza-
was studied. Carotenes and xanthophylls were tion 105°C/25 s) neither produced a considerable
extracted with acetone and identified by UV-vis- variation in the isomeric profile nor in the caro-
ible, infrared spectroscopy, and chemical tests; tenoid content. Treatment at 110°C/30 s produced
quantitation was carried out by UV-visible spec- the degradation of 45% of β-carotene while the
trometry. It was reported that fast drying induces isomers formed were in decrement order): 13-cis-
the carotenoid destruction and increments were > 15-cis- > 9-cis- > 13,15-di-cis-β-carotene, α-
observed by slow drying of pepper. It was pro- Carotene pattern was similar, but the main isomer
posed that an adequate drying process could con- was 15-cis-α-carotene. Lutein degradation was
duce carotenoid synthesis: in bola pepper, incre- around 30%, and the main isomers produced were
ments of red carotenoids were observed 13-cis > 9-cis. At 120°C/30 s; 48% of
(capsanthin, capsorubin, and capsolutein); how- β-carotene was lost. In the canning process (121°C/
ever, this effect was not observed in agridulce 30 min), an increment in β-carotene content was
pepper. Also, it was mentioned that during mill- observed, and the main isomers were 9-cis- and
ing process a high reduction in carotenoid content 13,15-di-cis-β-carotene. α-Carotene was reduced
227
in 60% and isomers were degraded after the can- It was shown that cooking in the presence of fat,
ning. Destroyed lutein was around 50%. Color carotenoid, and vitamin A reductions are lower
diminution was higher in more drastic treatments. because fat may dissolve and protect carotenoids.
Also, vitamin A destruction showed a similar It was recommended that blanching for a short
pattern: 55.7% of β-carotene was lost during can- time (5 min) and cooking in a pressure cooker are
ning.99 In contrast, it has been reported that can- better processing conditions for the retention of
ning of vegetables (e.g., broccoli, cantaloupe, car- β-carotene.512
rots, etc.) produces an isomerization all-trans- to During egg dehydration and storage, the ef-
cis-isomers of carotenoids. In sweet potatoes the fect of α-tocopherol supplementation was stud-
highest isomerization was observed and was the ied. It was shown that gas heater drying produces
lowest in broccoli. In processed red, yellow, and higher loses than electric resistance drying. It is
orange vegetables, the 13- and/or 13′-cis-isomer explained that during gas drying higher quantities
are favored, while in green vegetables it is the 9- of nitrogen oxides and other free radicals are
cis-isomer. The preponderance of the 9-cis-caro- formed, pepper oleoresin acts as a prooxidant,
tenoids in green vegetables was assigned to the and α-tocopherol stabilizes carotenoids and prob-
effect of chlorophyll as a sensitizer. Interestingly, ably permits the antioxidant action of caro-
the total carotenoid content was maintained after tenoids.269
canning and the provitamin A concentration was In the processing of saffron, drying tempera-
higher. Later observation may be due to an in- tures lower than 30°C or higher than 60°C pro-
creased extraction efficiency due to the disruption duced a greater carotenoid degradation. The opti-
of carotenoprotein complexes, inactivation of mum temperature of drying was 40 ± 5°C,
carotene-oxidizing enzymes, and/or loss of soluble independently of type of drying (sun or furnace
solids into the liquid canning medium.277 drying). Losses of 56.9 to 70.2% after 1 year of
Blanching (98°C /5 min), cooking (98°C for storage were observed (15 to 35°C, 10 to 12%
15, 30, and 60 min) and sun drying (25°C ± 6°C) moisture).386
are traditional processing practices. These meth- To preserve color in carotenoid pigmented-
ods were used in the processing of Tanzanian foods, the antidiscoloring effect of green tea
vegetables used for human consumption (ama- polyphenols [epigallocatechin 3-gallate (EGCg),
ranth, cowpea, peanut, pumpkin, and sweet po- gallocatechin (GC), gallocatechin 3-gallate (GCg),
tato leaves). In general, blanching resulted in a epigallocatechin (EGC), -)-epicatechin EC) or (+)-
reduction of β-carotene and in an increment of catechin (C)] in beverages and in margarine was
α-carotene concentration. Cooking enhanced the assayed. In general, it was observed that tea
quantity of carotenoids extracted, and sun drying polyphenols and several tea catechins have stron-
reduced the concentration of carotenoids in all the ger effects against the discoloration of β-carotene
evaluated vegetables. Also, it was shown that than L-ascorbate (widely used to prevent discol-
thermal processing increased the vitamin A activ- oration) in the aqueous (beverages) and oily (mar-
ity of all vegetables, but in amaranth leaves a garine) systems. The strength of antidiscoloring
reduction was observed. Thus, blanching and activity was GCg, EGC, EGCg, GC >> gallic acid
cooking could be considered as advantageous (GA) > L-ascorbate, EC, C. According to the ob-
processes for increasing the amount of provita- served order, it was established that activity de-
min A available when vegetables are consumed. pends on the presence of an hydroxyl group at the
It was suggested that consumption of 100 g of dry 5′ position, and it was suggested that the hydro-
weight of these vegetables cover the recommended gen radical from the phenolic hydroxyl group of
daily intake for vitamin A for both children and catechin stabilizes the peroxide radical formed
adults.333 In the processing of bathua (Chenopo- during the oxidation process. Thus, tea polyphe-
dium album) and fenugreek (Trigonella foenum nols might be used as excellent antidiscoloring
graecum) leaves that are consumed in India, compounds of β-carotene.479
blanching, cooking (using hydrogenated fat), and Poultry feed pigmented with marigold oleo-
sun drying resulted in the reduction of carotenoids. resin (lutein is the main carotenoid) was exposed
228
to sunlight. Poultry fed with sun-exposed feed ionic interactions, consequently, protein activ-
showed better pigmentation than control feed ity is also modified. Another suggestion indi-
(maintained in darkness). It was proposed that cates that membranes are modified and the cell
sunlight induces conversion of lutein to redder content is lost.
pigments (e.g., astaxanthin).149 However, in our Kinetic studies of carotenoid degradation have
lab it was shown that sunlight illumination pro- been also carried out in food systems and in gen-
duces a favorable equilibrium toward all-trans- eral, as in model systems, they are characterized
lutein isomer (trans-isomer showed redder hues by first-order kinetics. When carrot juice was
and high pigmenting efficiencies), but redder caro- used as model, photodegradation and photo-
tenoids were not observed. Additionally, we isomerization reaction were slower than in syn-
showed a better egg yolk-pigmenting efficiencies thetic models, thus some protective factors must
of marigold meals exposed to sunlight than con- be present in juice. In carrot juice, the main iso-
trols; and also we demonstrated that other compo- mer was 13-cis-β-carotene.367 In other studies,
nents different to carotenoids are participating in carrot juice was acidified, pasteurized, and then
pigmentation efficiency.268 subjected to light or dark storage at different tem-
Marigold pigments have also been used in peratures (4, 25, and 35°C) for 3 months. Tem-
the pigmentation of shrimp (Penaeus vannamei). perature had a negative effect on lutein content
It was found that saponified extracts were better (higher degradation at 35°C), and it was observed
pigmenting agents than esterified marigold ex- that the formation of 13-cis-lutein is favored.
tracts; its study suggested that lutein and zeax- Under light conditions the behavior was similar,
anthin (main carotenoids of marigold) can be although lutein degradation was higher than
metabolized into astaxanthin and deposited in in darkness storage. α-Carotene showed
shrimp and better colorations could be obtained a similar behavior than lutein; however,
than with astaxanthin supplemented feed.487 On 13-cis-α-carotene is formed preferentially
the other hand, in the pigmentation of white- in dark conditions (although the 9-cis-
fleshed fish, carotenoids must be eliminated from isomer increases also), and 9-cis-α-carotene
the corn gluten meal (CGM) used in feeds. In the is the preferred isomer in light conditions. The
corn gluten bleaching, it was established that concentration change of β-carotene and its iso-
carotenoid extraction with ethanol is a good pro- mers was similar to the other carotenoids, but β-
cess, but economical benefits could be enhanced carotene was more susceptible to degradation.
by using soybean flour (5% substitution level) as Vitamin A degradation showed a correlation with
a bleaching agent by its content of lipoxygenase carotenoid degradation.101 In saffron, pigment deg-
and because the high content of protein in soy- radation showed a first-order reaction with the
bean.359 effect of light, heat, and pH.475 During olive pro-
The use of high pressure (9000 times over cessing, lutein and β-carotene did
atmospheric pressure) to kill contaminating or- not show variations, while violaxanthin and
ganisms in food has been proposed.210 It was neoxanthin content decreased in parallel with
reported that sensorial characteristics, such as auroxanthin and neochrome increments. By con-
flavor and color, are unscathed. In Japan, this sidering that total carotenoids remained constant,
methodology has been used in the preparation thus processing produced a conversion of 5,6-
of fruit jams, yoghurts, and yomogimochi epoxyde carotenoids (violaxanthin, neoxanthin)
(steamed rice paste mixed with wild herbs). to 5,8-epoxydes (auroxanthin, neochrome) with
However, some vegetables showed undesirable first-order kinetics.317
characteristics after pressure treatment: grapes Koplas-Lane and Warthesen262 studied spin-
were hardened, cabbage were softened, and ach and carrot photostability (2000 l × /8 days).
mushroom and potato were oxidized. It was Carotenoids were degraded with first-order kinet-
suggested that the killing of organisms by pres- ics. Lutein was the most and violaxanthin the
sure could be assigned to modifications of pro- least stable carotenoids. Carrot carotenoids were
tein structure by affecting ionic bonds and/or not affected by light and showed higher stability
229
than spinach carotenoids. Interestingly, during intensity and quickly rising to 190 µmol m-2 s-1
carrot storage in darkness/6°C, chromogenesis was permitted obtaining the maximum carotenoid con-
observed and samples showed 11% more total tent in H. lacustris. Also, echineone and
carotenoids after 60 days of storage.262 canthaxanthin were identified in the cultures, sup-
Stability of carotenoid extracts from saffron porting the idea that astaxanthin is synthesized
(Crocus sativus L.) was studied at different water from β-carotene via echineone and canthaxanthin.520
activities (0.11, 0.23, 0.33, 0.43, 0.53, 0.64, and Other studies in vivo518 and in vitro110 have shown
0.75) and temperatures (25, 40 and 60°C). Caro- that high astaxanthin production required high lev-
tenoid degradation under all storage conditions els of oxygen (aerobic conditions). Additionally, it
and with a first-order kinetics was observed. Also, has been shown that cell growth requires low
a higher rate of degradation associated with incre- C/N ratios and astaxanthin production of high C/N
ments in water activities was shown. This result ratios. The results showed that pigment production
contrasts with those obtained with oil-soluble caro- by X. dendrorhous is affected by fermentation,
tenoids. Crocin, the main carotenoid in saffron, is respiration, and anabolism. It was suggested that
water soluble, and it was suggested that more high O2 levels are required for NADH oxidation,
available water could enhance the mobility of inhibiting the ethanol production (fermentation)
solution components (carotenoids and free radi- and enhancing respiration. On the other hand,
cals), inducing a higher degradation. Also, it was astaxanthin production required large amounts of
mentioned that higher stability of carotenoids NADPH and high C/N ratios, and high oxygen
might be reached at temperatures below the glass levels favor this condition. Thus, to optimize the
transition temperature, where solution components astaxanthin production, it is necessary to optimize
are at fixed positions and degradation reactions the synthetic pathway of astaxanthin and optimize
are more difficult.474 the metabolic pathway anabolic and respiratory),
and, consequently, it was proposed that optimal
astaxanthin production is reached by a two-stage
c. Production of Carotenoids in Bioreactors cultivation: the growth stage (first) in medium with
low C/N ratio and the astaxanthin production stage
An important approach for carotenoid produc- (second) in high C/N ratio.518 Also, it was sug-
tion consists of the culture of algae and bacterial gested that the addition of ethanol during the sec-
strains. The fungus Blakeslea trispora is a well- ond stage enhanced the production of astaxanthin
known β-carotene producer, and it has been shown 2.2 times more than control without ethanol.517
that cell growth and β-carotene production are Pine (Pinus pinaster) wood meals were used in the
enhanced by using surfactant agents such as Span production of carotenoids. Pine meals were treated
and Triton, except with Triton X-100.254 Another with acid and enzymatic hydrolysis. The enzymes
interesting model for β-carotene production is used were cellulases from Trichoderma reesei
Phycomyces blakesleeanus, and mutants carS have (Celluclast) and cellobiase from Aspergillus niger
been reported that accumulate up to 100 times the (Novozym). This material was incubated with
wild-type level (2 to 5 mg β-carotene/g dry myce- preproliferated broths of Xanthophyllomyces
lium). CarS mutants were exposed to N-methyl- dendrorhous (formerly Phaffia rhodozyma), and it
N′-nitrosoguanidine and the mutant S276 was ob- was determined that enzymatic hydrolysates be-
tained. S276 contained 9.2 ± 1.6 mg of β-carotene/ have as suitable culture media for proliferation of
g dry mycelium, and it was suggested that mutated X. dendrorhous, reaching good growth rates (up to
gene product probably activates carotenogenesis.307 0.07 h-1) with high cell yield (0.47 g biomass/g
The alga Haematococcus lacustris contains large consumed glucose) and carotenoid concentrations
amounts of astaxanthin esters and has been consid- up to 1.8 mg carotenoids/l. Thus, it was suggested
ered as a potential source of astaxanthin. Recently, that wood hydrolyzates are potential substrates for
it was established that higher light intensities re- carotenoid production with X. dendrorhous.357
sulted in the accumulation of more astaxanthin Astaxanthin production was also assayed in thin
esters in the cells: culture at 90 µmol m-2 s-1 of light stillage as substrate and a nitrosoguanidine mutantof
230
FIGURE 15. Basic structure of anthocyanidin pigments in which Rx could be H, OH, or OCH3 depending of the
considered pigment. The most common accepted nomenclature for numbering carbons is indicated inside the
structure.
X. dendrorhous called JB2. The carotenoid pro- the Greek anthos, a flower, and kyanos, dark blue)
duction was increased 2.3 times in relation to the are flavonoids (flavan like), and consequently
wild-type cells (1.54 µg carotenoid/mg dry weight). based on a C15 skeleton with a chromane ring
Thus, it was suggested that a strain like JB2 has bearing a second aromatic ring B in position
potential for commercial production of astaxanthin 2 (C6-C3-C6) and with one or more sugar mol-
from corn byproducts.50 Also, Haematococcus ecules bonded at different hydroxylated positions
pluvialis has been suggested for the production of of the basic structure (Figure 15). Anthocyanins
astaxanthin, and recently compactin-resistant mu- are substituted glycosides of salts of phenyl-2-
tants were isolated (compactin is an inhibitor of the benzopyrilium (anthocyanidins).87,455
HMGR that strongly blocks cholesterol formation).
Selected mutants showed growth comparable with
wild-type cells, while the astaxanthin content was 2. Classification
increased in 1.4 to 2.0 times. The specific HMGR
activity was reduced in 14 to 27%, and it suggested The basic C6-C3-C6 anthocyanin structure is
the presence of specific HMGR for astaxanthin the source of an infinity of colors produced by its
production.109 chemical combination with glycosides and/or acyl
groups (Figure 15) and by its interaction with
other molecules and/or media conditions. 62
B. Anthocyanins Harborne and Gryer195 mentioned the existence of
17 anthocyanidins, with differences in the num-
1. Definition ber and position of hydroxyl groups and/or me-
thyl ether groups, but six of them are the most
Anthocyanins are the most important group common anthocyanidin constituents of this kind
of pigments, after chlorophyll, that are visible to of pigments (Table 4). From these 17 structures
the human eye.195 Chemically, anthocyanins from combinations have arisen with at least one sugar
231
TABLE 4
Anthocyanidins Found in Nature
Substituted with a characteristic hydroxyl group
Namea Position of substitution Some of the produced colors
Apigeninidin 5,7,4’ Orange

Aurantinidin 3,5,6,7,4’ Orange
Cyanidin 3,5,7,3’,4’ Magenta and Crimson
Delphinidin 3,5,7,3’,4’,5’ Purple, mauve and blue
6-Hydroxycyanidin 3,5,6,7,3’,4’ Red
Luteolinidin 5,7,3’,4’ Orange
Pelargonidin 3,5,7,4’ Orange, salmon
Triacetidin 5,7,3’,4’,5’ Red
Substituted with a characteristic methyl ether group
Capensinidin 5,3’,5’ Bluish red

Europenidin 5,3’ Bluish red

Hirsutidin 7,3’,5’ Bluish red
Malvidin 3,5’ Purple
5-Methylcyanidin 5 Orange red
Peonidin 3’ Magenta
Petunidin 3’ Purple
Pulchellidin 5 Bluish red
Rosinidin 7,3’ Red
a Underlined names represent the most common anthocyanidins.

Adapted from Refs. 157, 193.
molecule to obtain anthocyanin compounds. Thus, ber of hydroxyl groups tend to deepen the color to
anthocyanins have also been classified in agree- a more bluish shade. On the other hand, incre-
ment with the number of sugar molecules that ments in the number of methoxyl groups increase
constitute their molecules (i.e., monosides, redness (Table 4). With all of these facts in mind,
biosides, triosides) and, interestingly, the number it must be not difficult to understand the gamut of
of probable compounds is greatly increased by colors observed in nature that is produced from a
taking into account the sugar diversity and all the single structure.
possible structural points of glycosylation, al-
though the order of sugar occurrence in natural
anthocyanins is glucose, rhamnose, xylose, galac- 3. Distribution
tose, arabinose, and fructose. Additionally, many
anthocyanins have shown in their structures ester Anthocyanins are responsible for many of
bonds between sugars and organic acids i.e., the attractive colors, from scarlet to blue, of
acylated anthocyanins), and in nature the most flowers, fruits, leaves, and storage organs.193,195
common acyl groups are coumaric, caffeic, feru- They are almost universal in higher plants, but
lic, p-hydroxy benzoic, synapic, malonic, acetic, in general anthocyanins seem absent in the liv-
succinic, oxalic, and malic.157 Moreover, substitu- erworts, algae, and other lower plants, although
tion of hydroxyl and methoxyl groups influences some of them have been identified in mosses
the color of anthocyanins. Increments in the num- and ferns.455 The type of anthocyanins in plants
232
is so variable that some ornamental plants 4. Biosynthesis: Biochemistry and
present only one main type of anthocyanin Molecular Biology
(Dianthus, Petunia), whereas others have mix-
tures (Rosa, Tulipa, and Verbena). On the other a. Biochemistry
hand, some fruits are a source of one anthocya-
nin: cyanidin in apple, cherry, fig, and peach; The phosphorylated compounds phospho-
delphinidin in eggplant and pomegranate; some enolpyruvate (glycolytic pathway) and erythrose-
fruits have two main anthocyanins such as 4-phosphote (pentose phosphate respiratory path-
cherry sweet and cranberry (cyanidin and way and Calvin cycle) are the precursors of
peonidin), while others have several anthocya- shikimic acid. Shikimic acid and acetate are the
nins (grape). In general, the anthocyanin con- precursors of the primary aromatic building stones
centration in most of the fruits and vegetables of many phenolic compounds, including antho-
goes from 0.1 up to 1% d.w.455 cyanins. Shikimate pathway gives several organic
Anthocyanins are vacuolar pigments and acids, such as cinnamic, p-coumaric, caffeic, feru-
in this organelle the presence of membrane lic, chlorogenic, and phenylalanine. By using
bound bodies called anthocyanoplasts has been chemogenetic studies and labeled precursors, it
proposed; these structures are formed while was shown that the shikimic acid product, pheny-
pigment synthesis is in operation, and eventu- lalanine, is incorporated in the C6-C3 (B aromatic
ally they are dispersed to produce a totally rings and carbons corresponding to the central
pigmented vacuole. In flowers, anthocyanins pyran ring) portion of the basic flavonoid struc-
are almost exclusively located in epidermal ture (Figure 3). On the other hand, ring A and the
cells, and only occasionally in the mesophyll. oxygen of the central pyran ring are provided by
On the other hand, in the leaves of rye (Secale acetyl-CoA. In addition, it is remarkable that elu-
cereale) they are restricted to the mesophyll cidation of anthocyanin biosynthesis and genetics
cells.195 Interestingly, flavones co-occur with have been carried out mainly by using undifferen-
anthocyanins and participate in the color of tiated cells of parsley, maize, snapdragon, and
plants as copigments (substances that petunia.193,195,455
contribute to anthocyanin coloration by pro- Anthocyanin biosynthesis pathway could be
tecting the anthocyanin molecules). The divided into two main parts: (A) precursors of the
copigmentation mechanism is unique to the general phenylpropanoid metabolism, and (B) spe-
anthocyanin family. Anthocyanins also react cific steps toward flavonoid biosynthesis (Figure
with alkaloids, amino acids, benzoic acids, 17). In the first part, phenylalanine is converted to
coumarin, cinnamic acids, and a wide variety p-coumaryl-CoA by effect of three enzymes: phe-
of other flavylium compounds. This weak nylalanine ammonia-lyase (PAL), cinnamate-4-
association is termed intermolecular co- hydroxylase (C4H), and 4-coumaryl-CoA ligase
pigmentation. Intramolecular copigmentation is (4CL). p-Coumaryl-CoA is the main precursor of
due to the acylation in the molecule, and it is flavonoids, lignin, and other phenylpropanoids
more effective than intermolecular; in acylated (Figure 17A). In the second part (Figure 17B),
anthocyanins, it is suggested that acyl groups chalcone synthase (CHS), which is considered
interact with the basic anthocyanin structure, the key enzyme in flavonoid biosynthesis, cata-
avoiding the formation of the hydrated spe- lyzes the condensation of three molecules of
cies. The basic role of copigments is to protect malonyl-CoA with 4-coumaryl-CoA to form the
the colored flavylium cation from the nucleo- intermediate chalcone. Chalcone synthase
philic attack of the water molecule.28,410 Other has been isolated and characterized
important phenomena that show a great con- from several sources (Dianthus, Verbena,
tribution to color is the self-association of Anthirrhinum). In the next step, chalcone shows a
anthocyanins, and in some models the contri- stereospecific isomerization to naringenin by the
bution of metal complexation have been sug- enzyme chalcone isomerase (CHI). Different CHI
gested (Figure 16). 63,146,157,193, 417 and tissue-specific isoenzymes have been charac-
233
FIGURE 16. Suggested mechanism of formation for the complex anthocyanin-metal-ascorbic acid. (Adapted from
Ref. 417.)
234
FIGURE 17. Anthocyanin biosynthesis pathway. (A) General phenylpropanoid metabolism. Enzymes involved: PAL, phenylalanine amonia lyase; C4H, cinnamate-
4-hydroxylase; 4CL, 4-coumaryl:CoA ligase. (B) Specific steps of anthocyanin biosynthesis. Enzymjes involved: CHS, chalcone synthase; CHI, chalcone isomerase;
F3H, F3′H, F3′5′H, flavonol hydroxylases; DFR, dihydroflavonol-4-reductase; ANS, anthocyanin synthase; 3GT, glucosyl transferase; MT, methyl transferase.
(Adapted from Refs. 193, 216, 328.)
235
terized. Narigenin is the common precursor to great specificity, thus multiple methylation re-
flavonoids and isoflavonoids; naringenin fla- quires multiple enzymes. In Petunia hybrida flow-
vanone) is converted to dihydrokaempferol fla- ers at least four methyltransferases have been
vone), by a dioxygenase or a monooxygenase, involved (Mt1, Mf1, Mt2, and Mf2).
depending on the tissue. In petunia, the enzyme On the other hand, it has been proposed that
flavanone-3-hydroxylase (F3H) (dioxygenase) is acyl transferases show high specificity, and
involved. In the next stage, dihydrokaempferol in some instances acylation must precede
is converted to leucoanthocyanidin by glycosylationin the acylation of anthocyanins. Acyl
dihydroflavonol-4-reductase (DFR), which is transferases have been described in Silene,
NADPH or NAD dependent according to the plant Matthiola, Callistephus, Dendranthema, and Zin-
source. This intermediate has been demonstrated nia; they catalyze the transfer of 4-coumaryl or
by using acyanic flowers produced by different caffeoyl groups to the corresponding acylated an-
gene blocks and by the characterization of the thocyanins, and succinyl transfer is
flavan-3,4-cis-diol-4-reductase (catalyzes the cat- a noncommon event. An acyltransferase from blue
echin synthesis). Further ahead in the pathway, flowers of Centaurea cyanus was isolated. This
leucoanthocyanidins are transformed to the col- enzyme catalyzes the transfer of the succinyl moi-
ored anthocyanidins. This conversion is not well ety from succinyl-CoA to 3-glucosides of cyanidin
characterized, but involves an oxidation and a and pelargonidin, but not to

dehydration step; gene mutations corresponding 3,5-diglucosides. Also, it was reported that the
to these activities have been identified in several same enzyme catalyzes the malonylation at similar
plant species (A2 in maize, Candi in snapdragon, rates of succinylation.513 Another important modi-
ant17 in petunia), and this enzymatic activity has fication is sulfation, and sulfate transferase cDNA
been called as anthocyanidin synthase ANS). The was isolated from Flaveria bidentis.6 The enzymes
route is followed by the anthocyanidin-anthocya- of anthocyanin synthesis are probably cytoplasmic
nin conversion that involves a glycosylation reac- and attached to the vacuolar membranes, and it is
tion. In this process the best described enzyme is likely that after the glycosylation, anthocyanins are
the UDP-glucose:flavonoid 3-O-glucosyl-trans- transported into the vacuole.151,193,195,216
ferase (GT). Glycosyl transferases have shown a In anthocyanin-producing cells of sweet po-
pronounced specificity with regard to substrate, tato were identified vacuoles containing large
position, and sugar to be transferred. Recently, amounts of pigment and protein. This protein of
the first glycosyltransferase that catalyzes 24 kDa (VP24), which accounted for more than
the transfer of a sugar galactose) other than glu- 50% of the total protoplast protein showed a simi-
cose to anthocyanidins was reported. lar pattern of accumulation to that of anthocyanin.
The galactose:cyanidin galactosyltransferase It was observed that VP24 began to appear in
(CGT) was characterized from a cell suspension vacuoles at nearly the same time as the accumu-
culture of Daucus carota L. In this system a lation of anthocyanin became detectable, and it
xylosyltransferase activity that is separated was suggested that VP24 is involved in the
by anion-exchange chromatography of the intravacuolar accumulation of anthocyanin.345
galactosyltransferase activitywas also observed,
suggesting the presence of two separate enzymes,
each catalyzing a specific glycosyl transfer. Also, b. Biosynthesis Regulation
it was proposed that glycosyltransferases act in
sequential reactions, initiating with the Little is known about the regulation of the
galactosyltransferase and followed by the reac- biosynthetic enzymes of flavonoid synthesis, and
tion catalyzed by xylosyltransferase.401 Hydroxy- much of the knowledge has emerged from mo-
lation and methylation occur at different stages of lecular biology studies.62,328 The pronounced sub-
flavonoid biosynthesis. In particular, anthocyanin strate specificity of the 4-coumarate:CoA ligase
methylation is a late step, and the isolated fla- of different plants has been shown, indicating that
vonoid O-methyltransferases (MT) have shown a this enzyme is an important regulation point to
236
establish the distribution of CoA esters in plants. individual members.238 On the other hand, differ-
Additionally, external and developmental factors ential display was used to identify four new chs
showed an interdependent response of flavonoid genes from morning glories.163 In addition, two
biosynthetic enzymes. At least three photorecep- clones with high homology with flavonol syn-
tors have been suggested that control flavonoid thase and anthocyanidin synthase were identified
biosynthesis (red/far red, blue UV-A, or by using A. thaliana-expressed sequence tag
cryptochrome and UV-B), and anthocyanin bio- (EST). This EST permitted the isolation of the
synthesis showed different responses depending first flavonol synthase gene.366 CHS is encoded
on the light quality. In parsley plants, enzyme by a multigene family in most of the species
activities of flavonoid synthesis are high only in examined to date. Only Arabidopsis and Antirrhi-
young cotyledons and leaves, and a decrement num appear to contain single gene copies. It was
was observed in the later stages of develop- suggested that the evolution of CHS genes is a
ment.195,216 In Arabidopsis, mutants in several of highly fluid gene family where copy number var-
these photoreceptors have been identified and ies greatly among plant lineages and where func-
some of them involve products of the Cop, Det, tional divergence may occur repeatedly.130 Inter-
and Fusca genes.326 In Petunia, mutations at four estingly, another chs-related cDNA was isolated
loci (An1, An2, An4, and An11) have regulatory from Gerbera hybrida (Gchs2). Gchs2 did not
effects on transcription of at least six structural show correlation with the flavonol and anthocya-
anthocyanin biosynthetic genes.216 nin accumulation; its pattern of expression and
accepted substrates were different. Thus, it was
suggested that Gchs2 represents a new family of
c. Molecular Biology of Anthocyanin genes that have a common evolutionary origin
Biosynthesis with chs genes.205 In addition, it was proposed that
Gchs2 emerged as the result of the duplication of
Flavonoid biosynthesis has been selected as a a single gene, followed by subsequent differentia-
favorite pathway to carry out studies at the mo- tion during the evolution of Gerbera.206
lecular biology level; thus, this is the best known Other studies have evaluated the temporal
pathway of secondary metabolism. Many cDNA and spatial control of flavonoid biosynthetic genes.
and genomic clones involved in flavonoid synthe- Some genes showed organ-specific and others an
sis have been characterized, and some of them stage-specific regulation, and several researchers
have been cloned in bacteria, providing an excel- have been characterizing the gene promoter re-
lent model to produce the corresponding protein gions.141,409 In maize it has been suggested that
and to induce the in vitro production of valuable changes in kernel colors resulted from changes in
flavonoid metabolites.151 In 1983, chalcone syn- cis regulatory elements and not changes in either
thase gene was the first gene cloned of flavonoid regulatory protein function or in the enzymatic
biosynthesis by using differential screening, which loci, suggesting that differences in 5′ or 3′ regu-
was also employed to isolate PAL, 4CL, and F3H latory sequences or methylation patterns could be
from parsley. Moreover, antibodies were used in involved.191 The influence of DNA methylation
an expression library to clone the genes CHI and was also demonstrated in transformed petunia
F3H. Interestingly, flavonoid biosynthesis path- plants. Petunia plants that show dihydrokaempferol
way was a model to design a new technique for accumulation (defective in dfr gene) were trans-
cloning genes transposon tagging), and between formed with the corresponding enzymes of maize
others several regulatory genes were isolated.125,481 (A1) and gerbera (gdfr). gdfr transformants showed
In addition, new methodologies have been used to higher pigmentation than A1 transformants, and,
clone anthocyanin genes. Rapid amplification of particularly, it was observed that A1 transgene
cDNA Ends (RACE) by PCR was used to isolate inactivation was associated with a higher GC
the chalcone synthase gene 2 (chs2) from potato, content and an increased methylation state.134 In-
and by Southern analysis it was shown that chs terestingly, studies on chalcone synthase (CHS)
genes of potato comprise a family of at least six promoter of French bean showed that exists a
237
differential utilization of cis regulatory elements banylus can direct the biosynthetic pathway to-
for stress-induced (elicitors, wounding, and in- ward different metabolites.3 It is clear that regula-
fection) and tissue-specific expression, and it was tion is a very complex process, and, as mentioned
indicated that this multiple combination of dis- above developmental (maturation, germination)
tinct cis elements contributes to the complex ex- and external signals (light, temperature) are in-
pression pattern of CHS genes.140 It has been es- volved: VP-1 is a pleiotropic protein that acti-
timated that at least 30 genes are required for vates C1 specifically, during seed maturation of
flavonoid biosynthesis, and within these different maize, by interacting synergistically with one or
mutations have defined regulatory proteins whose more ABA-regulated transcription factors. How-
absence prevents the synthesis of multiple en- ever, there is no evidence that VP-1 can bind
zymes (probable transcription factors). It has been directly to DNA, and it has been proposed that
shown that chalcone synthase activity is under the protein-protein interactions have a principal role
control of other genes (f in Matthiola, niv in An- in the activation of promoters of downstream
tirrhinum, and c2 in Zea mays). In maize two genes.304 In maize and petunia, it was shown that
different families of regulatory genes have been duplicated sequences of regulatory genes exert a
established, C1 and R. The expression of one of critical role in gene regulation. Therefore, the
these genes induced a pleiotropic effect that in copy number must be strictly controlled by as yet
some instances is observed as an increment in the unidentified mechanisms, but indicating that DNA
anthocyanin biosynthesis, and it has been ob- methylation is involved.467 Moreover, it has been
served that maize anthocyanin synthesis requires established that maize-anthocyanin biosynthetic
the expression of at least one gene of each fam- pathway can be considered as cold-regulation
ily.125,328 Additionally, it has been observed that genes that in addition are related with water stress
regulatory systems of anthocyanin synthesis are but not with heat stress. It was observed that a
conserved between plant families: transformation moderate cold (10ºC) enhances the transcription
of petunia plants with genes of the C1 and R and/or stabilities for the anthocyanin biosynthesis
families induces the accumulation of anthocya- genes; however, at 5ºC the biosynthetic ability is
nins. In petunia three regulatory mutants damaged. At 10ºC stress, it was observed a dra-
(an1, an2, an11) showed accumulation of matic increase in transcript abundance for antho-
dihydroflavonols, and it has been suggested that cyanin regulatory and structural genes, but only a
An2 gene is an R-homologue, while An1 is C1- slight increase in anthocyanin pigmentation that
homologue.125 Also, it was shown that the Anti- is increased severalfold after plants are shifted
rrhinum del gene, tomato Lc gene, and the maize back to higher temperatures, showing that 10ºC
R gene family are structurally and functionally stress impairs postranscriptional processes impor-
homologous.175,178 When plants of two Solanaceae tant for anthocyanin biosynthesis.108 By contrast,
species (tomato and tobacco) were transformed low temperature enhances the pigmentation and
with the del gene, an increment in anthocyanins chalcone synthase gene expression in petunia flow-
was observed in the pigmented regions, but ers, similarly to the observed in apple skin and in
this effect was not found in del-transformed flower buds.326,442
Arabidopsis, indicating that some genes may not In eggplant hypocotyl-tissue was identified a
be able to function effectively in all plant hosts.329 P450 cDNA clone that is inducible by white light.
To complicate more the understanding of the At the same time, white light induces the accumu-
anthocyanin biosynthesis regulation, negative lation of CHS and DFR mRNA in a coordinated
regulators have been identified in Arabidopsis fashion, and it is proposed that this P450 enzyme
seeds (fus and banylus). The presence of fus and catalyzes the hydroxylation of the B-ring in fla-
banylus products prevents the accumulation of vonoids, that is, the P450 isolated clone encodes
pigments. Additionally, other negative regulators for a 3′,5′-hydroxylase that acts during anthocya-
have been isolated (eluta from Antirrhinum flower, nin biosynthesis.468 Interestingly, it has been shown
C1-1 from maize aleurone layer, fusca from that phytochrome stimulates anthocyanin biosyn-
Arabidopsis embryo). It has been proposed that thesis through the induction of 3′,5′ cyclic mono-
238
phosphate (cGMP) acting alone or together with The world market of flowers is very impor-
calcium.55 In Perilla frutescens, all the anthocya- tant, and the knowledge of the biosynthetic route
nin biosynthetic genes were coordinately induced of anthocyanins and molecular biology techniques
by strong light (chs, f3h, dfr, ldox, 3gt, aat).176 In allows to modifying flower colors.215,220,243 Petu-
Arabidopsis, it was shown that light-induced sig- nia DFR can not reduce dihydrokaempferol, thus
nals converge on the fusca genes that relay the wild type petunias do not produce pelargonidin
information to different plant responses (cell ex- pigments. Petunia plants were transformed with
pansion, chloroplast differentiation, tissue-specific maize DFR and mutant petunias that accumulate
changes).322 In addition, it the hy4 gene that codes pelargonidin were obtained.312 On the other hand,
for the CRY1 protein and have the characteristics petunia flowers with reduced pigmentation were
of a blue-light photoreceptor was identified. Thus, obtained by transforming with the chs gene of
CRY1 must be responsible for anthocyanin bio- petunia in anti-sense. 482 White-flowering
synthesis under UV-light irradiation.283 It was transgenic chrysanthemums have been produced
found that UV-A and UV-B induce different re- by trans-inactivation of the main chs gene, and a
sponses. UV-A induction of CHS expression does similar strategy was used to obtain a range of
not appear to involve calmodulin, whereas UV-B color for carnation and rose flowers.88,188 Interest-
response does. Consequently, the light signaling ingly, the effect of anthocyanins on sexual pro-
pathway showed differences between them and cess has been proposed to be manipulated by
with phytochrome-mediated response.27,107 On the molecular biology as an alternative to produce
other hand, the gibberellin GA3 stimulates the male sterile hybrid crops.326 In alfalfa f3h corre-
anthocyanin synthesis on petunia corollas, while lates with flavonoid accumulation, and particu-
it is completely inhibited in cultured carrot cells. larly f3h expression in pollen grains and ovary
In petunia corollas, GA3 is essential for the in- probably could be associated with plant fertility.95
duction of chs, chi, and dfr expression, probably It was also demonstrated in tobacco that flavonoids
by effect of a regulatory protein receptor), be- do not only determine flower color, but also play
cause after GA3 addition exists a lagging stage an essential role in the development of male ga-
until the response can be observed. Interestingly, metophyte. The stilbene synthase that competes
it was observed that the presence of sucrose is for primary precursors with chalcone synthase
required to observe the effect of GA3.325,326,502 was overexpressed, and, consequently, flavonoid
Similarly, it has been shown that the cytokinin production was impaired (reduced color) and male-
benzyladenine (BA) treatment induces a red pig- sterile plants were obtained.147 On the other hand,
mentation in Arabidopsis seedling, showing that the rapid quality improvement of some cultivars
CHS and DFR appear to be transcriptionally ac- requires of selection at the seedling stage, and
tivated, while PAL and CHI are controlled molecular biology provides excellent tools to
postransciptionally.116 develop this selection. Random-amplified poly-
The last step in anthocyanin biosynthesis in- morphic DNA (RAPD) was used to identify red-
volves the transfer and deposition of the red and der apple cultivars, and one marker was identified
purple pigments in vacuoles. In maize, this step is for this characteristic. It was suggested that this
carried out by the protein encoded by the bronze- marker is associated with a regulatory gene of the
2 gene (bz2), and it was shown that bz2 codes for anthocyanin biosynthesis.102
a glutathione-S-transferase (GST) with activity in Another interesting function was suggested
maize, transformed A. thaliana plants, and E. coli. by transforming a wine yeast strain with the an-
In bz2 mutants, cyanidin-3-glucoside is accumu- thocyanin-β-glucosidase gene of Candida
lated in cytoplasm, whereas in bz2 plants antho- molischiana. Transformed S. cerevisiae produces
cyanins are transferred to vacuole. It was sug- wines with quality characteristics comparable to
gested that anthocyanin import into vacuoles could those obtained with the wild-type strain. This new
occur as anthocyanin-glutathione conjugates, par- strain is of industrial relevance mainly in the pro-
alleling the mechanism by which plants eliminate duction of white wines with lower red color, al-
herbicides and xenobiotics.296 though they were obtained from red grape variet-
239
ies. Additionally, transformed S. cerevisiae was nomic markers, although this goal has not been
proposed for the production of wines with higher achieved yet because of nowadays only a limited
levels of resveratol and terpenols from the hy- number of species levels within families have
drolysis of their glycosidic precursors.414 As pre- been investigated. However, interestingly, the
viously mentioned, food industry is interested in preferred presence of cyanidin has been suggested
pigments with higher stability and acylation of as a marker of ancestral plants.193,195,455 In this
anthocyanins produces more stable pigments. aspect, anthocyanins with substitution (sugar or
Anthocyanidin-3 glucoside rhamnosyltransferase acid) in the sugar 3-position have only been re-
(UFGT) of Antirrhinum majus was used to transported in the genus Allium;153 in the genus Bego-
form lisianthus (Eustoma grandiflorum Grise.). nia a pattern of six anthocyanins as marker (from
Transformed lisianthus plants were able to pro- a survey of 200 begonia cultivars)was suggested,105
duce a novel 3-O-glucosylated flavonols. Inter- and acylated anthocyanins with 5-O-
estingly, C-3 glucosylated anthocyanins have (6-O-hydroxycinnamoylglucoside), where
never been observed in this plant.433 On the other hydroxycinnamic acids are coumaric or caffeic
hand, sometimes it is recommendable to diminish acids, have only been reported in the Gentiana
the quantity of anthocyanins and to increase the genus.223
content of other metabolites. The presence of con-
densed tannins (CT) in alfalfa has supported a

higher sheep productivity higher weight and wool b. Marker for Good Manufacturing
production) than CT-free alfalfa. Recently, it was Practices in Food Processing
shown that cultivars with higher CT content could
be obtained by molecular biology. It has been Anthocyanins have been used to evaluate the
established that dfr is involved in CT biosynthe- adulteration of some pigmented food products.56
sis, and some Lotus corniculatus plants trans- Prune juice is a product in which brown color is
formed with the Antirrhinum majus dfr cDNA developed by the reaction of phenolic compounds
showed increased CT levels. and/or anthocyanins, and it is possible the adul-
teration of prune juice with other fruit juices im-
prove its color. To control this possible source of
5. Functions adulteration, it is believed that prune juice can
have only traces of anthocyanins, while the adul-
a. Color and Ecological Functions terated juice will show increased levels.485 Also,
anthocyanin profiles have been used to determine
As flavonoids, anthocyanins are benzopyran the authenticity of fruit jams. With this kind of
derivatives. Thus, anthocyanins also showed simi- analyses, it was determined that labeled black
lar functions in plants to those described in the cherry jams in reality were prepared with com-
corresponding section for flavonoids: antioxidant, mon red cherries (less expensive fruit). In addi-
photoprotection, defense mechanism, as well as tion, it was suggested that adulteration of black-
other ecological functions (symbiosis phenom- berry jams with strawberries can be detected with
ena). In particular, anthocyanins are the most analysis of the relation between pelargonidin and
important pigmenting compounds between the cyanidin 3-glucoside. Also, it was pointed out
flavonoids, and consequently they show an inter- that this methodology is very efficient because
esting role in several reproductive mechanisms of anthocyanins are pretty stable during jam manu-
plants such as pollination, seed dispersal, and facture.165
antifeedant. Interestingly, it has been observed
that cyanidin-3-glucoside is inhibitory to the lar-
val growth in tobacco budworm Heliothis c. Pharmacological Effects
viriscens, and consequently anthocyanins could
be considered agents of biological control. Addi- Reports on biological activities of anthocya-
tionally, anthocyanins have been proposed as taxo- nins are scarce, but considering the wide distribu-
240
tion of anthocyanins, it is reasonable to assume aglycones of high number of OH groups in the
that humans are well conditioned to large con- B-ring, opposite to that observed with other fla-
sumptions of these compounds. In a survey with vonoids, while O2- scavenging is independent of
Italian subjects, anthocyanin daily intake was in the glycosylation state but also increases with the
the range 25 to 215 mg/person, depending on number of hydroxyl groups, similar to the ob-
gender and age, and this intake is largely enough served with other flavonoids.476 In the red colorant
to induce pharmacological effects. The consump- extracted from Anonia a mixture of anthocyanins
tion of wine flavonoids has been correlated with (cyanidin, cyanidin-3-glucoside and cyanidin-3,5-
low incidences of coronary hearth diseases (French diglucoside) and polyphenol substances called
paradox), and, similarly, chokeberry (Anonia bioflavonoids (leucoanthocyanidins, catechins,
melanocarpa) extracts have shown very strong and flavonols) has been identified. In particular,
nutraceutical properties. Moreover, anthocyanins bioflavonoids have shown activities to improve
possesses bactericidal, antiviral, and fungistatic the permeability and strength of capillaries, to
activities. They exhibit a strong antioxidant activ- accelerate the ethanol metabolism, and to reduce
ity that prevents the oxidation of ascorbic acid, inflammatory and edematic reactions.286 Similar
provides protection against free radicals, shows effects have been observed with crude extracts of
inhibitory activity against oxidative enzymes, and Rubus occidentalis, Sambucus nigra, and
Vaccinium myrtillus.157,195
has been considered as important agents in reduc-

ing the risk of cancer and heart disease.58 Also,
there is information indicating that 3′- and 4′-OH
in the B ring structure are determinants for the 6. Methodological Aspects
radical scavenging potential in flavonoids with a
saturated 2,3-double bond, and that different pat- a. Extraction
terns of hydroxylation and glycosylation may
modulate their antioxidant properties.499 It has Anthocyanin, like flavonoids in general, have
been mentioned that an increased number of hy- aromatic rings containing polar substituent groups
droxyl substituents on the B-ring produce higher (hydroxyl, carboxyl, and methoxyl) and glycosyl
antioxidant activities when are glucosides, but the residues that altogether produce a polar molecule.
activity is weaker with aglycones.476 These two Consequently, they are more soluble in water than
hydroxyl groups are important in protecting ascor- in nonpolar solvents, but depending on the media
bic acid against oxidation by chelating metal ions. conditions anthocyanins could be soluble in ether
When the anthocyanin-metal chelate is formed, a at a pH value where the molecule was unionized.
20- to 25-nm shift in the visible spectra is ob- These characteristics aid in the extraction and
served, which in the presence of ascorbic acid separation of anthocyanin compounds.193 Conven-
induces a further shift of 10 to 15 nm. It was tional methods of pigment extraction usually
proposed the formation of the stable complex employ dilute hydrochloric acid in methanol.
anthocyanin-metal-ascorbic acid, where ascorbic Methanol containing 0.001% HCl was the most
acid acts as a copigment. Thus, improved colors effective, but HCl is corrosive, and methanol pro-
are obtained with the intrinsic protection of ascor- duces toxic effects after human exposition; con-
bic acid from oxidation (Figure 16).417 sequently, food scientists frequently prefer the
The effect of pure anthocyanins against lipid use of other extraction systems. Among other
peroxidation has been studied. Liposomes were solvents, one finds ethanol and water, 80 and 27%
used to evaluate the inhibition in the production as effective as methanol, respectively. Addition-
of malondialdehyde. All evaluated anthocyanins ally, it must be taken into account that aromatic
were better agents against lipid peroxidation than acyl acid linkages are relatively stable in dilute
α-tocopherol (up to seven times). Also, it was HCl/MeOH mixtures, but aliphatic dicarboxyl acyl
demonstrated that anthocyanins have scavenging groups (malonic, malic, oxalic) are susceptible to
properties against ·OH and O2-. In addition, it was diluted acids, and different methodologies must
mentioned that ·OH scavenging is better with be considered. It is recommended to use weaker
241
acids (acetic, formic, perchloric) during extrac- vonoids show high absorbance in the range of 250
tions and to monitor the acidity during the pro- to 270 nm (UV region), and, particularly, antho-
cess. With methanol, citric acid is the most effec- cyanins have an intense absorption in the range of
tive organic acid followed by tartaric, formic, 520 to 560 nm (visible region). It has been sug-
acetic, and propionic; with water, the best acids gested that UV absorption could be assigned
are acetic acid, citric, tartaric, and hydrochlo- mainly to ring A, while the visible to the pyran
ric.58,157,195,455 Recently, an aqueous extraction pro- and ring B. With this methodology, researchers
cess for anthocyanins from sunflower hulls was suppose that identity between the isolated pig-
evaluated. It was shown that extraction with sulfu- ments and those found in living tissue is the same;
rous water (1000 ppm SO2) was better than the however, the main problems are the generation of
traditional extraction with ethanol:acetic- artifacts during the extraction process. To solve
acid:water system. Also, it was mentioned that this problem, model systems such as anthocya-
1 h of extraction was enough to reach a complete nins stored in synthetic vacuoles have been devel-
extraction of pigments. It was suggested that pos- oped. With UV-visible spectroscopy, it is also
sible reasons for the improved extraction with possible to detect glycosylation on B-ring, be-
SO2 are the interaction of anthocyanins with HSO3- cause of the spectra show an hypsochromic shift
ions, which improves the anthocyanin solubility in relation to the unglycosylated B-ring. Antho-
and the diffusion through cell walls.164
cyanin acylation is also observed by this method-

ology: in the presence of AlCl3 a bathochromic
shift is observed, only if the 3′- and 5′-OH groups
b. Separation are free (nonacylated).462 Moreover, acylated an-
thocyanins show a maximum absorbance in the
The initial attempts for the separation of an- 310-nm region, and it has been found that the
thocyanins were based on the adsorption in paper ratio of absorbance at the acyl maximum wave-
chromatography or in other suitable adsorbents.455 length to the absorbance at the visible maximum
Nowadays, thin layer chromatography (TLC) is wavelength (Amaxacyl/ Amaxvisible ratio) is a mea-
widely used, because this technique has shown sure of the molar ratio of acyl group to anthocya-
continuous innovations and still keeps its advan- nin.173 Additionally, visible absorption is the best
tages (practical and very cheap). For preparative tool to observe the copigment effect: visible spec-
work, droplet counter current chromatography has tra of anthocyanins show a hyperchromic effect,
been applied to separate the anthocyanins of black increment of the intensity of this maximum re-
currant. On the other hand, a general patent for sulting in a more colored species, and a
the purification of anthocyanins involves selec- bathochromic shift by a solvation effect.62,63,195
tive absorption on a finely divided oxide such as Heating causes a hypsochromic and hypochromic
silicic acid, titanium oxide, or alumina, which is effect that is restored by copigment regeneration
coated with a styrene polymer.157 However, un- after cooling in the malvin-rutin systems; how-
doubtedly, the main developments of recent years ever, the reversibility was not observed in the
in the research of anthocyanins is the introduction malvin-quercetin system. However, in both sys-
of HPLC for their separation and quantitation.193,195 tems the copigment reaction was a spontaneous
Interestingly, it is possible to distinguish zwitteri- thermodynamic process (negative ∆G).20 On the
onic anthocyanins by their HPLC chromatographic other hand, visible absorption and RAMAN spec-
separation.195 troscopy have been used in the study of pigments
in the living tissue. RAMAN spectroscopy has
been used to show the anthocyanin substitution
c. Characterization pattern. The presence of phenyl ring substitutions
on benzopyrylium produces clear spectral modi-
Spectroscopy. In general, color is evaluated fications. On the other hand, the anthocyanin spec-
by spectrophotometry.157 Isolated pigments have tral features are modified by glycosylation, show-
been studied by UV-visible spectroscopy. All fla- ing that perturbations are dependent of the sugar
242
TABLE 5
Some Chemical Tests for Anthocyanin Characterization
Detection
Reaction conditions Structural characteristic Observed characteristic

(anthocyanin in)
Ethanol dissolved with HCl γ-benzopyrone structure (all Colors from red to green
and Mg (Wilstatter reaction) flavonoids)
Ethanolic solution with Flavonoids with o-hydroxyl Formation of a silver mirror
AgNO3 (12% in water) groups
Paper chromatography Flavonoids Dark or fluorescent colors
and treatment with ammoniac
vapors
Sodium methoxide (2.5% in Flavonoids with hydroxyl Bathochromic change of
methanol) groups at 3- and/or 4’- spectra and if intensity of
positions visible band do not decrease
thus a 4’-OH is present
Sodium acetate (solid) Hydroxyl at 7-position Bathochromic change of the
UV band
AlCl3 (5% in methanol) Flavonoids with o-hydroxyl Bathochromic change of

and/or 5-OH and/or 3-OH spectra
AlCl3 (5% in methanol) with Flavonoids with o-hydroxyl The bathochromic change is
HCl solution reverted after HCl addition
nature. Interestingly, 5-glycosides present higher an intense peak for the molecular ion, something
modifications than 3-glycosides.63,309 The coupling difficult with the previously designed detectors;
of the diode array detector (DAD) into the HPLC anthocyanins are unstable and with low volatility.
methodology has permitted the tentative identifi- Nowadays, methodological advances have been
cation of the separated anthocyanins, as was de- conducted on other mass detectors that inclusively
scribed with carotenoids. In addition, with the are coupled with the HPLC equipment (see above
introduction of innovated methodologies such as discussion about carotenoids).19 Interestingly, new
NMR and mass spectrophotometry, anthocyanin structures have been elucidated, and anthocya-
compounds have been identified conclusively. nins with caffeoylglucose and malonyl substitu-
Proton NMR has been used to study the self- tions are perhaps the most notable.18,291 Another
association of anthocyanin molecules, while car- methodology used to study the copigmentation
bon-13 NMR spectroscopy has been used to de- phenomena is circular dichroism.62,193,195
fine the sequence, position, and configuration of Chemical tests. A large number of chemical
sugar residues in flavonoid glycosides. NMR tests have been developed to determine the antho-
methodology has been used with heteronuclear cyanin structure (some of them are shown in Table
shift correlation through multiple quantum coher- 5), and a general procedure could be envisioned,
ence (HMQC) to show the configuration of an- but this must be modified depending on the ana-
thocyanin glycosides. β configuration was lyzed material.193 After separation, isolated antho-
observed with glucosyl, galactosyl, and cyanins were hydrolyzed with mineral acid
xylanopyranosyl groups and α configuration with and glycoside bonds were disrupted, then
rhamnosyl and arabinopyranosyl groups. More- anthocyanidins were methylated, and after other
over, 1H-1H COSY and DIFNOE have been used acidic hydrolysis the nature of the anthocyanidin
in structure characterization to assign the NMR and the positions of glycosylation were deter-
proton signals.365 In addition, mass spectrometry mined.455 When anthocyanin molecules show ali-
increased its potential with the introduction of the phatic acids in their structures, a zwitterion is
FAB-mass detector, which permitted observing observed that can be detected by electrophore-
243
sis.157 Additionally, to determine the occurrence ber of methoxyl groups. In addition, the
of phenolic acids as esters, the extract is concen- glycosylation level showed a clear effect on sta-
trated and resolubilized with 6N HCl and hydro- bility, diglucosides are more stable than
lyzed (100°C/60 min), the solution is extracted monoglucosides, but browning is more favorable
with ethyl acetate and then evaporated under in diglucosides by the presence of the additional
vacuum and the residue prepared to be analyzed sugar molecule. Also, it has been showed that
by HPLC.19 The presence of B-ring-substituted hydroxylation at position 4 changes the colors
sugars is easily detected by an hypsochromic spec- toward red tones.157
tral shift.157 The equilibrium colored-uncolored anthocya-
Evaluation of pigmenting efficiency. In nin structures affects the stability of products on
the evaluation of visual color, reflectance storage, because the R+ form is the most stable.
colorimetry has been the better approach be- R+ + H2O 
→ ROH + H +
cause it shows a good correlation with the
chemical composition, and as above men- Colored catonic Uncolored
tioned, lightness has been used to determine form pseudobase form
the pigment concentration; saturation (purity)
reflects the spectral properties and the chro- The reactive species was found to be the an-
thocyanin carbonium ion (R+), which reacts with
maticity coordinates give an appreciation of

the various mixed pigments. 62,157 In this sense, a bisulfite ion to form a colorless 2-bisulfite ad-
a method has been developed to assess the duct product.127
anthocyanin concentration by reflectance In solution, anthocyanin molecules are
measurements in red raspberry fruit, and it is present in an equilibrium between the colored
reported that the best predictor is the hue cationic form and the colorless pseudobase.
parameter (r = 0.73). 330 This equilibrium is directly influenced by pH.
Acidic pH is favorable for the colored form
that diminished with pH increments. Some
7. Importance as Food Colors — anthocyanins are red in acid solutions, violet
Stability, Processing, and Production or purple in neutral solutions, and blue in al-
kaline pH. This is the reason that most colorants
a. Stability in Model Systems containing anthocyanins can only be used at
pH values below four. 58 Acylation hinders
Anthocyanins are of great economic impor- hydrolysis of the red flavylium cationic form,
tance as fruit pigments and thus also as pigments allowing preferential formation of the blue
of fruit juices and wines.195 Interestingly, one of quinonoidal bases. Interestingly, acylated pig-
the main claims of the food industry is for natural ments retain more color at the higher pH val-
colorants to replace synthetic red dyes, and antho- ues than unmodified anthocyanins. In addi-
cyanins are the principal candidates, but, interest- tion, acylated anthocyanins show an improved
ingly, enocyanin and lees (sediment of the grape resistance to other factors such as heat, light,
juice tanks) preparations are the only anthocyanin and SO 2. In particular, in Vitis vinifera wines
sources approved by FDA (in the U.S.) to be used were identified the anthocyanins vitisins in
for human food, while the main use is in the which C-4 positions of their structures are
production of beverages and soft drinks.58,157 How- substituted, and consequently these pigments
ever, anthocyanin instability limits their use, and give greater colors, higher resistance to color
different preparations have been evaluated to avoid loss with sulfur dioxide, and higher pH val-
anthocyanin degradation.87 The color of antho- ues.18 Thus, this group of anthocyanins has
cyanins is provided by its resonating structure, been proposed as potential food colorants.
but resonance phenomena also confers its intrin- Moreover, it is also very important that acyla-
sic instability. Moreover, it has been established tion produces a major absorption band at pH
that instability has a direct relation with the num- values above four, and, consequently, acylated
ber of hydroxyl groups and indirect with the num- anthocyanins will be highly colored at pH > 4,
244
contrasting with the conventional compounds acylation groups permitted anthocyanins to
that are uncolored at these condi- adopt a folded conformation, which protects
tions.58,127,157,161,193 As a matter of fact, a great the basic anthocyanin structure of degradative
number of studies have been carried out to factors.146,458 To corroborate the effect of acy-
find new acylated pigments, and several ones lation on anthocyanin stability, anthocyanin
with novel characteristics not previously re- extracts of Sambucus nigra and S. canadiensis
ported) have been characterized (Table 6). In- were compared. Anthocyanins of S. nigra have
terestingly, malonylated anthocyanins are pro- a free hydroxyl group on C-5, while in S.
duced in purple sunflower seeds, which is one canadiensis a glucosyl residue acylated with
of the most important oilseed crops, and, con- p-coumaroyl is present. It was observed that
sequently, the purple-hulled residue obtained S. canadiensis extracts were more stable than
after oil extraction from seeds provides an those of S. nigra. It was shown that C-5
excellent source of red colorant (cyanidin de- glucosylation does not represent a crucial fac-
rivatives) that has been suggested as a colorant tor on heat stability, but acylation does. How-
of food, pharmaceutical, and cosmetic prod- ever, it was also shown that C-5 glycosidation,
ucts.303 In addition, several of the acylated as well as acylation, has an important
anthocyanins have shown highly desirable sta- contribution to color stability for light. In ad-
bility, and the structures of some of them are dition, the following decreasing order
presented in Figure 18. In particular, the im- of stability tolight was reported: acylated
proved stability of the Ajuga reptans antho- diglucosides > nonacylated diglucosides >
cyanins was assigned to the presence of two monoglucosides.232 A comparable high stabil-
cinnamic acid groups in the anthocyanin struc- ity of C-5 acylated anthocyanins was also ob-
ture,461 and it has been suggested that long served with the pigments from red radish. 173
TABLE 6
Reports on Acylated Anthocyanins
Plant model Remarks Ref.
Verbena flowers First report of a diacylated anthocyanin: 469

(Verbena hybrida) Pelargonidin (3-O-(6-O-(malonyl)-β-D-
glucopyranoside)-5-O-(6-O-(acetyl)-β-D-
glucopyranoside)
Blue flowers of Identification of anthocyanins with a cis-p-coumaric 222

Hyacinthus orientalis acid as acylglucosyl moiety
Geranium flowers First structure elucidation of a monoacetylated 7

(Geranium sylvaticum) anthocyanin 3,5-diglycoside with identical sugars
Gentiana flowers Identification of anthocyanins in which the 5-O- 224

(Gentiana makinoi) glucose attached to anthocyanidin is acylated with
hydroxycinnamic acid
Stem of Allium First report on acylation of the 3”-position in the sugar 8

victorialis moiety of any anthocyanin. Malonylation at this
position provides better stability than 6”-acylation
Water lily First report involving a diacylation, which involves 154

(Nymphaéa X gallic acid as one of the acyl moieties and produces a
morliacea) bathochromic shift (5 nm) on the maxima of the
visible spectra
245
FIGURE 18. Acylated anthocyanins with improved stability under different process conditions. These pigments were
obtained from (A) Ajuga reptans, (B) Bletilla striata, (C) Eichhornia crassiped, (D) Ipomoea purpurea, and (E)
Tradescantia pallida.
246
TABLE 7
Processing and Stability Studies on Anthocyanin Pigments in Different Food Systems
Model Conditions Effects Ref.
Blackberry Different Disappearance rate followed a first-order 114

juice temperatures and kinetics and was higher in the presence of
addition of aldehydes aldehyde and dependent of temperature
Quick frozen Addition of different Sugar addition stabilizes the pigment 511
strawberries levels of sucrose content ad produce a reduction in
browning reactions. The protective effect
was assigned to inhibition of degradative
enzymes and to steric interference with
condensation reactions
Grape musts Addition of Quinones are involved in anthocyanin 103

glutathione degradation because of this is inhibited by
glutathione
Barley Heating (40–100ºC) Hordeumin anthocyanin showed an 347

improved stability because of it is

constituted by a molecular complex
between anthocyanins and polyphenols
Marashino cherries Brined cherries The coloration imparted by RAE and 173
pigmented with red FD&C Red No. 40 was similar; the
radish anthocyanin kinetics of degradation followed first-
extracts (RAE) order kinetics
On the other hand, anthocyanin production in ence of acetaldehyde 15ºC/4 months) was ob-
leaves of Ajuga pyramidalis was compared served. Three new pigments were observed and
with that produced in cell cultures. The main their visible spectra showed a bathochromic dis-
anthocyanin was 3-O-(6-O-(E)-ferulyl)-2-O- placement, in relation to the anthocyanin by the
[(6-O(E)-ferulyl)-β- D -glucopyranosyl-β- D - condensation of these compounds. Interestingly,
glucopyranosyl]-5-O-(6-O-malonyl)-β- D - these compounds showed an improved stability.155
glucopyranosylcyanidin; and it was determined Compounds with similar characteristics to the
that anthocyanins from cell cultures showed synthesized ones have been detected in red wines.
higher stability than those obtained from in
vivo extracts. It was explained that in cell cul-
tures copigmenting agents such as flavonols, b. Processing and Stability in Foods
phenolic acids, and tannins could accumulate,
which may be absent in plant.292 Anthocyanin pigments can be destroyed easily
Condensation reactions have been reported during the processing of fruits and vegetables, and
by the reaction of acetaldehyde, anthocyanins, considering food color as an appealing characteris-
and flavan-3-ols, producing the increment of color tic, many studies have been carried out to under-
(up to seven times). It is believed that acetalde- stand the anthocyanin properties and to obtain bet-
hyde forms a bridge between the two flavonoids, ter products with minimal degradation (Table 7).
and consequently condensation reactions could High temperature, increased sugar level, pH, and
proceed and contribute to the polymeric ascorbic acid can affect the rate of destruction.157,510
color.157,193,242 Moreover, the formation of new Temperature has been reported to induce a loga-
anthocyanins by the reaction of malvidin 3- rithmic destruction of pigment with time of heating
monoglucoside and procyanidin B2 in the pres- at a constant temperature. Bleaching by effect of
247
heat occurs because of the above-described equi- nificantly affected by different CO2 treatments,
librium is changed toward the uncolored forms. It evaluated by reflectance colorimetry; however,
has been suggested that flavonoid structure is internal color decreased markedly. Color disap-
opened to form chalcone, which is degraded fur- pearance was correlated with low anthocyanin
ther to brown products.157 However, interestingly, levels. Additionally, it was suggested that this
it has been observed that optimal conditions permit opposite behavior (skin and internal) could be
the regaining of color on cooling if there is suffi- produced by different anthocyanin profiles in the
cient time (several hours) for the reconversion. analyzed structures; higher concentrations of cya-
In slightly alkaline solutions (pH 8 to 10) nidin 3-glucoside are observed in external tissue
highly colored ionized anhydro bases are formed. (higher stability), while pelargonidin glycosides
At pH 12, these hydrolyze rapidly to fully ionized are in the internal tissue. Moreover, the accumu-
chalcones.58 lation of phenolic compounds is greater in exter-
Knowledge of anthocyanin characteristics have nal tissue, in agreement with the protective role
allowed the development of a large number of prod- assigned previously.170 In addition, other method-
ucts and processing conditions that obtain high- ologies have been proposed to change metabolic
quality colored products. Pomace extracts were freeze pathways to reach a higher color preservation. In
dried on DE 20 maltodextrin, and this preparation frozen strawberries, it was shown that sugar addi-
showed good shelf life (2 months at 50°C/0.5 aw tion has an stabilizing effect on the total mono-
and more than 5 years with aw< 0.3). Also, improved meric anthocyanins, suggesting that sugar can be
stability was obtained in concentrated extracts of employed to enhance the shelf life of colored
fermented elderberries. The described process was products (Table 7).511 Litchi is a tropical fruit and
as follows: berries were homogenized, fermented within 2 to 3 days after harvest its pericarp be-
with C. cereviceae var. malaga (room temperature, comes desiccated and turns brown. To diminish
pH = 4.5), and a concentrated juice is obtained (4% the losses, litchis were coated with chitosan
d.w. of anthocyanins). Additionally, the use of this (1.0 to 2.0%) and stored (4ºC/90% relative hu-
product to enhance the color of spiced wines is midity). The use of chitosan delayed changes of
mentioned. Four patents have been developed to contents of anthocyanin, flavonoid, and total phe-
extract the colorant from blue corn by using a simple nolics. Interestingly, the activities of polyphenol
acidified aqueous extraction. A patent to obtain the oxidase and peroxidase, which have been involved
sorghum colorant involves alcoholic extraction and in anthocyanin degradation, are inhibited. It is
purification on a silica gel column. While other suggested that a plastic coating forms a protective
patents have been developed for anthocyanin ex- barrier on the surface of the fruit and reduces the
traction from black mulberries, elderberries, Vibur- supply of oxygen for enzymatic oxidation of phe-
num berries, rowanberries, red sweet potatoes (Ipo- nolics.523 Also, anthocyanin profiles have been
moea batatas), and for a variety of plant sources. In proposed as an indication of inadequate process-
addition, a successful extraction procedure to isolate ing conditions. In red raspberry juices, it was
pigments using 350 ppm SO2 in water and ion ex- found that bad processed samples showed higher
change chromatography was reported. Also, a puri- levels of polymeric color instead of the mono-
fication process was implemented that involves se- meric anthocyanin pigments, and also this was
lective absorption on inorganic oxides such as silica observed as a product of poor quality.56 Another
gel, titanium oxide, or alumina, all of them coated important factor to preserve color during fresh-
with a styrene polymer.157 fruit storage is the elimination of mold contami-
During processing and commercialization of nation, which has a deleterious effect on
fresh products, color is the main factor of choice, anthocyanin polymerization. 405 In general,
and several attempts have been carried out to it is considered that light has deleterious effects
preserve the “fresh” appearance of fruits when on anthocyanin stability and light exposure of
stored. In fresh strawberry fruits, CO2 treatments natural colored beverages must be avoided. It has
have been assayed to preserve their attractive been reported that other flavonoids flavone,
appearance. It was found that skin color was sig- isoflavone, and aurone sulfonates) increase the
248
photostability.157 On the other hand, light inten- Gallotannin will inactivate phenolase through a
sity has a profound effect on an apple’s color, direct condensation system. The destruction of
because the light-exposed peel contains twice as anthocyanins by enzymatic activity could be im-
much anthocyanin as a shaded peel. Interestingly, portant in the design of an extraction procedure
a correlation was shown between UFGT and perhaps in the final formulation in a food.157
(UDPGal:flavonoid-3-O-glycosyltransferase) ac-
tivity and anthocyanin accumulation with an
UFGT activation by ethephon; it suggests a regu- c. Production of Anthocyanins by Plant
latory importance of UFGT in anthocyanin syn- Tissue Culture
thesis in apple.245 Also, in apple it has been ob-
served that methyl jasmonate is associated with Plant tissue culture is a potential source of
the ethylene production, and thus with anthocya- production of anthocyanins, in view of the quoted
nin accumulation; this is the explanation for the price ($1250 to $2000/kg) and the expanding mar-
application of methyl jasmonate during the initial ket of natural anthocyanins. Moreover, plant cell
stages of apple development to produce better culture could ensure a continuous supply of uni-
colored fruits.142 To modify the apple fruit appear- form-quality anthocyanin pigments that cannot
ance, the use of magnesium and urea as a source be produced by other biotechnological approaches;
of nitrogen was attempted, and a better final col- however, to date no food colorant obtained by this
oration was observed, but this was mainly af- system has been commercialized, and the main
fected by the increase in chlorophyll and caro- bottleneck is the low yield of secondary metabo-
tenoid content, while the anthocyanin level lites.58,157,202
remained the same.393 Oxygen has a negative ef- It has been considered that bioreactor produc-
fect on anthocyanin stability, while ascorbic acid tion is prohibitive because present technological limi-
could have a negative or positive effect, depend- tations make the process very expensive; a commer-
ing on the media conditions. Additionally, it has cial pigment production by plant cell tissue culture
been suggested that flavonols act as free radical could be achieved only when a fully automated
scavengers to protect anthocyanin molecules. predictable process with a more advanced technol-
Anthocyanins are very reactive toward metals, ogy could be established.442 One of the main prob-
and they form stable complexes with tin, copper, lems with plant cell suspensions is the heterogeneity
and iron; it has been proposed that metal com- of media due to the existence of cells as large aggre-
plexes could be used as colorants.417 gates that cause problems of oxygen diffusion, mass
The overall color changes may be due to a transfer, cell ejection of the liquid media, and re-
number of factors such as pigment degradation, duced growth rates. Additionally, the conditions
pigment polymerization, reactions with other com- favoring high growth do not favor production, and
ponents of the formulation, nonenzymatic brown- frequently high production conditions are detrimen-
ing, oxidation of tannins, and other reactions com- tal for growth. In particular, maximal growth of
pletely unrelated to the added colorant. 157 strawberry cells in suspension cultures was found at
Anthocyanins can be degraded by a number of 30ºC, but lower temperature induced an increased
enzymes found in plant tissue: glycosidases, level of anthocyanin pigment (the maximum at
polyphenoloxidases, and peroxidases. Glycosi- 20ºC).524 Thus, processes by stages have been devel-
dases produce anthocyanidins and sugars, and oped (a first stage for cell growth and a second one
anthocyanidins are very unstable and rapidly de- for anthocyanin production), and models have been
graded. Polyphenoloxidase catalyzes the oxida- designed for batch and semicontinuous anthocyanin
tion of o-dihydrophenols to o-quinones that fur- production to introduce the concept of primary and
ther react to brown polymers; however, this secondary metabolism because of the continuous
reaction is more favorable for other phenols. Thus, competence for precursors.441
pigment loss can be reduced by blanching to in- Anthocyanin production by cell cultures has
activate these enzymes. Addition of 30 ppm SO2 been evaluated from grapes (2 to 3 mg/g fresh
will inhibit phenolase activity in sour cherry juice. weight),514 Euphorbia milli,516 and carrot,492 among
249
others. In general, stable production by plant cell nutrient limitation induces the activities of PAL
cultures has not been reached. However, some and CHS enzymes, suggesting an association of
cultures of Ajuga reptans have produced antho- phenylalanine addition with enzyme induction,
cyanins for more than 10 years, but the analyses and a similar behavior was observed with straw-
of five lines showed quantitative differences be- berry cells.115,246,524 With Fragaria anansa cell
tween the lines, and it was observed that 5′-subcultures, riboflavin addition had a 3.2 times greater
stituted anthocyanins seem to decrease more eas- production than in control medium, but only un-
ily during repeated subcultures.67 In wild carrot der light conditions.331 In carrot cell, suspension
(Daucus carota), clonal variability has been ob- cultures were found that favor fructose growth,
served to obtain low- and high-pigmented cells. while anthocyanin production is better with glu-
Interestingly, it was shown that high-pigmented cose, suggesting quantitative differences in the
cells have an intermediate limitation reflected by metabolism of these two sugars.530 In cultured
a considerable increment in pigment production cells (e.g., Vitis vinifera, Aralia cordata, Fragaria
after supplementation with dihydroquercetin, anansa), high sucrose and low nitrate are
naringenin, and 4-coumarate.492 desireable for anthocyanin production. It has been
Remarkably, anthocyanin production is light considered that although sucrose is an essential
dependent. Darkness cell cultures showed an in- nutrient, it also acts as an osmotic agent when
tense pigmentation after illumination,418 although
used at high concentrations. Interestingly, it was

darkness cultured cells, Aralia cordata among observed that high ammonium concentration
others, have shown anthocyanin accumulation. (24 mM) promotes the anthocyanin acylation,
Cultured cells of Perilla frutescens need light which is important for the food industry based on
irradiation for 7 days from the initial cultivation the improved stability of acylated pig-
period for the formation of anthocyanin pigments.213,331,332,411,418 It has been proposed that re-
ments.526 Interestingly, it was observed that light duced nitrate favors anthocyanin synthesis by
cultured cells are positively influenced by the favoring the uptake into vacuoles by a tonoplast-
treatment with auxins and cytokinins, while cul- associated transport mechanism.213
tures in darkness are not affected.411 In callus cul- Recently, the use of extracts of cultures or
tures of Oxalis linearis, anthocyanin production conditioned media to improve the anthocyanin pro-
was promoted by cytokinins but repressed by duction by tissue cultures has proposed. Culture
auxins such as NAA and 2,4-D.311 Another factor filtrates and cell extracts of B. cereus,
analyzed in plant cell pigment production is the S. aureus and E. coli (among others) were used as
pH, and considering the stability characteristics elicitors of anthocyanin production in carrot cell
of anthocyanins the optimum pH is mainly acidic cultures, obtaining increments up to 77%.454 It has
(pH < 7), but it was found that suspension cul- been found that conditioned media (CM) from
tures of strawberry cells showed the highest an- strawberry cell cultures promote anthocyanin ac-
thocyanin production at pH 8.7. It was observed cumulation in cell suspensions of strawberry. The
that the time profile ratio of pigmented cells did anthocyanin accumulation was sixfold higher in
not change with the variation in initial medium CM than in control media. On the other hand, rose
pH, suggesting that initial pH affected anthocya- cells were transferred to CM from strawberry cells,
nin synthesis by changing the anthocyanin con- and it was observed that anthocyanin accumulation
tent inside the pigmented cells only.525 was induced. Thus, it the presence of some un-
Several substrates have been assayed to in- known material(s) released (heterologous condi-
duce increments in anthocyanin production. In tioning factor) during culture that contributed to
cell suspension cultures of Vitis, phenylalanine anthocyanin accumulation was proposed.332,412
addition induces the cessation of cell division and
the induction of anthocyanin biosynthesis. Addi-
tionally, it was shown that Vitis cell division is
limited by nutrients in medium, such as sucrose,
phosphate, and nitrate or ammonium, and that
250
C. Betalains Betacyanin structures (Figure 20A) show some
variations in the acyl groups and sugar moieties,
1. Definition while betaxanthin (Figure 20B) exhibits the same
dihydropyridine moiety but show conjugation with
The term “betalains” was introduced by Mabry several amines and amino acids.
and Dreiding;288 this was supported by structural Betanidin is the basic structural unit of most
and biogenetic considerations. In a strict sense, of the betacyanins, followed by its C15 epimer, the
betalains do not belong to alkaloids because they isobetanidin.374 A considerable number of differ-
are acidic in nature due to the presence of several ent betacyanins can be obtained with glycosidation
carboxyl groups. Originally, betalains were called of one of the hydroxyl groups located at positions
“caryophyllinenroth” and successively renamed 5 and 6 (Figure 20A).
“rübenroth” and “chromoalkaloids”.371,395 Betaxanthins are constituted of different
Chemically, betalain definition embraces all proteinogenic and nonproteinogenic amino acids, as
compounds with structures based on the general well as biogenic amine-conjugated moieties of
formula shown in Figure 19; therefore, they are betalamic acids. More than 200 amino acids found
immonium derivatives of betalamic acid.375,451 The in plants may potentially give rise to betaxanthin
chromophore of betalains can be described structures.451 The archetypal compound represent-
as a protonated 1,2,4,7,7-pentasubstitued ing betaxanthins is the indicaxanthin, isolated from

1,7-diazaheptamethin system.371 prickly pear (cactus fruits of Opuntia ficus-indica).371
FIGURE 19. Betalain general formula. (A) Betalamic acid moiety is present in all betalain molecules. (B) The
structure will represent a betacyanin or a betaxanthin, depending on the identity of the R1 and R2 residues. (Adpated
from Ref. 46.)
251
FIGURE 20. Betanidin is an example of betacyanins (A), while miraxanthin II is of betaxanthins (B). (Adapted from
Refs. 375, 451.)
252
2. Classification ized, and they are usually assigned in agreement
with their botanical genus.371 In the betacyanin
They can be divided into two structural groups, group, amaranthin-I was obtained from
the yellow betaxanthins (from Latin beta, red beet Amaranthus tricolor, betanin from Beta vulgaris,
and Greek xanthos, yellow) and red-purple and gomphrenin-I from Gomphrena globosa.
betacyanins (kyanos, blue color), depending on While in the betaxanthin group, miraxanthin oc-
R1-N-R2 moieties. More than 50 betalains are well curs in flowers of Mirabilis jalapa, vulgaxanthin-
known, and all of them have the same basic struc- I and II have been found in root of Beta vulgaris,
ture, in which R1 and R2 may be hydrogen or an and portulaxanthin has been isolated from the
aromatic substituent. Their color is attributable to petals of Portulaca grandiflora.375
the resonating double bonds.46 Conjugation of a Up to date more than 50 structures of natu-
substituted aromatic nucleus to the rally occurring betalains have been identified. A
1,7-diazaheptamethinium chromophore shifts the considerable number of different betacyanins may
absorption maximum from 480 nm in yellow be derived from two basic compounds, betanidin
betaxanthins to 540 nm in red-purple (2S, 15S) and isobetanidin (2S, 15R) by
betacyanins.451 glycosidation of one of the hydroxyl groups lo-
In some earlier papers, the terms “betalaines”, cated at position 5,374 for example, betanin, which
“betacyanines”, and “betaxanthines” were used; occurs as the 5-O-glucoside, and the less-occur-
the terminal letter “e” was added by Fisher and ring position 6, for example, gomphrenin-II,
Dreiding148 so the names would conform to the which is a 6-O-glucoside. No betacyanin is
I.U.P.A.C. nomenclature; at present, these terms known to have both positions substituted with
can be used without the terminal “e”. sugar residues. 375 A few biosides are
Betacyanins and betaxanthins can be classi- known: amaranthin the betanidin 5-O-[2–O-
fied using their chemical structures. Betacyanin (β- D -glucopyranosyluronic acid) β- D -gluco-
structures show variations in their sugar (e.g., pyranoside], and its epimer isoamaranthin is in
5-O–D-Glucose) and acyl groups (e.g., feruloyl), the leaves of A. caudatus. 38 Moreover, two
whereas betaxanthins show conjugation with a epimeric betacyanins, bougainvillein-r-I and
wide range of amines (e.g., glutamine) and amino isobougainvillein-r-I, have been isolated from B.
acids (e.g., tyrosine) in their structures. Table 8 glabra.372
shows some well-studied betalains. The isomeric 5-O-β-cellobiosides have been
produced by deacylation of the pigments aleracin-
I and II from P. oleracea.45 The only known
3. Distribution 6-(2 G-glucosylrutinosides) of betanidin and
isobetanidin have been obtained by deacylation of
Among higher plants the occurrence of bougainvillein-V. Betalain glycosides can be es-
betalains is restricted to the Caryophyllales289 and terified with hydroxycinnamic acids (ferulic and p-
those found in certain higher fungi such as coumaric acids), for example, celosianin-I
Amanita, Hygrocybe, and Hygrosporus. 451 4-coumaroylamaranthin) in Chenopodium rubrum
Betalains of higher plants are in different organs.402 and Lampranthus sociorum;48 in addition, sulfuryl
They produce red, yellow, pink, and orange col- and malonyl betacyanins are known, for example,
ors in Aizoaceae and Portulacaceae flowers,473 rivianin (betanin-3′-sulfate) from Riviana humilis
and purple pigmentation in Cactaceae fruits and and phyllocactin (malonic acid 6′-half-ester of
in red-beet root (Chenopodiaceae).370 Betalains betanin) from Phyllocactus hybridus. 370 The
are in bracts, for example, Bougainvillea decarboxybetanidin, isolated from Carpobrotus
(Nyctagynaceae) possesses a wide range of col- acinaciformis, is unique among betacyanins in
ors;372 they are also in seeds of Amaranthus,37 in containing a modified aglycone moiety.371
leaves of Teloxis and in stems.12 There are about 15 naturally occurring
Common names and classification of differ- betaxanthins; the indicaxanthin from Opuntia fi-
ent betacyanins and betaxanthins are standard- cus-indica was the first crystallized.375 In total,
253
TABLE 8
Some Fully Identified Naturally Occurring Betalains
Betalaina Residueb Ref.
Aglycones
Betanidin — 375
Betanin group
Betanin 5-O-Glc 46
Phyllocactin 5-O-Glc 373
Lampranthin-I 5-O-Glc 48
Amaranthin group
Amaranthin 5-O-Glc-2-O-GlcU 451

Celosianin II 5-O-Glc-2-O-GlcU 449
n-
Bougainvillein
Bougainvillein 5-O-Glc-2-O-Glc 372
Gomphrenin group
Gomphrenin-I 6-O-Glc 316
Betaxanthins
DOPAxanthin DOPA 371

Indicaxanthin Proline 371
Portulaxanthin-II Glycine 473
Vulgaxanthin-I Glutamic acid 375
a Names were standardized by Strack et al.451

b Abbreviations: Glc β-D-Glucose; GlcU β-D-Glucoronic acid; DOPA 3,4-dihidroxyphenylalanine.
eight of the naturally occurring betaxanthins con- pically labeled precursors and in vitro cell cul-
tain non-protein amino acids.472 tures were important tools in such discovering.46,375
However, very few enzymes involved in betalain
synthesis have been purified and character-
4. Biosynthesis: Biochemistry and ized.211,273,434,451 Figure 21 summarizes the pro-
Molecular Biology pounded biosynthetic pathway. In detail, betalains
are considered secondary metabolites; they de-
rive from shikimic acid and from the tyrosine
aminoacid.371 In their basic structure, the phenyl
a. Biochemistry group is bonded to a lateral n-propyl chain giving
place to a C6-C3 unit.35 Biogenesis of betalains
Initial stages (Figure 21A). The determina- from tyrosine has not been thoroughly under-
tion of the chemical structure of betalains and of stood, and only a few enzymes involved in the
their biosynthetic intermediates contributed to the biosynthetic route have been identified.47,273
establishment the corresponding biosynthetic path- Two molecules of tyrosine are required in the
way; in addition, feeding experiments with isoto- biosynthesis of one molecule of betacyanin or
254
FIGURE 21. Pathway proposed for betalain biosynthesis. (A) Intial stage, (B) betacyanin biosynthesis. Betacyanins
may be glycosylated at position R1 and R6 and or acyglycosylated at postion R3 or R4. (B) Betaxanthin biosynthesis.
R5 and R6 represent lateral chains of amine compounds. Some of the enzymatic activities involved in betalain
biosynthesis are (1) DOPA 4,5-dioxygenase, (2) glycosyltransferase, and (3) acyltransferase. (Adapted from Refs.
46, 451.)
255
betaxanthin. Initially, two molecules of 3-hydroxy- fission of catechols, and the last reactions are
L-phenylalanine (L-DOPA) are formed.335,375 The catalyzed by specific dioxygenases. These en-
hydroxylation of tyrosine to L-DOPA is recog- zymes contain Fe ion (both ferric and ferrous
nized as the first step in the biogenesis of betalains states) as an essential cofactor. Girod and Zrÿd171
because experiments with radioactive precursors isolated the DOPA 4,5-dioxygenase from
have shown good incorporation of labeled [C14]- A. muscaria. This enzyme as other extradiol-
tyrosine into amaranthin and betanin, the major cleaving dioxygenases is an oligomer that cata-
betacyanins in Amaranthus tricolor seedlings and lyzes the 4,5-extradiol disruption of L-DOPA
red-beet (Beta vulgaris), respectively.135 The as- leading, via 4,5-secoDOPA to betalamic acid.463
sumption has been that the first enzyme is a phe- It was shown by affinity chromatography that
nol-oxidase complex catalyzing both the conver- DOPA 4,5-dioxygenase is composed of a vary-
sion of tyrosine to L-DOPA by a monophenol ing number of identical 22-kDa subunits.
oxidase and the dehydrogenation of the latter to Another DOPA dioxygenase (DOPA 3,4-
O-quinone by a diphenol oxidase. dioxygenase) was also extracted from A.
Recent work on the enzymology of betalain muscaria; this enzyme catalyzes the 2,3-ring-
biosynthesis has focused on the toadstool “fly opening reaction that yields the muscaflavin pig-
agaric” (Amanita muscaria) mushroom of the ment, a compound that has never been found in
Agaricales. In this basidomycete, the accumula- plants. The betalamic acid may condense with
tion of betalain pigments is restricted to the cu- the imino-group of cyclo-DOPA to produce the
ticle of the cap and its subject to developmental red-purple betacyanins (Figure 21B) or with the
regulation.451 There is a rapid increase in betalain imino or amino group of amino acids to give the
levels during the process of development from yellow betaxanthins (Figure 21C).451
young fungi, which are still entirely covered by Betacyanin biosynthesis (Figure 21B).
the veil, to mature specimens. Recently, Müeller Betacyanins are formed through the reaction of
et al.335 characterized a tyrosinase from pileus of cyclo-DOPA with betalamic acid followed by
Amanita muscaria. This enzyme was located only glycosylation, or by condensation of the cyclo-
in the colored parts of the fungi, and it was dem- DOPA glycosides with betalamic acid.209 In gen-
onstrated that tyrosinase catalyzes the reaction of eral, it could be mentioned that condensation of
tyrosine hydroxylation to L-DOPA, confirming betalamic acid with cyclo-DOPA to betanin for-
its involvement in betalain biosynthesis. Tyrosi- mation and the subsequent glucosylation reaction
nase also shows diphenolase activity, and it seems to betanidin 5-O-β-glucoside, main compound
to be an heterodimer of two subunits with mo- of red-beet, remain unknown. Complete
lecular weights of 27 and 30 kDa, something glycosylation takes place with cyclo-DOPA fol-
unusual for tyrosinases. lowed by condensation with betalamic acid. On
On the other hand, one L-DOPA molecule is the other hand, free betanidin can be stored as the
transformed into DOPA-quinone, which is spon- main component in betalain-producing cells, and
taneously converted to cyclo-DOPA, while it has been shown that betanidin can be the main
betalamic acid is formed from the second mol- receptor of UDP-glucose from glucosyl-transferase
ecule of L-DOPA by means of a reaction cata- during betanin biosynthesis.208,209 It may be pos-
lyzed by DOPA-dioxygenase.336 In the proposed sible that the sequence of reactions depends on
metabolic pathway betalamic acid arises from L- the plant genus.
DOPA. L-DOPA is cleaved at the C4-C5 bond of Betanin glucosylation catalyzed by uridine
the aromatic ring to form an intermediate 5′-diphospho-glucose: betanidin 5-O-β-
(4,5-seco-DOPA) that is cyclized, forming an het- glucosyltransferase (5-GT) was demonstrated by
erocyclic system. It has been suggested that Heuer and Strack,208 who described the occur-
4,5-seco-DOPA is produced by an “extradiol- rence of one of the two proposed pathways of
cleavage” of L-DOPA.463 betacyanin formation, the transfer of glucose to
The oxidative disruption of the L-DOPA ring 5-hydroxy group of betanidin in the formation of
appears to be analogous to the aromatic rings betanin.
256
On the other hand, Heuer et. al.209 described routes.451 A spontaneous condensation between
a new glucosyltransferase, the 6-O- betalamic acid and an amine group inside the
glucosytransferase (6-GT) that catalyzes the vacuole has been based on genetic and biochemi-
regiospecific transfer of glucose to the 6-hydroxy cal studies on clones of P. grandiflora. In a recent
group of betanidin in the formation of gomphrenin work, Hempel and Böhm207 found two new
I, analogs to 5-GT. Both GT were extracted from betaxanthins in hairy root cultures of Beta vul-
cell cultures of Dorontheanthus bellidiformis. The garis var. Lutea, vulgaxanthin III, and IV, when
5-GT showed three isoforms and the 6-GT only the culture media was supplemented with the
one; both enzymes are monomers with a molecu- corresponding L-amino acids. This feeding ex-
lar weight near 55 kDa. Nowadays, it has been periment provides arguments for a spontaneous
shown that both enzymes catalyze the indiscrimi- condensation of betalamic acid with amino acids
nate glucose transfer from UDP-glucose to hy- or amines in the course of betaxanthin biosynthe-
droxyl groups of betanidin, flavonols, anthocya- sis.
nins, and flavones. Interestingly, it was observed In other experiments, the administration of
that GT catalyzes the formation of 7-O-gluco- L-DOPA to violet petals of flowers in Portulaca
sides, but in a minor extent.493 Based on these grandiflora led to the biosynthesis of betaxanthins
results, it could possibly be that 5-GT and 6-GT not present in natural plants. These results show
have a phylogenetic relation with flavonoid that L-DOPA administration elicits the formation
glucosyltransferases. Subsequently, acylation of of betaxanthins, which are absent in untreated
glycosylated betanidins is through acyl group flowers.397 In addition, nine L- and D-isomers of
donation from 1-O-acylglucosides. It is important amino acids were supplied to seedlings and indi-
to point out that this reaction catalyzed by an vidual hairy roots strains of Beta vulgaris, var.
acyltransferase seems to be an exclusive biosyn- Lutea; the increment of betaxanthin concentra-
thetic mechanism of betalain-producing plants, tion and also the appearance of new betaxanthins
contrasted with the analogous reaction in the bio- was observed.207 Therefore, the theory of a spon-
synthesis of flavonoids where acylated flavonoids taneous condensation of betalamic acid with amino
are produced by the hydroxycinnamoyl-coenzyme- acids or amines in the course of betaxanthin bio-
A pathway.47 synthesis is supported.
Another enzyme involved in betacyanin for- On the other hand, the activity of a betacyanin
mation is an acyltransferase (HCA), which cata- and betaxanthin-decoloring enzymes has been pos-
lyzes the transfer of hydroxycinnamic acids from tulated in Beta vulgaris,271,437 Amaranthus tri-
1-O-hydroxycinnamoyl-β-glucose to the C2 hy- color,135 and Phytolacca americana;265 the results
droxy group of glucuronic acid of betanidin 5-O- suggest there is an enzyme complex with two
glucuronosylglucose (amaranthin) in Chenopo- acidic and two basic enzymes that contain a metal
dium rubrum; 48,49 the products formed are ion in the active site. Earlier works described this
celosianin I (4-coumaroylamaranthin) and complex like a “peroxidase” enzyme.231 Parra and
celosianin II (feruloylamaranthin), as shown in Muñoz361 confirmed that horseradish peroxidase
Figure 22. This enzyme exhibits a molecular catalyzes the oxidation of betanin. HPLC analysis
weight near 70 kDa. showed a red intermediate and several yellow
HCA could also catalyze the formation of products (presumably of a polymeric nature) be-
4-coumaroyl and feruloyl-derivatives in Beta vul- ing betalamic acid, one of the final products.
garis (lampranthin II), Gomphrena globosa Zakharova et al.521 described the occurrence of a
(gomphrenin III), Lampranthus sociorum betalain oxidase in three species of Amaranthus.
(celosianin I and II), and Iresine lindenii This enzyme was found mainly in cell walls of
(lampranthin II).47 A. caudatus, and it was suggested that pigment
Betaxanthin biosynthesis (Figure 21C). yield can be increased by regulating the activity
Little is known about the betaxanthin biosynthe- of this enzyme during plant tissue extraction.
sis.472 However, it has been suggested the inter- Watson and Goldman500 reported dominant
change of basic compounds as one of the main alleles at two tightly linked loci (R and Y) of red
257
258
FIGURE 22 Amaranthin acylation catalyzed by the enzyme 1-0-hydroxycinnamoyl-transferase (HCA) in the biosynthesis of celosianin-II, an acylated betacyanin.
(Adapted from Ref. 49.)
beet. These alleles are involved in the production yellow and red phenotypes. The selective expres-
of betalain pigment. In addition, it has been shown sion of betaxanthin and betacyanin appeared to
that several alleles in these loci influence the occur through a limited number of discrete, stable,
production and distribution of betalains. The au- and differentiated states, because only four col-
thors also suggested the existence of genes that ored phenotypes were isolated.171
play an important role in betalain synthesis. In absence of specific information, it is only
possible to speculate about the regulatory mecha-
nism affecting the gene expression during pheno-
b. Biosynthesis Regulation typic transitions of plant cell cultures. It has been
proposed that DNA transposition or specific rear-
In plants, betalain biosynthesis is subject to rangements such as translocations, inversions,
complex regulation. Pigments are accumulated breakage, and fusion are involved. It has been
only in certain tissues and at specific stages of also suggested that such rearrangements could
development. Their synthesis has been shown to result in the relocation of particular genes, either
be regulated by light and cytokinins.135 It has been within the same chromosome or to another chro-
shown that effects of DOPA, light, and kinetin mosome. Moreover, it is possible that regulation
exposure are different between models producing of pigment synthesis is so tightly coupled to cel-
betacyanins or betaxanthins. The betanin synthe- lular morphology that an enhancement of gene
sis was only induced under the influence of DOPA expression never occurs in the yellow and red
or with the combination of kinetin and light. On cells.171
the other hand, betaxanthins were synthesized only Some inhibitors of cell division were used in
when the seedlings were fed with DOPA, and a Phytolacca americana suspension cultures, and a
significant increment of this production was ob- reduced betacyanin accumulation was observed,
tained by light and kinetin supplementation.37 even though their modes of action are known to
The presence of free betalamic acid in plants, be different. It was suggested that inhibitory ef-
which produce betaxanthins or betacyanins and fects on betacyanin accumulation are due to the
betaxanthins, and its absence in plants, which inhibition of cell division as a result of lack of
produce only betacyanins, suggest a regulatory DNA synthesis and that betacyanin biosynthesis
mechanism during its biosynthesis.451 A coordi- and accumulation are correlated not only with
nated condensation mechanism of betalamic acid DNA synthesis, but also with the progression of
with cyclo-DOPA or glycolized-cyclo-DOPA has the cell cycle.212
been suggested in plants that produce betacyanins;
then the accumulation of betalamic acid is ar-
rested. On the other hand, control mechanisms in c. Molecular Biology of Betalain
betaxanthin-producing plants must be different to Biosynthesis
allow the accumulation of betalamic acid (Mueller
et al., 1997b).46 Regulatory mechanisms on betalain Up to now, very few of the enzymes involved
formation are largely unknown, but the involve- in betalain synthesis have been purified and char-
ment of photoregulation and hormone control has acterized, despite the importance of betalains as
been suggested.395 natural food colorants.211 The only enzyme activi-
Cell lines produced from red beet showed a ties that have been described from higher plants
range of cell colors via specific induction meth- are enzyme preparations that catalyze the
ods. Phenotypic color ranged from white/green glycosylation of betanidin and enzymes involved
through yellow, orange and red to deep violet, in betalain degradation, but work regarding genes
representing all types of pigments found in red- is scarce.
beet plant. Differences in callus phenotypes were Two clones encoding polyphenol oxidase were
associated with cells of markedly different mor- isolated from a cDNA library constructed from a
phologies; cells were classified into two groups: log-phase suspension culture of Phytolacca
white, orange, and violet phenotypes, and green americana, producing betalains. Spatial and tem-
259
poral expressions were investigated by Northern the relationship between betalains, glucosinolates,
blot analysis of total RNA from various organs of and polyacetylenes in flowering plants.
Phytolacca plants. Transcripts of the two clones This remarkable correlation between chemi-
were found to be 2.1 and 2.3 kb. Both transcripts cal and morphological characters has led to pro-
were present only at substantial levels in the rip- pose that the order Centrospermae, including
ening of betalain-containing fruits.244 Cactaceae, must be reserved for betalain-contain-
Hinz et al.211 described the cloning and regula- ing families, while the anthocyanin-containing
tion of the gene dodA from basidiomycete ones (Caryophyllaceae and Molluginaceae) must
A. muscaria. This constitutes the first effort to be separated into the related but distinct order
clone genes of the betalain biosynthetic pathway. Caryophyllales.375
dodA codes for a DOPA dioxygenase. The cDNA The totally different chemical structure of
library was constructed from the cap tissue of young betalains and anthocyanins,158 the fact that they
specimens where betalain synthesis had not been are mutually exclusive, and the restricted distri-
yet begun. Southern blot analysis showed that bution of betalains are good arguments in favor of
DOPA dioxygenase is encoded by a single-copy the paramount taxonomic significance of these
gene in A. muscaria.211 Müeller et al.334,336 trans- pigments.
formed white petals of Portulaca grandiflora with Such arguments are not lessened by the find-
the dodA gene of A. muscaria, and the formation of ing of their occurrence in the Basidiomycetes of
yellow and violet spots that contained betalain and higher fungi, which do not have any phylogenetic
muscaflavin pigments was observed, indicating that relation to flowering plants. This coincidental
the fungal enzyme DOPA dioxygenase was ex- occurrence could be a case of chemical conver-
pressed in an active form in plants. gence under evolutionary phase.371 Considering
Notwithstanding these promissory results, it the crucial dependence of a living system on the
is obvious that much more research work is needed economy of energy and our growing knowledge
in order to unravel the processes involved in bio- about the chemoecology of secondary products in
synthesis and regulation of betalains. nature, this assumption can be feasible.451
5. Functions b. Ecological and Physiological Aspects
a. Taxonomic Markers As in the case of other secondary metabolites,

it is impossible to assign a definite function to
Even before the structure of betalains was betalains in the economy of the organisms that
evident, the importance of betalain pigments produce them.375 When pigments are in flowers or
in plant taxonomy and systematic distribution fruits they may have a role as attractants for vec-
was clear. Betalains are in eight tors (insects or birds) in the pollination process
families: Amaranthaceae, Aizoaceae, Basellaceae, and in seed dispersal by animals, such as antho-
Chenopodiaceae, Cactaceae, Nyctaginaceae, cyanins.371,503
Phytolaccaceae, and Portulacaceae. Nowadays, The occurrence in other plant parts (e.g.,
it is known that 9 of 11 families of the leaves, stem, root) may be devoid of immediate
Caryophyllales order contain betalains.402 The function. However, it has been suggested that
recent addition to the list of betalain families is betalain accumulation in red beet root is related to
Didieraceae, a small family from Madagascar.451 the storage of carbohydrates as a physiological
Only two families of Caryophyllales, viz., response under stress conditions.260 In addition,
Caryophyllaceae and Molluginaceae, lack the transient coloration of many seedlings and the
betalains and possess anthocyanins. This suggests reddening of senescent leaves of several plants of
an early differentiation of the Caryophyllales into Caryophyllales order (e.g., Kochia scoparia) have
groups with different kinds of pigments. Rosendal- no obvious physiological or ecological reasons.
Jensen et al.402 constructed a phenogram that shows Whatever its significance, the process resembles
260
the analogous phenomenon observed in antho- villea bracts mixed with honey is used widely for
cyanin-producing species. Betalains are also pro- a cough. However, in both cases the action mecha-
duced in injured tissues, normally not pigmented, nisms are still unknown.
possibly as a defense mechanism against infec- Finally, in a recent work, the importance of
tion. This physiological response was only ob- some natural pigments as nutraceutical ingredi-
served in plants possessing specific factors that ents was reviewed.382 It was suggested that
have been associated with two novel antifugal betalains like anthocyanins, β-carotene, and vari-
proteins.263 ous vegetable and fruit extracts must be used for
Interestingly, it must be mentioned that their potential health benefits. For example, yel-
betalains from Beta vulgaris (betanin and low betaxanthins, in addition to their potential
vulgaxanthin) are effective inhibitors of indoleace- role as natural food colorant, may be used as a
tic acid (IAA) oxidase and that betanin counter- means of introducing essential dietary amino ac-
acts the inhibitory effect of IAA on wheat root ids into foodstuffs, giving rise to an “essential
elongation.375 It means that betalains could be dietary colorant”.273 Another new interesting area
considered as potential modifiers of auxin me- includes foods that can generate their own light
tabolism; notwithstanding, there is no evidence (“biolume products”) in which betalains could
that supports the betalains role as in vivo regula- play an important role, suggesting how exciting
tors of IAA activity. the area of natural colorants has become. As other
topics, it is necessary to emphasize the need of
more research in these new areas.
c. Pharmacological Effects
Although structurally related to alkaloids, 6. Methodological Aspects

betalains have no toxic effects in the human body,
as can be deduced from the fact than they are a. Extraction
present in considerably high amounts in certain
foodstuffs, such as red-beet, prickly pear fruits, Betalains-containing material (raw plant or
and Amaranthus seeds.46 Therefore, betalains rep- cell culture) are generally macerated or ground.
resent a safe natural alternative to some synthetic Pigments can be extracted with pure water, cold,
color additives that are currently in use. Interest- or at room temperature, although in most cases
ingly, there is no upper limit to the recommended the use of methanol or ethanol solutions (20 to
daily intake.158 50% v/v) is necessary to achieve complete extrac-
On the other hand, in betanin tested for mu- tion. 375 Sometimes, the necessity of an
tagenic and carcinogenic activity an absence of aerobic juice fermentation (e.g., Saccharomyces
mutagenicity in five Salmonella typhimurium cerevisiae, Aspergillus niger) in order to reduce
strains was observed, and it did not initiate or free sugars and then to increase the betacyanin
promote hepatocarcinogenesis in levels of 50 content has been reported.379 In both procedures,
mg/kg of weight of pure betanin or diets contain- the inactivation of degradative enzymes by a short
ing 2000 mg/kg of betacyanin.429,431 Notwithstand- heat treatment of the extract (70ºC, 2 min) could
ing, after ingestion of these products (particularly be desirable, although this may destroy some of
red beet), betanin occasionally appears in the urine, the pigments. Betacyanins can be precipitated by
an effect known as beeturia or betaninuria. The a slight acidification with hydrochloric acid or
etiology and mechanism of this disorder are still with acidified ethanol (0.4 to 1% HCl); subse-
controversial.371 There are a very few pharmaco- quently, by the addition of 95% aqueous ethanol
logical applications of betalains. Recently, they yields betaxanthins.39,373
have received attention because betanin has shown Degradation of betanin may occur very fast
antiviral and antimicrobial activities (e.g., Pythium and destruction of complex pigments should also
debaryum, a pathogenic fungi in red-beet). In be avoided, because acid-acylated betacyanins are
some places in Mexico, an infusion of Bougain- rapidly deacylated and such pigments can be over-
261
looked.451 In such a situation, extraction should be terization of betalains; however, time analysis is
carried out with cold water for long-term and longer, but it could be shorten if separation is
darkness conditions. focused on the two major red pigments, leaving
the aglycones unseparated. Betaxanthins can also
be quantitated by CZE with an acceptable resolu-
b. Separation tion.
By contrast, thin layer chromatography (TLC)
Ion-exchange and column chromatography. is not widely used for betalains because its low Rf
Ionic-exchangers are the most widely used values; however, Bilyk41 developed a preparative
adsorbents in fractionation, as much as in separa- TLC system in a 0.5-mm cellulose-coated plate
tion; then gel filtration is used.374 In a simple and using two different mobile phases: isopropanol-
rapid procedure, plant extract must be stirred with ethanol-water-acetic acid in a ratio of 6:7:6:1
the ion-exchanger resin (e.g., Dowex 50W-X2, (v/v) in the first solvent mixture and a 11:4:4:1 (v/
Merck I, DEAE-Sephadex A25, etc.), which v) ratio in the second one. When acid is incorpo-
adsorbs the betalains (nonionic interaction). Sub- rated in the developing solvent, betalain mobility
sequently, resin is washed with aqueous HCl (0.1% on the TLC plate is facilitated due to protonation
v/v) and pigments are eluted with water followed of the betacyanin carboxyl group. The acid anion
by final separation on a chromatographic column provides an electrically neutral system by its in-
(e.g. Polyamide, Polyclarc-AT, or polyvinylpyr- teraction with the quaternary nitrogen. The
rolidone, Sephadex G-15 and G-25). same effect occurs with betaxanthins.41 Good
The chromatographic and electrophoretic betaxanthin separations was also obtained on
properties from unknown plant materials can be diethylaminoethyl cellulose plates using isopro-
compared with those reported in the literature for panol-water-acetic acid (13:4:1 v/v).451 No indi-
known pigments (e.g., Piatelli and Minale,374 for cator is needed for visualization of the separated
betacyanins; Minale et al.316 for acylated betalains; pigments because they are clearly observed on the
Piatelli and Imperato; 370 von Elbe et al.; 496 TLC plate with their natural colors.
Piatelli;371 and Steglich and Strack449 for betalains High-performance liquid chromatography
in general). (HPLC). The HPLC technique has become the
Electrophoresis and thin layer chromatog- method of choice for chromatographic separa-
raphy (TLC). Paper electrophoresis using pyri- tion, rapid quantification, and tentative identifica-
dine and formic or acetic acid as solvents or in tion of betalains. The first application was done
cellulose are common and reliable methods for by Vicent and Scholz488 using a C18 column with
betacyanin detection,380 because they migrate first a gradient run using tetrabutylammonium in paired
as immobile zwitterions (pH 2), followed as ion system as the mobile phase. The most useful
monoanions (pH 2 to 3.5), and finally as bis- column supports are C8 and C18 reversed phase
anions (pH 3.5, 7.0). In the case of betaxanthins, (e.g., Nucleosil, LiChrosorb, µBondpack, etc.),
the mobility may be related to indicaxanthin, and with particle sizes between 3 to 10 µm, while the
betacyanins are related with the mobility of most used solvents are water-methanol or water-
betanin.374 Electrophoresis can be carried out us- acetonitrile mixtures, acidified with acetic, for-
ing pyridine-citric acid solvent, voltage gradient mic, or phosphoric acid.451 HPLC elution order of
of 5.6 volts/cm, and a temperature of 4ºC.496 Re- pure crystalline pigments was as follows: betanin,
cently, capillary zone electrophoresis (CZE) has betanidin, isobetanin and isobetanidin.432 This
been used for the analysis of betalains, particu- evidence was based on an acid hydrolysis of the
larly from Beta vulgaris.452 This technique was glycosides to yield aglycones and isomerization
carried out with a fused-silica capillary at 15ºC of betanin to isobetanin occurring.488
and at a constant voltage of -22 kV, and it has More recently, Pourrat et al.379 analyzed a
permitted the separation of betanin, isobetanin, fermented red-beet root extract using a reversed-
and their corresponding aglycones. CZE has been phase C18 column and ion-pairing and methanol-
used successfully for the separation and charac- water as mobile phase, and the elution order was
262
TABLE 9
Differentiation between Anthocyanins and Betalains
Test Anthocyanins Betalains

Addition KOH, NaOH Final color changes to blue-green Color changes to yellow
Electrophoresis Movement toward cathode Movement toward anode
Addition hot-aqueous HCl Color-stable Destruction of color
Extraction with amyl alcohol Yes, at low pH Does not enter at any pH
Thin layer chromatography Moderate mobility None

· n-butanol-acetic acid-water
(BAW) Low/intermediate mobility High mobility
· Aqueous solvents
Column chromatography Elution with water Elution with methanol/HCl mixtures

· cationic resins
Adapted from Refs. 375, 451.
betanin, isobetanin, betanidin, isobetanidin, and Spectroscopy. Betalain analysis as that of

prebetanin for the betacyanins and vulgaxanthin I other colored compounds has been based basi-
followed by vulgaxanthin II for the betaxanthins. cally on UV-visible spectroscopy. As a matter of
Another good example of betaxanthin character- fact, red violet betacyanins absorbs around λmax
ization was carried out by Trezzini and Zrÿd.472 = 540 nm, while yellow betaxanthins at λmax =
Betalamic acid was conjugated with both protein 480 nm, and the starting studies of betalain iden-
and non-protein amino acids to yield a series of tification were supported in this methodology. In
betaxanthins. They described retention character- addition, structural modifications of betalains have
istics for 15 naturally occurring pigments such as been followed by UV-visible spectroscopy.287,288,375
portulaxanthin-I, miraxanthin-II, vulgaxanthin-I, However, in the 1980s spectroscopy showed enor-
among others. Such products could be used as mous progress and nowadays chemical character-
HPLC standards for unknown pigments. ization must be carried out considering at least
HPLC separation and UV-visible, MS, and NMR
spectroscopies: rigorous characterization of
c. Characterization betanin, lampranthins, cellosianins, neobetanin,
among others, was established thanks to these
General procedures. In most cases it is im- methodologies.449,451,452
possible to distinguish between anthocyanins Chemical tests. Chemical methods for the
and betalains visually. However, the extract source synthesis and degradation of pigments are very
is an indication for the presence of betalains or important in betalain research. Some of these es-
anthocyanins because in plants the presence of tablished methods are well described in more detail
one is mutually exclusive to the other. It is impor- by Strack et al.,451 and they are briefly summa-
tant to remember that betalains are characteristic rized in this section. A number of color reactions
pigments in plants members of Caryophyllales. based on changes in pH have been proposed to
Preliminary tests have been developed to easily distinguish between betalains and anthocyanins.
distinguish between betacyanins and anthocya- Acid hydrolysis (dilute aqueous HCl) of
nins (Table 9) using the color exhibited at differ- betanin gave a mixture of both aglycones,
ent pHs and their temperature.451 betanidin, and its 15R epimer isobetanidin; this
263
mixture is easily separated by chromatographic methylneobetaninidin dimethyl ester in good yield
methods. On the other hand, enzyme-catalyzed (Figure 23), exhibiting an absorption at 403 nm,
hydrolysis produces only betanidin.374 Moreover, which can be shifted, with the addition of acid, to
heating by prolonged time produces the cleavage 513 nm.375 Also, neo-derivatives can be obtained
of betanin into betalamic acid and cycloDOPA from betaxanthins on treatment with
5-O-glycoside.371 After alkali fusion, betanidin was diazomethane, whereas esterification with
split into 4-methylpyridine-2,6-dicarboxylic acid, CH3OH-HCl or CH3OH-BF3 affords the normal
5,6-dihydroxy-2,3-dihydroxyindol, and formic esters.
acid; together, these fragments revealed the car- Quantification and pigmenting efficiency.
bon structure of betanidin. Epimerization at C15 is Nilsson342 developed a method to measure both
also observed by betanidin treatment with diluted red and yellow betalains of Beta vulgaris without
alkali or citric acid solution (5% aqueous); in both prior separation of betacyanins and betaxanthins.
reactions a betanin-isobetanin ratio of 3:2 was Their quantitative determination mainly involved
observed, while the isobetanin produced a 2:3 spectrophotometry, where the absorbance at the
ratio under the same treatment conditions. In ad- maximum wavelength (λ) is translated into con-
dition, alkaline treatment, for example, alkaline centration by means of the appropriate absorptivi-
hydrolysis during deacylation of gomphrenin-II ties. Another method is based on electrophoretic
to gomphrenin-I, a 1:1 betacyanin-isobetacyanin separation of individual pigments followed by the

ratio can be obtained. measurement of the color intensity of the sepa-
On the other hand, betaxanthin analyses in- rated bands in a densitometer. The result was
volve methodologies for amino acid analysis. They expressed as peak area in cm2, which was deter-
are hydrolyzed with 1 N aqueous HCl or 0.6 N mined with the aid of an integrator after correct-
ammonia to obtain betalamic acid and free amino ing the baseline; thus, the results are translated to
acids.375 The reaction of betanin in ammonia alka- concentration comparing them with a betanin stan-
line solution with an excess of amino acids are dard curve.496
used in betaxanthin synthesis. This reaction is A computer-aided determination, based on
followed by monitoring the increments of the previously reported absorptivity values, has been
betaxanthin maximum (absorption at 475 nm) or performed by Saguy et al.408 This method uses a
decrements of betanin maximum at 540 nm. Thus, nonlinear curve fitting of the spectrum with a
vulgaxanthin II can be obtained mixing betanin in predicted function of the individual pigments (e.g.,
0.6 ammonia solution with an excess (10 M) of betanin, betalamic acid, vulgaxanthin-I). The pro-
glutamic acid; this base exchange method has posed procedure is rapid and accurate, avoiding
also been applied for the synthesis of the laborious and time-consuming separation steps.
indicaxanthin, miraxanthin, and other Schwartz and von Elbe432 developed a method to
betaxanthins. However, nonnaturally occurring quantify individual betalains by HPLC using the
betaxanthins can be obtained with serine, pheny- molar absorptivity of each pigment instead of
lalanine, threonine, and lysine.472 absorptivity values. This method provides a more
Indicaxanthin oxidation with peroxyacetic acid accurate determination of the total betalain con-
yields L-aspartic acid, a reaction used to demon- tent.
strate the 11S configuration of this betaxanthin. It must be pointed out that discrepancies
Betanin has the 15S configuration at the between spectrophotometric and HPLC meth-
dihydropyridine ring, as indicaxanthin was ob- ods have been observed. Differences up to 15%
tained via amino acid exchange from betanin. have been reported after an extended heat treat-
This is in accord with the isolation of S-betalamic ment of betalains. Such differences have been
acid after the degradation of many betaxanthins attributed to degradation products or interfer-
with alkali treatment.451 The formation of neo- ing substances formed during processing.430 Pre-
derivatives demonstrated the tendency of the vious fermentation of red beet juice is recom-
dihydropyridine ring to amortize. Thus, treatment mended for betalain quantification because free
of betanidin with diazomethane gives di-O- sugars are degraded and betalain content of the
264
FIGURE 23. Aromatization of dihydropyridine ring to form a neoderivated. (Adapted from Ref. 451.)
265
extract is enriched; thus, HPLC is more easily vulgaxanthin I was most stable between pH 5.0 to
performed.379 6.0, and it was more stable in juice than in puri-
Interestingly, it has been shown that betalain fied extracts, while optimal pigment stability in
quantitation by capillary zone electrophoresis is reconstituted powders was noted at pH 5.7.440
in close agreement with the HPLC determina- Temperature. Temperature also shows a clear
tion.452 effect on betalain stability.129 Thermal kinetic deg-
On the other hand, pigment efficiency is usu- radation of betanin has been evaluated by several
ally measured in terms of CIELAB parameters. It authors.4,129,225,226,408,495 It has been reported that
means that triestimulus colorimetry is the best thermostability of betanin solutions is pH depen-
methodology to carry out such measurements.158,230 dent and partially reversible. Heating of betanin
Values provided by universal colorimeters are solutions produces a gradual reduction of red color,
described in previous sections (“L”, “a”, and “b”). and eventually the appearance of a light brown
Sapers and Hornstein416 reported the Hunter color. von Elbe et al.495 observed a first-order
color values for standardized dilutions of juice reaction kinetics for betanin degradation by heat-
from 48 red beet cultivars. “a” values varied over ing.
a relatively narrow range being slightly lower in Saguy et al.408 developed a thermal kinetic
samples with higher “b” values. The analyses of degradation model using a nonlinear least-square
the 20 most highly pigmented cultivars produced technique; this model enables one to predict the
values in the range 8.5 to 17.2. Interestingly, it retention of betalains under variable conditions of
was reported that “b” values could be used in the temperature and time. Thermal degradation of
estimation of the betaxanthin-betacyanin. betalains produced activation energies EA) in the
range 17 to 21 Kcal·mol-1 for the forward reac-
tion, whereas the reverse reaction showed values
7. Importance as Food Colors — between 0.6 to 3.5.225,408 It was also reported that
Stability, Processing, and Production EA values showed a pH dependence. Betalamic
acid and cycloDOPA-5-O-glycoside have been
a. Stability in Model Systems reported as the probable intermediates for betanin
degradation.226,408 Interestingly, the regeneration
When betalains are used as food colorants, (reverse reaction) involves a Schiff’s base con-
color stability is a major concern. There are sev- densation of the amine group of cycloDOPA-5-
eral factors that have been recognized to affect O-glycoside with the aldehyde group of betalamic
the stability of these pigments: acid; betanin is rapidly formed when both com-
pH. The hue of betalains is unaffected at the pH pounds are mixed in solution.226 Altamirano et
between 3.5 to 7; the values of most foods are in al.4 reported the lowest stability of betanin and the
this range. Betalain solutions in this pH range lowest EA in a water-ethanol model system, sup-
showed a similar visible for betacyanins and porting the idea that the first step of the thermal
betaxanthins. Betacyanin maximum is in the wave- betanin degradation is the nucleophilic attack on
length (λ) range 537 to 538 nm, while betaxanthin the >N+ = CH- structure of betanin. Ethanol has
maximum is between 475 to 477.497 Below pH a high electron density on the oxygen atom; there-
3.5, λ shifts toward a lower wavelength, and fore, it is a strong nucleophilic agent that dimin-
above 7 the change is toward a longer wave- ishes the betanin stability.
length; out of the pH range 3.5 to 7 the intensity Light. Von Elbe et al.495 found that rate of betanin
of the visible spectra decreases.225 degradation increased 15.6% after pigment day-
Stability of betanin solutions is pH depen- light exposure at 15ºC. Degradation of light-ex-
dent. Huang and von Elbe225,226 have shown that posed betalains followed a first-order kinetic. In
optimal pH for maximum betanin stability in the addition, it was observed that degradation was
presence of oxygen is between 5.5 to 5.8. Red higher at pH 3.0 (k = 0.35 days-1) than at pH 5.0
beet solutions showed their maximum stability at (k = 0.11 days-1), when betacyanins were exposed
pH 5.5, the normal pH for beets. In addition, to fluorescent light. On the other hand, at dark-
266
ness conditions betacyanins were most stable (k molecular oxygen, producing pigment degrada-
= 0.07 days-1).416 Attoe and von Elbe16 showed tion in air-saturated solutions.15 Degradation ki-
an inverse relationship between betalain stability netic under air atmosphere follows a first-order
and light intensity in the range 2200 to 4400 lux). model, but deviates from first-order in the ab-
It is explained that visible light absorption ex- sence of oxygen. As mentioned above betanin
cites π electrons of the pigment chromophore to degradation is a partially reversible reaction,15
a more energetic state (π*). This would cause a and it has been reported that in order to increase
higher reactivity or a lowered activation energy the recovering of pigment it is necessary to have
for the molecule (EA = 25 Kcal·mol-1 in darkness the samples under low levels of oxygen. Thus,
and 19.2 in illumination). The effect of UV and heated betanin solutions (pH 4.75, 130 min, 15ºC)
gamma irradiation on betanin stability was re- under low oxygen levels showed an increased
ported by Aurstad and Dahle;17 total pigment betanin retention from 54 to 92%.225 Reaction
destruction was reported by the treatments with reversibility was responsible for the deviation from
120 h of UV radiation or with 100 krad of gamma the first-order degradation kinetics of betanin in
radiation. Nevertheless, these results, the the absence of oxygen.
photodegradation mechanisms for betalains re- Several methods have been reported to prevent
main to be determined. the destruction or to improve the stability of pig-
Water activity. Recognizing the importance of ments, including degassing, addition of antioxidants
water in many degradation reactions, it is not and stabilizers, control of pH, minimal heath treat-
surprising that water activity (aw) is included ment, among others,5,15,40,190,363 and these efforts have
among the primary factors affecting the betalain been directed to their application in food products.
stability and/or color of a food product contain-
ing these pigments.494 Because the degradation
reaction does involve water, the greatest stability b. Processing and Stability in Foods
of betalains has been reported in foods or model
systems of low moisture and aw.82 Pigment deg- The sensitivity of betalains to different fac-
radation follows first-order kinetics, and stabil- tors suggests that their application as food colorants
ity increases with decreasing aw.407 It has been is limited. Based on these properties, betalains
established that aw has a pronounced exponential can be used in foods with a short shelf-life, pro-
effect on pigment stability. Pigment stability deduced by a minimum heat treatment, and pack-
creases in one order of magnitude when aw was aged and marketed in a dry state under reduced
increased from 0.32 to 0.75.82 levels of light, oxygen, and humidity.390,497
On the other hand, Simon et al.439 studied the Betalains have several applications in foods,
influence of aw on the stability of betanin in vari- such as gelatins desserts, confectioneries, dry
ous water-alcohol model systems. In all cases, it mixes, poultry, dairy, and meat products.87,497 Table
was observed a rate-constant dependence with 10 summarizes some applications of betalain pig-
aw.439,494 The increase in stability of betanin with ments in food products. The amount of pure pig-
decreasing aw may be attributable to reduced ment required in these foods groups to obtain the
mobility of reactants or limited oxygen solubility. desired hue is relatively small and for most appli-
Consequently, high moisture content pro- cations does not exceed 50 ppm of betalains, cal-
duces a high degradation rate. Furthermore, speci- culated as betanin. Problems associated with
fication of aw alone without the moisture content betalain degradation and pigment recovery dur-
is not enough to predict pigment stability. ing the processing operations are of economic
Oxygen. Oxygen causes a product darkening importance and must be solved to betalains dis-
and loss of color. Von Elbe et al.495 stored buff- place the application of synthetic dyes in some
ered betanin solutions at pH 7 under atmosphere food products. The effectiveness of commercial
of air and nitrogen for 6 days at 15ºC; it was betalains depends largely on a continuous avail-
observed that color degradation increases up to ability of highly pigmented sources, the use of
15% due to air conditions. Betanin reacts with cold and modified storage atmospheres prior to
267
TABLE 10
Applications of Beet Root Powder as Natural Color in Food Products
Food products Shade Level
Dairy products:
· Strawberry yogurt Rose–pink 0.09%

· Ice creams Pink 0.25%
Rose–pink 0.20%
Meat products:
· Sausages Pink 600 mg/100 g

· Cooked ham Pink–brown 0.17%
Dry powder beverages Strawberry 1.2%

Raspberry 1.5%
Blackcurrant 1.0%
Water ices Strawberry–red 0.5 to 1.0%

Raspberry 0.5 to 1.0%
Marzipan Pastel–red 0.4%

Bluish–red 2 mg/cm2
Baked goods Pink–brown 2.5%
Biscuit creams Pink 0.28%

Brown 1.6%
Hard candies Pink 0.1%
Jellies Raspberry–red 0.2%
Fruit cocktails Raspberry–red 2.0%
Adapted from Ref. 87.
processing, efficient enzymatic control, handling thermore, this value is increasing as advanced
practices, extraction procedures, purification, con- selection is developed.382
centration, and finishing operations e.g., freeze, Commercial preparations of beet pigment
spray, and vacuum drying). for use as food colorants are available as either
Nowadays, beet roots represent the main com- juice concentrates (produced by concentrating
mercial source of betalains (concentrates or pow- juice under vacuum to 60 to 65% total solids) or
ders).382 Many factors during the pre- and post- powders (produced by freeze or spray drying).
harvest period and during processing influence These preparations contain from 0.3 to 1% of
the recovery of these natural beet colorants. In pigment.42,76,77,497 They show a variety of colors,
addition, recent efforts are centered around the depending on their content of yellow pigments,
betalain content in red beets through selective and may have a beet-like odor and flavor. The
breeding. Initially, high pigment content is very remainder of the solids is mainly sugars (75 to
important. The average pigment content of beets 80%), ash (8 to 10%), and protein (10%). On a
is approximately 130 mg/100 g fresh laboratory scale, betalains can be obtained by
weight,416,496,497 but new red beet varieties produce employing reverse osmosis, 276 ultrafiltra-
around 450 to 500 mg/100 g fresh weight. Fur- tion,30,391,392 solid -liquid extraction,275,505 and dif-
268
fusion.504 These processes have been shown to betaxanthins. Some models do not produce
be efficient on the recovery of betalains from betaxanthins. It has been also established that
raw beet tissue when compared with conven- main betacyanin in most of the studied models is
tional hydraulic techniques.391 As approximately betanin. Schwitzguébel et al.434 observed that
80% of beet juice solids consist of fermentable individual Beta vulgaris callus cultures contained
carbohydrates and nitrogenous compounds, a fer- cells exhibiting a variety of colors either non-
mentation process to remove these materials has pigmented, yellow, orange, red, or purple. The
been widely employed.128 The yeast Candida range of observed pigmentation was due to the
utilis and Saccharomyces cerevisiae have been presence of the deep red-purple betacyanins and
used in the fermentative process, whereas a strain to the yellow betaxanthins contained within the
of Aspergillus niger not only destroyed free sug- cell vacuole.171
ars but also enriched the colorant in betanin.379 Recently, plant hairy roots have become of
The powder obtained from fermented juice con- interest as an alternative for cell culture because
tained five to seven times as much as the their infinite and active proliferation in a phyto-
betacyanin obtained in the powder from raw juice hormone-free medium and their ability to synthe-
(on a dry weight basis). size and accumulate valuable betalains at compa-
rable levels to those found in plants.459 Extracellular
production of betalains accompanied by pigment

c. Production of Betalains by Plant Tissue release has been obtained in hairy root cultures
Culture under oxygen starvation.255 However, both plant
cell and hairy root cultures are influenced by a
Cell tissue culture has been a very useful tool variety of physical and chemical factors impli-
in the study of various aspects of biochemistry, cated in the production of betalain pigments, and
enzymology, genetics, and biosynthesis of some of them, such as growth regulators, light,
betalains,273 and, interestingly, betalain produc- nitrogen, carbon, and microelements, are widely
tion by plant cell culture will represent an excel- discussed by Böhm and Rink46 and Leathers, et
lent option in the future; it has a number of advan- al.273
tages over conventional procedures. Mainly with In addition, it is important to consider that
this methodology, it is possible to control quality betalain production by cell or hairy root cultures
and availability of pigments independently of are mainly empirical, and it is not supported by a
environmental changes.126 Nevertheless, the pro- strong knowledge of the underlying mechanisms
ductivity of the bioreactor systems must be in- of biosynthesis and regulation. Nevertheless, in
creased over 0.168 mg/g dry weight/day240,446 and some instances, such as in the production of
the cost reduced below $0.15 U.S. dollars/l in B. vulgaris betalains, it is possible to obtain cul-
order to be considered economically feasible.241 tures producing specific pigments in comparable
Thus, a successful betalain production will de- or even larger quantities than in the tissues of the
pend on process optimization to maximize yields, original plant.446
and, consequently, suitable downstream recovery Recently, Hempel and Böhm207 adminis-
techniques must be available.382 trated nine L-amino acids to hairy root cultures
Betalain production has been detected in cell of Beta vulgaris var. Lutea. Two betaxanthins,
cultures of plant species belonging to five fami- portulaxanthin II and vulgaxanthin I, were pro-
lies of Caryophyllales.46 Betalain accumulation duced predominantly, while minor quantities
of betalains in beet callus culture was reported of muscaauri-VII, dopaxanthin, and
by Constabel and Nassif-Makki85 and also has indicaxanthin were synthesized de novo. These
been demonstrated in cell cultures of P. grandi- results are very important because the possibil-
flora, 137 A. tricolor, 37 O. microdasys, 239 Ch. ity of betaxanthin production at commercial
rubrum,35 and P. americana.212 Plant cell cul- level is opened, and, interestingly, red beet is
tures are generally deep-red or purple colored; it one of the best known betalain production mod-
means that betacyanins are dominant over els, being easiest the methodology standardiza-
269
tion. Nowadays, the selection of a bioreactor broad range of natural colors, and importantly
and cultivation techniques for optimal culture with better stability characteristics.
growth and betalain production is one of the It is convenient now to point out that plant
most important issues to be solved.241 Much pigment production is limited, being that pig-
data are now available about the growth and ments are secondary metabolites. Furthermore,
production kinetics in suspension cultures;273 agriculture must continue focusing on food pro-
however, most of them are not suitable for the duction for a growing population, and, undoubt-
design of large-scale processes, and the growth edly, food color does not matter for millions of
and production kinetics must be studied under people all over the world as much as basic food
well-defined conditions and at different steady production. However, the demand for better natu-
states using the type of bioreactor selected for ral-colored foods by an important sector of the
the large-scale process.241 society will be increased because it is highly likely
that future studies will increase people’s con-
science about the positive health benefits of natu-
V. FUTURE TRENDS ral pigment consumption. Consequently, better
production systems for natural pigments will be
Since the 1960s people have shown a clear required higher productivities in smaller areas).
preference for natural products, including pig- Then, research interests will evolve, and two ar-
ments, because more nutritious and healthy char- eas will be the future of natural pigment produc-
acteristics have been associated with them. Re- tion: the generation of crops with improved char-
markably, food scientists consider that this acteristics, and pigment production at the
consumer trend will be maintained in the future. industrial level and under controlled conditions.
In this sense, food technologists will continue An interesting goal of food research will continue
affronting the problems of availability and stabil- being the production of highly consumed crops
ity of natural pigments in order to replace the (e.g., rice, corn, wheat, oat, bean) with improved
synthetic ones. Moreover, one of the main prob- chemical composition, plus better functional per-
lems to be solved, that natural colors approved by formance, and at the same time favoring their
FDA and European Union do not cover all ranges nutraceutical properties, and genetic-engineered
of colors (e.g., blue). Thus, many research groups plants with higher productivity and with modified
are looking for new sources of natural pigments; biosynthetic pathways as well. In order to pro-
however, these efforts have vanished, because duce such crops, the understanding of the in-
under the current legislation the FDA or the Eu- volved metabolic pathways must be wider and
ropean Union approval of new natural sources of deeper. In this sense, more studies must be carried
pigments is very difficult, contrasting with poli- out to have a complete vision of the biosynthesis
tics followed in Japan. Consequently, it is ex- and regulation of carotenoid and betalain plant
pected that the world global market must contrib- production, while nowadays those performed on
ute to the implementation of more realistic laws. the anthocyanin biosynthetic pathway will con-
On the other hand, it has been clearly established tinue focusing on regulatory aspects. Remark-
that technology development has introduced new ably, the most impressive advances in these as-
methodologies or processes to avoid the intrinsic pects have been reached using molecular biology
instability and solubility problems of natural pig- techniques, and this will be so in the near future.
ments, and today most of these problems can be Interestingly, model systems for pigment pro-
solved through technological processes. In addi- duction under controlled conditions are now avail-
tion, the pigment research area is very wide, and able, but production at the industrial level has not
natural pigments with improved characteristics been feasible yet. Thus, carotenoid production by
(new colors and improved stability) have been yeasts, bacteria and fungi, and anthocyanin and
discovered, being that their application differed betalain production by plant tissue cultures re-
by the above-mentioned reasons. Thus, a more quire the development of better biotechnological
adequate and updated legislation will provide a approaches.
270
ACKNOWLEDGMENTS 11. AOAC. Official Methods of Analysis, Association of
Official Analytical Chemists. Washington, D.C., 1980.
12. Arenas, O. M., Sánchez, R. M., del Villar, M. A.,
FDV, ARJ, and OPL acknowledge the finan- Jiménez, A. A., and Dávila, O. G., Obtención de
cial support received from the Consejo Nacional pigmentos de interés alimentario mediante el cultivo
de Ciencia y Tecnología, México (CONACYT); de tejidos vegetales a partir de epazote (Teloxis
ARJ also acknowledges the support from the ambriosoides L.), Biotecnología, 1993; 3(3,4): AV1–
Instituto Politécnico Nacional (IPN) on natural AV9.
13. Armstrong, G. A., Genetics of eubacterial carotenoid
pigment studies.
biosynthesis: a colorful tale, Ann. Rev. Microbiol.,
1997; 51: 629–659.
14. Armstrong, G. A. and Hearst, J. E., Genetics and
REFERENCES molecular biology of carotenoid pigment biosynthe-
sis, FASEB J., 1996; 10: 228–237.
1. Adewusi, S. R. A. and Bradbury, J. H., Carotenoids in 15. Attoe, E. L. and von Elbe, J. H., Oxygen involvement
Cassava: comparison of open-column and HPLC in betanine degradation: effect of antioxidants, J. Food
methods of analysis, J. Sci. Food Agric., 1993; 62: Sci., 1985; 50: 106–110.
375–383. 16. Attoe, E. L. and von Elbe, J. H., Photochemical deg-
2. Al-Babili, S., Lintig, J. V., Haubruck, H., and Beyer, radation of betanine and selected anthocyanins, J.
P. A novel soluble form of phytoene desaturase from Food Sci., 1981; 46: 1934–1937.
17. Aurstad, K. and Dahle, H. K., The effect of heath
Narcissus pseudonarcissus chromoplasts is Hsp70-

complexed and competent for flavinylation, mem- treatment UV and gamma radiation on some beet root
brane association and enzymatic activation, Plant J., pigments, Z. Lebensm. Unters-Forsch., 1973; 151:
1996; 95): 601–612. 171–174
3. Albert, S., Delseney, M., and Devic, M., BANYLUS, 18. Bakker, J. and Timberlake, C. F., Isolation, identifica-
a novel negative regulator of flavonoid biosynthesis tion, and characterization of new color-stable antho-
in the Arabidopsis seed coat, Plant J., 1997; 11: 289– cyanins occurring in some red wines, J. Agric. Food
299. Chem., 1997; 45: 35–43.
4. Altamirano, R. C., Drdák, M., Simon, P., Rajniakova, 19. Baldi, A., Romani, A., Mulinacci, N., Vincieri, F. F.,
A., Karovicová, J., and Preclík, L., Thermal degrada- and Casetta, B., HPLC/MS application to anthocya-
tion of betanine in various water alcohol model sys- nins of Vitis vinifera L., J. Agric. Food Chem., 1995;
tems, Food Chem., 1993; 46: 73–75. 43: 2104–2109.
5. Altamirano, R. C., Drdák, M., Simon, P., Smelík, A., 20. Baranac, J. M., Petranovic, N. A., and Dimitric-
and Simko, P., Stability of red beet pigment concen- Markovic, J. M., Spectrophotometric study of antho-
trate in maize starch. J. Sci. Food Agric., 1992; 58: cyan copigmentation reactions. II. Malvin and the
595–596. nonglycosidized flavone quercetin, J. Agric. Food
6. Ananboranich, S., Gulik, P., and Ibrahim, R. K., Fla- Chem., 1997; 45: 1694–1697.
vonol sulfotransferase-like c-DNA clone from Flaveria 21. Barber, J., Nield, E. P., Zheleva, D., and Hankamer,
bidentis, Plant Physiol., 1995; 107: 1019–1020. B. 1997. The structure, function and dynamics of
7. Andersen, O. M., Viksund, R. I., and Pedersen, photosystem two. Physiol. Plant., 100: 817–827.
A. T., Malvidin 3-(6-acetylglucoside)-5-glucoside 22. Barth, M. M., Zhou, C., Kute, K. M., and Rosenthal,
and other anthocyanins from flowers of Gera- G. A., Determination of optimum conditions for
nium sylvaticum, Phytochemistry, 1995; 38: 1513– supercritical fluid extraction of carotenoids from car-
1517. rot (Daucus carota L.) tissue, J. Agric. Food Chem.,
8. Andersen, O. M. and Fossen, T., Anthocyanins with 1995; 43(11): 2876–2878.
an unusual acylation pattern from stem of Allium 23. Bartley, G. E. and Scolnik, P. A., Molecular biology
victorialis, Phytochemistry, 1995; 40: 1809–1812. of carotenoid biosynthesis in plants., Ann. Rev. Plant
9. Ang, H. L., Deltour, L., Hayamizu, T. F., Zgombic- Physiol. Plant Mol. Biol., 1994; 45: 287–301.
Knight, M., and Duester, G., Retinoic acid synthesis 24. Bartley, G. E. and Scolnik, P. A., Plant carotenoids:
in mouse embryos during gastrulation and craniofa- pigments for photoprotection, visual attraction, and
cial development linked to class IV alcohol dehydro- human health, Plant Cell, 1995; 7: 1027–1038.
genase gene expression, J. Biol. Chem., 1996; 27(116): 25. Bartley, G. E., Schmidhauser, T. J., Yanofsky, C., and
9526–9534. Scolnik, P. A., Carotenoid desaturases from
10. Anton, R., Flavonoids and traditional medicine. In: Rhodobacter capsulatus and Neurospora crassa are
Cody, V., Middleton Jr., E., Harborne, J.B., and Beretz, structurally and functionally conserved and contain
A., Eds. Progress in Chemical and Biological Re- domains homologous to flavoprotein disulfide oxi-
search Vol. 280. Alan R. Liss Inc., New York, 1988, doreductases, J. Biol. Chem., 1990; 265(26): 16020–
423–449. 16024.
271
26. Bartley, G. E., Viitanen, P. V., Pecker, I., Chamovitz, 41. Bilyk, A., Thin-layer chromatographic separation of
D., Hirshberg, J., and Scolnik, P. A., Molecular clon- beet pigments, J. Food Sci., 1981; 46: 298–299
ing and expression in photosynthetic bacteria of a 42. Block, J. E., Amundson, C. H., and von Elbe, J. H.,
soybean cDNA coding for phytoene desaturase, an Energy requirement of beet colorant production, J.
enzyme of the carotenoid biosynthesis pathway, Proc. Food Proc. Eng., 1981; 5(1): 67–75.
Natl. Acad. Sci. USA, 1991; 88: 6532–6536. 43. Boege, F., Straub, T., Kehr, A., Boesenberg, C.,
27. Batschauer, A., Rocholl, M., Kaiser, T., Nagatani, A., Christiansen, K., Anderson, A., Jakob, F., and Köhrle,
Furuya, M., and Schäfer, E., Blue and UV-A light- J., Selected novel flavones inhibit the DNA binding or
regulated CHS expression in Arabidopsis independent the DNA religation step of eukaryotic topoisomerase
of phytochrome A and phytochrome B, Plant J., 1996; I, J. Biol. Chem., 1996; 271(4): 2262–2270.
9: 63–69. 44. Böhm, F., Edge, R., Land, E. J., McGarvey, D. J., and
28. Baublis, A.J. and Berber-Jiménez, M.D., Structural Truscott, T. G., Carotenoids enhance vitamin E anti-
and conformational characterization of a stable antho- oxidant efficiency, J. Am. Chem. Soc., 1997; 119:
cyanin from Tradescantia pallida, J. Agric. Food 621–622.
Chem., 1995; 43: 640–646. 45. Böhm, H. and Böhm, L., Portulaca grandiflora hook.
29. Bauernfeind, J. C., Natural food colors. In: and P. oleracea L.: formation of betalains and unsat-
Bauernfeind, J. C., Ed., Carotenoids as Colorants and urated fatty acids, Biotech. Agric. Forestry, 1996; 37:
Vitamin A Precursors, Academic Press, New York, 335–354.
1981, 1-45. 46. Böhm, H. and Rink, E., Betalains. In: Cell culture and
30. Bayindirli, A., Yildiz, F., and Özilgen, M., Modeling somatic cell genetics of plants. Academic Press, New
of sequential batch ultrafiltration of red beet extract, J. York, 1988, 449–463.
Food Sci., 1988; 53(5): 1418–1422. 47. Bokern, M., Heuer, S., and Strack D., Hidroxycinnamic
31. Belbin, A. A., Colors in oils. INFORM, 1993; 4(6): acid transferases in the biosynthesis of acylated
648–654. betacyanins: purification and characterization from
32. Bell, A. A. and Wheeler, M. H., Biosynthesis and cell cultures of Chenopodium rubrum and occurrence
functions of fungal melanins, Ann. Rev. Plant Physiol., in some other members of the Caryophillales, Bot.
1986; 24: 411–451. Acta, 1992; 105: 146–151.
33. Bell, C. M., Sander, L. C., Fetzer, J. C., and Wise, S. 48. B o k e r n , M . a n d S t r a c k , D . , S y n t h e s i s o f
A., Synthesis and characterization of extended length hydroxycinnamic acid esters of betacyanins via
alkyl stationary phases for liquid chromatography with 1-O-acylglucosydes of hydroxycinnamic acids by
application to the separation of carotenoid isomers, J. protein preparations from cell suspension cul-
Chromatogr. A, 1996; 753: 37–45. tures of Chenopodium rubrum and petals of
34. Benosman, A., Oger, J. M., Richomme, P., Bruneton, Lampranthus sociorum, Planta, 1988; 174: 101–
J., Roussakis, C., Bösch, S., Sévent, T., Ito, K., Ichino, 105.
K., and Hadi, A. H. A., New terpenylated 49. Bokern, M., Wray, V., and Strack, D., Accumulation
dihydrochalcone derivatives isolated from Mitrella of phenolic acid conjugates and betacyanins, and
kentii, J. Nat. Prod., 1997; 60(9): 921–924. changes in the activities of enzymes involved in
35. Berlin, J., Sieg, S., Strack, D., Bokern, M., and Harms, feruloylglucose metabolism in cell-suspension cul-
H., Production of betalains by suspension cultures of tures of Chenopodium rubrum L., Planta, 1991; 184:
Chenopodium rubrum L. Plant Cell, Tiss. Org Cul. 261–270.
1986; 5(2): 163–174. 50. Bon, J. A., Leathers, T. D., and Jayaswal, R. K.,
36. Biacs, P. A. and Daood, H. G., High-performance Isolation of astaxanthin-overproducing mutants of
liquid chromatography with photodiode-array detec- Phaffia rhodozyma, Biotechnol. Lett., 1997; 19(2):
tion of carotenoids and carotenoid esters in fruits and 109–112.
vegetables, J. Plant Physiol., 1994; 143: 520–525. 51. Booker, F. L., Anttonen, S., and Heagle, A. S., Cat-
37. Bianco-Colomas, J. and Hugues, M., Establishment echin, proanthocyanidin and lignin contents of loblolly
and characterization of a betacyanin producing cell pine (Pinus taeda) needles after chronic exposure to
line of Amaranthus tricolor: inductive effects of light ozone, New Phytol., 1996; 132: 483–492.
and cytokinin, J. Plant Physiol., 1990; 136: 734–739. 52. Bosevska, M., Karlovic, D., Turkulov, J., and Pericin,
38. Bianco-Colomas, J., Qualitative and quantitative as- D., Quality of corn germ oil obtained by aqueous
pects of betalains biosynthesis in Amaranthus caudatus enzymatic extraction, J. Am. Oil. Chem. Soc., 1993;
L. var. pendula seedlings, Planta, 1980; 149: 176– 70(12): 1273–1277.
180. 53. Botling, J., Castro, D. S., Öberg, F., Nilsson, K., and
39. Bilyk, A., Extractive fractionation of betalaines, J. Perimann, T., Retinoic acid receptor/retinoid X recep-
Food Sci., 1979; 44: 1249–1251. tor heterodimers can be activated through both sub-
40. Bilyk, A., Kolodij, M. A., and Sapers, G. M., Stabili- units providing a basis for synergistic transactivation
zation of red beet pigments with isoascorbic acid, J. and cellular differentiation, J. Biol. Chem., 1997;
Food Sci., 1981; 46: 1616–1617 272(14): 9443–9449.
272
54. Bouvier, F., d’Harlingue, A., Hugueney, P., Marin, 70. Camara, B., Plant phytoene synthase complex: com-
E., Marion-Poll, A., and Camara, B., Xanthophyll ponent enzymes, immunology, and biogenesis,
biosynthesis. Cloning, expression, functional recon- Method. Enzymol., 1992; 214(part B): 352–365.
stitution and regulation of β-cyclohexenyl carotenoid 71. Camara, B. and Dogbo, O., Demonstration and solu-
epoxydase from pepper (Capsicum annuum), J. Biol. bilization of lycopene cyclase from Capsicum chro-
Chem., 1996; 271(46): 28861–28867. moplast membranes, Plant Physiol., 1986; 80: 172–
55. Bowler, C., Neuhaus, G., Yamagata, H., and Chua, N. 174.
H., Cyclic GMP and calcium mediate phytochrome 72. Camara, B. and Moneger, R., Carotenoid biosynthesis
phototransduction, Cell, 1994; 77: 73–81. in vitro conversion of antheraxanthin to capsanthin by
56. Boyles, M. J. and Wrolstad, R. E., Anthocyanin com- a chromoplast enriched fraction of Capsicum fruits,
position of red raspberry juice: influences of cultivar, Biochem. Biophys. Res. Commun., 1981; 99(4): 1117–
processing, and environmental factors, J. Food Sci., 1122.
1993; 58: 1135–1141. 73. Cano, M. P. and de Ancos, B., Carotenoid and caro-
57. Bramley, P. M., Analysis of carotenoids by high- tenoid ester composition in mango fruit as influenced
performance liquid chromatography and diode array by processing method, J. Agric. Food Chem., 1994;
detection, Phytochemical Anal., 1992; 3: 97–104. 4212): 2737–2742.
58. Bridle, P. and Timberlake, C. F., Anthocyanins as 74. Cano, M.P. and Marin, M.A., Pigment composition
natural food colour-selected aspects, Food Chem., and color of frozen and canned kiwi fruit slices, J.
1997; 58(1-2): 103–109. Agric. Food Chem., 1992; 40(11): 2141–2146.
59. Britton, G., General carotenoid methods, Method. 75. Carpenter, K. L. H., vander Veen, C., Hird, R., Den-
Enzymol., 1985; 111(part B): 113–149. nis, I. F., Ding, T., and Mitchinson, M .J., The caro-
60. Britton, G., Carotenoids, Method. Plant Biochem., tenoid β-carotene, canthaxanthin and zeaxanthin in-
1991; 7: 473–518. hibit macrophage-mediated LDL-oxidation, FEBS
61. Britton, G., Structure and properties of carotenoids in Lett., 1997; 401: 262–266.
relation to function, FASEB J., 1995; 9: 1551–1558. 76. Cerezal, M. P. and Núñez, L. D., Caracterización del
62. Brouillard, R., Dangles, O., Jay, M., Biolley, J. P., and colorante de remolacha roja (Beta vulgaris L.) en
Chirol, N., Polyphenols and pigmentation in plants. polvo, Alimentaria, 1996; enero/febrero: 91–94
In: Scalbert, A., Ed., Polyphenolic Phenomena. Paris: 77. Cerezal, M. P., Pino, A. J., and Salabarria, Y., Red
INRA, 1993, 41–47. beet (Beta vulgaris L.) colorant stability in the form of
63. Brouillard, R., The in vivo expression of anthocyanin a concentrated liquor, Tecnología Alimentaria, 1994;
colour in plants, Phytochemistry, 1983; 22: 1311– 293,4): 7–16
1323. 78. Cippitelli, M., Ye, J., Viggiano, V., Sica, A., Ghosh,
64. Brown, D. W. and Salvo, J. J., Isolation and charac- P., Gulino, A., Santoni, A., and Young, H. A., Retinoic
terization of sexual spore pigments from Aspergillus acid-induced transcriptional modulation of the human
nidulans, Appl. Environ. Microbiol., 1994; 60(3): 979– interferon-γ promoter, J. Biol. Chem., 1996; 27143):
983. 26783–26793.
65. Buckner, B., San Miguel, P., Janick-Buckner, D., 79. Clairmont, A., Tessmann, D., and Sies, H., Analysis
and Bennetzen, J. L., The y1 gene of maize codes for connexin 43 gene expression induced by retinoic
for phytoene synthase, Genetics, 1996; 143: 479– acid in F9 teratocarcinoma cells, FEBS Lett., 1996;
488. 397: 22–24.
66. Burkardt, P. K., Beyer, P., Wünn, J., Klöti, A., 80. Clydesdale, F. M., Color as a factor in food choice,
Armstrong, G. A., Schledz, M., von Lintig, J., and Crit. Rev. Food Sci. Nutr., 1993; 331): 83–101.
Potrykus, I., Transgenic rice (Oryza sativa) endosperm 81. Cogdell, R. J. and Gardiner, A. T., Functions of caro-
expressing daffodil (Narcissus pseudonarcissus) tenoids in photosynthesis, Method. Enzymol., 1992;
phytoene synthase accumulates phytoene, a key inter- 214(part B): 185–193.
mediate of provitamin A biosynthesis, Plant J., 1997; 82. Cohen, E. and Saguy, I., Effect of water activity and
115): 1071–1078. moisture content on the stability of beet powder pig-
67. Callebaut, A., Terahara, N., de Haan, M., and Decleire, ments, J. Food Sci., 1983; 48: 703–707
M., Stability of anthocyanin composition in Ajuga 83. Conkerton, E. J., Wan, P. J., and Richard, O. A.,
reptans callus and cell suspension cultures, Plant Cell Hexane and heptane as extraction solvents for cotton
Tiss. Org. Cult., 1997; 50: 195–201. seed: a laboratory-scale study, J. Am. Oil. Chem. Soc.,
68. Camara, B., Carotenoid biosynthesis in vitro conver- 1995; 72(8): 963–965.
sion of violaxanthin to capsorubin by a chromoplast 84. Connelly, J. P., Müller, M. G., Bassi, R., Croce,
enriched fraction of Capsicum fruits, Biochem. R., and Holzwarth, A. R., Femtosecond transient
Biophys. Res. Commun., 1980; 93(1): 113–117. absorption study of carotenoid to chlorophyll en-
69. Camara, B., Biosynthesis of keto-carotenoids in Cap- ergy transfer in the light-harvesting complex II
sicum annuum fruits, FEBS Lett., 1980; 118(2): 315– of photosystem II, Biochemistry, 1997; 36(2):
318. 281–287.
273
85. Constabel, F. and Nassif-Makki, H., Betalainbildung 98. Chen, B. H., Chen, T. M., and Chien, J. T., Kinetic
in Beta-Calluskulturen, Ver. Dtsch. Bot. Ges., 1971; model for studying the isomerization of α- and β-
84: 629–636 carotene during heating and illumination, J. Agric.
86. Corona, V., Aracri, B. Kosturkova, G., Bartley, G. E., Food Chem., 1994; 42(11): 2391–2397.
Pitto, L, Giorgetti, L., Scolnik, P. A., and Giuliano, 99. Chen, B. H., Peng, H. Y., and Chen, H. E., Changes
G., Regulation of a carotenoid biosynthesis gene pro- of carotenoids, color, and vitamin A contents during
moter during plant development, Plant J., 1996; 9(4): processing of carrot juice, J. Agric. Food Chem., 1995;
505–512. 437): 1912–1918.
87. Counsell, J. N., Jeffries, G. S., and Knewstubb, C. J. 100. Chen, C. W., Shu, C. K., and Ho, C. T., Photosensi-
Some other natural colors and their applications. In: tized oxidative reaction of 2,5-dimethyl-4-hydroxy-
Counsell, J. N. and Dunastable, J. A., Eds., Natural 32H)-furanone, J. Agric. Food Chem., 1996; 448):
Colors for Foods and Other Uses. Applied Science, 2361–2365.
London, 1979, 122–151. 101. Chen, H. E., Peng, H. Y., and Chen, B. H., Stability
88. Courtney-Gutterson, N., Napoli, C., Lemieux, C., of carotenoids and vitamin A during storage of carrot
Morgan, A., Firoozabady , E., and Robinson, K. E. P., juice, Food Chem., 1996; 57(4): 497–503.
Modification of flower color in florist’s chrysanthe- 102. Cheng, F. S., Weeden, N. E., and Brown, S. K., Iden-
mum: production of a white-flowering variety through tification of co-dominant RAPD markers tightly linked
molecular genetics, Bio/Technology, 1994; 12: 268– to fruit skin color in apple, Theor. Appl. Genet., 1996;
271. 93: 222–227.
89. Crouzet, J. and Kanasawd, P., Formation of volatile 103. Chenier, V., Souquet, J. M., Kontek, A., and
compounds by thermal degradation of carotenoids, Moutounet, M., Anthocyanin degradation in oxidising
Method. Enzymol., 1992; 213(part A): 54–62. grape musts, J. Sci. Food Agric., 1994; 66: 283–288.
90. Cunningham, F. X., Pogson, B., Sun, Z., McDonald, 104. Chester, T. L., Pinkston, J. D., and Raynie, D. E.
K. A., DellaPenna, D., and Gantt, E., Functional Supercritical fluid chromatography and extraction,
analysis of the β and ε lycopene cyclase enzymes of Anal. Chem., 1992; 64(12): 153R–170R.
Arabidopsis reveals a mechanism for control of cy- 105. Chirol, N. and Jay, M., Acylated anthocyanins from
clic carotenoid formation, Plant Cell, 1996; 8: 1613– flowers of Begonia, Phytochemistry, 1995; 40: 275–
1626. 277.
91. Cunningham, F. X., Sun, Z., Chamovitz, D., Hirshberg, 106. Christensen, F.M., Extraction by aqueous enzymatic
J., and Gantt, E., Molecular structure and enzymatic processes, INFORM, 1991; 211): 984–987.
function of lycopene cyclase from the cyanobacterium 107. Christie, J. M. and Jenkins, G. I., Distinct UV-B and
Synechococcus sp. strain PCC7942, Plant Cell, 1994; UV-A/blue light signal transduction pathways induce
6: 1107–1121. chalcone synthase gene expression in Arabidopsis cells,
92. Chandler, L. A. and Schwartz, S. J., HPLC separation Plant Cell, 1996; 8: 1555–1567.
of cis-trans carotene isomers in fresh and processed 108. Christie, P. J., Alfenito, M. R., and Walbot, V.,
fruits and vegetables, J. Food Sci., 1987; 52(3): 669– Impact of low-temperature stress on general
672. phenylpropanoid and anthocyanin pathways: en-
93. Chappell, J., Biochemistry and molecular biology of hancement of transcript abundance and anthocyanin
the isoprenoid biosynthetic pathway in plants, Ann. pigmentation in maize seedlings, Planta, 1994; 194:
Rev. Plant Physiol. Plant Mol. Biol., 1995; 46: 521– 541–549.
547. 109. Chumpolkulwong, N., Kakizono, T., Handa, T., and
94. Charleux, J. L., Beta-carotene, vitamin C, and vitamin Nishio, N., Isolation and characterization of compactin
E: the protective micronutrients, Nutr. Rev., 1996; resistant mutants of an astaxanthin synthesizing green
54(11): S109–S114. alga Haematococcus pluvialis, Biotechnol. Lett., 1997;
95. Charrier, B., Leroux, C., Kondorosi, A., and Ratet, P., 19(3): 299–302.
The expression pattern of alfalfa flavanone 3-hydroxy- 110. Chumpolkulwong, N., Kakizono, T., Ishii, H., and
lase promoter-gus fusion in Nicotiana benthamiana Nishio, N., Enzymatic conversion of β-carotene to
with the presence of flavonoids detected in situ, Plant astaxanthin by cell extracts of a green alga
Mol. Biol., 1996; 30: 1153–1168. Haematococcus pluvialis, Biotechnol. Lett., 1997;
96. Chauhan, N.P., Fatma, T., and Mishra, R.K., Protec- 19(5): 443–446.
tion of wheat chloroplasts from lipid peroxidation and 111. Dai, G. H., Andary C., Mondolot-Cosson, L., and
loss of photosynthetic pigments by quercetin under Boubals, D., Histochemical studies on the interaction
strong illumination, J. Plant Physiol., 1992; 140: 409– between three species of grapevine, Vitis vinifera, V.
413. rupestris, and V. rotundifolia and the downy midew
97. Che-Man, Y. B., Suhardiyono, A. B., Asbi, A. B., fungus Plasmopara viticola, Physiol. Mol. Plant
Azudin, M. N., and Wei, L. S., Aqueous enzymatic Pathol., 1995; 46: 177–188.
extraction of coconut oil, J. Am. Oil. Chem. Soc., 112. De Ritter, E. and Purcell, A.E., Carotenoid analytical
1996; 736): 683–686. methods. In: Bauernfeind, J.C., Ed. Carotenoids as
274
Colorants and Vitamin A Precursors. Academic Press,
New York, 1981, 815–882.
113. De-Azevedo, W. F., Jr., Mueller-Dieckmann, H. J., 127. Dougall, D. K., Baker, D. C., Gakh, E., and Redus,
Schulze-Gahmen, U., Worland, P. J., Sausville, E., M., Biosynthesis and stability of monoacylated antho-
and Kim, S. H., Structural basis for specificity and cyanins, Food Technol., 1997; 51: 69–71.
potency of a flavonoid inhibitor of human CDK2, a 128. Drdák, M., Altamirano, R. C., Rajniakova, A., Simko,
cell cycle kinase, Proc. Natl. Acad. Sci. USA, 1996; P., Karovicova, J., and Benkovska, D., Red beet pig-
93: 2735–2740. ment composition. Effects of fermentation by differ-
114. Debicki-Pospisil, J., Lovric, T., Trinajstic, N., and ent strains of Saccharomyces cerevisiae, J. Food Sci.,
Sabljic, A., Anthocyanin degradation in the presence 1992; 57(4): 935–936
of furfural and 5-hydroxymethylfurfural, J. Food Sci., 129. Drdák, M., and Vallová, M., Kinetics of the thermal
1983; 48: 411–416. degradation of betanine, Die Nahrung, 1990; 34: 307–
115. Dedaldechamp, F., Uhel, C., and Macheix, J. J., En- 310
hancement of anthocyanin synthesis and 130. Durbin, M.L., Learn, G. H., Jr., Huttley, G. A., and
dihydroflavonol reductase (DFR) activity in response Clegg, M. T., Evolution of the chalcone synthase gene
to phosphate deprivation in grape cell suspension, family in the genus Ipomoea, Proc. Natl. Acad. Sci.
Phytochemistry, 1995; 40: 1357–1360. USA, 1995; 92: 3338–3342.
116. Deikman, J. and Hammer, P. E., Induction of antho- 131. Durnford, D. G., Aebersold, R., and Green, B. R., The
cyanin accumulation by cytokinins in Arabidopsis fucoxanthin-chlorophyll proteins from a chromophyte
thaliana, Plant Physiol., 1995; 108: 47–57. alga are part of a large multigene family: structural
117. Delgado-Vargas, F. and Paredes-López, O., Correla- and evolutionary relationships to other light harvest-
tion of HPLC and AOAC methods to assess the all- ing antennae, Mol. Gen. Genet., 1996; 253: 377–386.
trans-lutein content in marigold flowers, J. Sci. Food 132. Düsterhöff, E. M., Bonte, A. W., Venokamp, J. C.,
Agric., 1996; 72: 283–290. and Voragen, A. G. J., The role of fungal
118. Delgado-Vargas, F. and Paredes-López, O., Effects of polysaccharidases in the hydrolysis of cell wall mate-
enzymatic treatments of marigold flowers on lutein rials from sunflower and palm-kernel meals, World J.
isomeric profiles, J. Agric. Food Chem., 1997; 45(4): Microbiol. Biotechnol., 1993; 9: 544–554.
1097–1102. 133. Eichele, G., Retinoids: from hindbrain patterning to
119. Delgado-Vargas, F. and Paredes-López, O., Effects of Parkinson disease, Trends Genet., 1997; 13(9): 343–
enzymatic treatments on carotenoid extraction from 345.
marigold flowers (Tagetes erecta), Food Chem., 1997; 134. Elomaa, P., Helariutta, Y., Griesbach, R. J., Kotilainen,
583): 255–258. M., Seppänen, P., and Teeri, T. H., Transgene inacti-
120. Delgado-Vargas, F. and Paredes-López, O., Enzy- vation in Petunia hybrida is influenced by the proper-
matic treatment to enhance carotenoid content in de- ties of the foreign gene, Mol. Gen. Genet., 1995; 248:
hydrated marigold flower meal, Plant Foods Hum. 649–656.
Nutr., 1997; 50: 163–169. 135. Elliott, C. D., The pathway of betalain biosynthesis:
121. Delgado-Vargas, F., Paredes-López, O., and Avila- effect of cytokinin on enzymic oxidation and hydroxy-
González, E., Effects of sunlight illumination of mari- lation of tyrosine in Amaranthus tricolor seedlings,
gold flower meals on egg yolk pigmentation, J. Agric. Physiol. Plant., 1983; 59: 428–437.
Food Chem., 1998; 46(2): 698–706. 136. Emenhiser, C., Englert, G., Sander, L. C., Ludwig, B.,
122. De-Mello, J. P., Petereit, F., and Nahrstedt, A., Flavan- and Schwartz, S. J., Isolation and structural elucida-
3-ols and prodelphinidins from Stryphnodendron tion of the predominant geometrical isomers of a -
adstringens, Phytochemistry, 1996; 41(3): 807–813. carotene, J. Chromatogr. A, 1996; 719: 333–343.
123. Dogbo, O. and Camara, B., Purification of isopentenyl 137. Endres, R., Einfluss möglicher phosphodiesterase-
pyrophosphate isomerase and geranylgeranyl pyro- inhibitoren und cAMP auf die betacyan-akkumulation,
phosphate synthase from Capsicum chromoplasts by Phytochemistry, 1977; 16: 1549–1554
affinity chromatography, Biochem. Biophys. Acta, 138. Ershov, A. V., Lukin, W. J., and Bazan, N. G., Selec-
1987; 920: 140–148. tive transcription factor induction in retinol pigment
124. Domínguez, H., Nuñez, M. J., and Lema, J. M., Enzy- epithelial cells during photoreceptor phagocytosis, J.
matic pretreatment to enhance oil extraction from fruits Biol. Chem., 1996; 271(45): 28458–28462.
and oilseeds: a review, Food Chem., 1994; 49: 279– 139. Eskling, M., Arvidsson, P. O., and Åkerlund, H. E.,
286. The xanthophyll cycle, its regulation and components,
125. Dooner, H. K. and Robbins, T. P., Genetic and devel- Physiol. Plant., 1997; 100: 806–816.
opmental control of anthocyanin biosynthesis, Ann. 140. Faktor, O., Kooter, J. M., Loake, G. J., Dixone, R. A.,
Rev. Genet., 1991; 25: 173-199. and Lamb, C. J., Differential utilization of regulatory
126. Dörnenburg, H. and Knorr, D., Challenges and oppor- cis-elements for stress-induced and tissue-specific
tunities of metabolite production from plant cell and activity of a French bean chalcone synthase promoter,
tissue culture, Food Technol., 1997; 51(11): 47–54 Plant Sci., 1997; 124: 175–182.
275
141. Faktor, O., Loake, G., Dixon, R. A., and Lamb, C. J., flavonols, J. Agric. Food Chem., 1997; 45: 2262–
The G-box and H-box in a 39 bp region of a French 2266.
bean chalcone synthase promoter constitute a tissue- 156. Francis, F. J., Handbook of Food Colorant Patents,
specific regulatory element, Plant J., 1997; 11: 1105– 1st. ed., Food and Nutrition Press, Westport, CT,
1113. 1986.
142. Fan, X., Mattheis, J. P., Fellman, J. K., and Patterson, 157. Francis, F. J., Food colorants: anthocyanins, Crit.
M. E., Changes in jasmonic acid concentration during Rev. Food Sci. Nutr., 1989; 28(4): 273–314.
early development of apple fruit, Physiol. Plant., 1997; 158. Francis, J., Carotenoids as colorants, The World of
101: 328–332. Ingredients, 1995; Sept.-Oct.: 34–38.
143. Favati, F., King, J. W., Friedrick, J. P., and Eskins, K., 159. Frankel, E. N., In search of better methods to evaluate
Supercritical CO2 extraction of carotene and lutein natural antioxidants and oxidative stability in food
from leaf protein concentrates, J. Food Sci., 1988; lipids, Trends Food Sci. Technol., 1993; 4(7): 220–
53(5): 1532–1536. 225.
144. FDA/IFIC, Regulation of color additives, Food and 160. Fraser, P. D., Linden, H., and Sandmann, G., Purifi-
Drug Administration, FDA/IFIC Brochure, 1993. cation and reactivation of recombinant Synechococcus
145. Ferrali, M., Signorini, C., Caciotti, B., Sugherini, L., phytoene desaturase from an overexpressing strain
Ciccoli, L., Giachetti, D., and Comporti, M., Protec- of Escherichia coli, Biochem. J., 1993; 291: 687–
tion against oxidative damage of erythrocyte mem- 692.
brane by the flavonoid quercetin and its relation to 161. Freund, P. R.,Washam, C. J., and Maggion, M., Natu-
iron chelatin activity, FEBS Lett., 1997; 416: 123– ral color for use in foods, Cereal Foods World, 1988;
129. 33(7): 553–559.
146. Figueiredo, P., Elhabiri, M., Toki, K., Saito, N., 162. Frick, D. and Meggos, H., FD&C colors, Food
Dangles, O., and Brouillard, R., New aspects Technol., 1988; 7: 49–56.
of anthocyanin complexation, intramolecular 163. Fukada-Tanaka, S. H. A., Hisatomi, Y., Habu, Y.,
copigmentation as a means for colour loss?, Phy- Hasabe, M., and Iida, S., Identification of new chal-
tochemistry, 1996; 41: 301–308. cone synthase genes for flower pigmentation in the
147. Fischer, S., Böttcher, U., Reuber, S., Anhalt, S., and Japanese and common morning glories, Plant Cell
Weissenböck, G., Chalcone synthase in the liverworth Physiol., 1997; 38: 754–758.
Marchantia polymorpha, Phytochemistry, 1995; 39: 164. Gao, L. and Mazza, G., Extraction of anthocyanin
1007–1012. pigments from purple sunflower hulls, J. Food Sci.,
148. Fisher, N. and Dreiding, A.S., Biosynthesis of 1996; 61: 600–603.
betalaines. On the cleavage of the aromatic ring 165. García-Viguera, C., Zafrilla, P., and Tomás-Barberán,
during enzymatic transformation of DOPA into F. A., Determination of authenticity of fruit jams by
betalamic acid, Helv. Chim. Acta, 1972; 55: 649– HPLC analysis of anthocyanins, J. Sci. Food Agric.,
652 1997; 73: 207–213.
149. Fletcher, D. L., The effect of light exposure on feed 166. Gau, W., Ploschke, H. J., and Wünsche, Ch., Mass
in broiler pigmentation, Poultry Sci., 1981; 60: 68– spectrometric identification of xanthophyll fatty acid
75. esters from marigold flowers (Tagetes erecta) ob-
150. FNB, Food Colors, Food and Nutrition Board, Na- tained by high performance liquid chromatography
tional Academy of Sciences, Washington, D.C., 1971. and Craig counter-current distribution, J. Chromatogr.,
151. Forkmann, G., New naturally occurring plant polyphe- 1983; 262: 277–284.
nols, In: Scalbert, A., Ed. Polyphenolic Phenomena, 167. Geibel, M., Sensitivity of the fungus Cytospora
INRA, Paris, 1993, 65–71. persoonii to the flavonoids of Prunus cerasus, Phy-
152. Forrest, H. S., Pteridines: structure and metabolism. tochemistry, 1995; 383): 599–601.
In: Florkin, M. and Mason, H. S., Eds, Comparative 168. Geiss, F., Heinrich, M., Hunkler, D., and Rimpler, H.,
Biochemistry. A Comprehensive Treatise. Vol. IV. Con- Proanthocyanidins with (+)-epicatechin units from
stituents of Life-Part B. Academic Press, New York, Byrsonima crassifolia bark, Phytochemistry, 1995;
1962, 615–641. 393): 635–643.
153. Fossen, T., Andersen, O. M., Ovstedal, D. O., Pedersen, 169. Giannasi, D. E., Flavonids and evolution in the Di-
A. T., and Raknes, A., Characteristic anthocyanin cotyledons. In: Harborne, J.B., Ed., The Flavonoids,
pattern from onions and other Allium spp, J. Food Chapman and Hall, London, 1988.
Sci., 1996; 61: 703–706. 170. Gil, M. I., Holcroft, D. M., and Kader, A. A., Changes
154. Fossen, T. and Andersen, O. M., Acylated anthocya- in strawberry anthocyanins and other polyphenols in
nins from leaves of the water lily, Nymphaéa x response to carbon dioxide treatments, J. Agric. Food
marliacea, Phytochemistry, 1997; 46: 353–357. Chem., 1997; 45: 1662–1667.
155. Francia-Aricha, E. M., Guerra, M. T., Rivas- 171. Girod,P. A. and Zrÿd, J., Secondary metabolism
Gonzalo, J. C., and Santos-Buelga, C., New antho- in cultured red beet (Beta vulgaris) cells: differ-
cyanin pigments formed after condensation with ential regulation of betaxanthin and betacyanin
276
biosynthesis, Plant Cell Tiss. Org. Cult., 1991; 188. Gutterson, N., Anthocyanin biosynthetic genes and
25: 1–12 their application to flower color modification through
172. Giuliano, G., Bartley, G. E., and Scolnik, P. A., Regu- sense suppression, Hortscience, 1995; 30: 964–966.
lation of carotenoid biosynthesis during tomato devel- 189. Haila, K. M., Lievonen, S. M., and Heinonen, M. I.,
opment, Plant Cell, 1993; 5: 379–387. Effects of lutein, lycopene, annatto and γ-tocopherol
173. Giusti, M. M. and Wrolstad, R. E., Characterization of on autoxidation of triglycerides, J. Agric. Food Chem.,
red radish anthocyanins, J. Food Sci., 1996; 61: 322– 1996; 44(8): 2096–2100.
326. 190. Han, D., Kim, S. J., and Kim, D. M., Repeated regen-
174. Goldman, M., Horev, B., and Saguy, I., Decoloriza- eration of degraded red beet juice pigments in the
tion of β-carotene in model systems simulating dehy- presence of antioxidants, J. Food Sci., 1998; 63(1):
drated foods. Mechanism and kinetic principles, J. 69–72
Food Sci., 1983; 48: 751–754. 191. Hanson, M. A., Gaut, B. S., Stec, A. O., Fuerstenberg,
175. Goldsbrough, A. P., Tong, Y., and Yoder, J. I., Lc as S. I., Goodman, M. M., Coe, E. H., and Doebley, J. F.,
a non-destructive visual reporter and transposition Evolution of anthocyanin biosynthesis in maize ker-
excision marker gene for tomato, Plant J., 1996; 9: nels: the role of regulatory and enzymatic loci, Genet-
927–933. ics, 1996; 143: 1395–1407.
176. Gong, Z., Yamazaki, M., Sugiyama, M., Tanaka, Y., 192. Haralampu, S. G. and Karel, M., Kinetic models for
and Saito, K., Cloning and molecular analysis of moisture dependence of ascorbic acid and β-carotene
structural genes involved in anthocyanin biosynthe- degradation in dehydrated sweet potato, J. Food Sci.,
sis and expressed in a forma-specific manner in Pe- 1983; 48: 1872–1873.
rilla frutescens, Plant Mol. Biol., 1997; 35: 915– 193. Harborne, J. B., The flavonoids: recent advances. In:
927. Goodwin, T. W., Ed., Plant Pigments. Academic Press,

177. González-de-Mejía, E., Loarca-Piña, G., and Ramos- London, 1988, 298–343.
Gómez, M., Antimutagenicity of xanthophylls present 194. Harborne, J. B., New naturally occurring plant polyphe-
in Aztec marigold (Tagetes erecta) against 1- nols. In: Scalbert, A., Ed. Polyphenolic Phenomena,
nitropyrene. Mut. Res., 1997; 389: 219–226. Paris, 1993, 9–21.
178. Goodrich, J., Carpenter, R., and Cohen, E. S., A com- 195. Harborne, J. B. and Grayer, R. J., The anthocyanins.
mon gene regulates pigmentation pattern in diverse In: Harborne, J.B., Ed. The Flavonoids. London:
plant species, Cell, 1992; 68: 955–964. Chapman and Hall Ltd, 1988, 1-20.
179. Goodwin, T. W., Biochemistry of the Carotenoids. 196. Hari, R. K., Patel, T. R., and Martin, A. M., An
Vol. 1: Plants., 2nd. ed., Chapman and Hall, New overview of pigment production in biological sys-
York, 1980, 1–95. tems: functions, biosynthesis, and applications in food
180. Goodwin, T. W., Biosynthesis of carotenoids: an over- industry, Food Rev. Int., 1994; 10(1): 49–70.
view, Method. Enzymol., 1992; 214part B): 330–340. 197. Hasegawa, T., Anti-stress effect of β-carotene. In:
181. Goodwin, T. W. and Britton, G., Distribution and Canfield, L. M., Krinsky, N. I., and Dunastable, J. A.,
analysis of carotenoids. In: Goodwin, T.W., Ed., Plant Eds., Carotenoids in Human Health. Annals of the
Pigments. Academic Press, London, 1988, 62–132. New York Academy of Sciences, New York, Vol.
182. Gordon, H. T. and Bauernfeind, J. C., Carotenoids as 691, 1993, 281–283.
food colorants, Crit. Rev. Food Sci. Nutr., 1982; 18: 198. Haslam, E., Natural polyphenols vegetable tannins) as
59–97. drugs: possible modes of action, J. Nat. Prod., 1996;
183. Gottfried, D. S., Steffen, M. A., and Boxer, S. G., 59(2): 205–215.
Large protein-induced dipoles for a symmetric caro- 199. Hatano, T., Yamashita, A., Hashimoto, T., Ito, H.,
tenoid in a photosynthetic antenna complex, Science, Kubo, N., Yoshiyama, M., Shimura, S., Itoh, Y.,
1991; 251(Feb. 8): 662–665. Okuda, T., and Yoshida, T., Flavan dimers with lipase
184. Granado, F., Olmedilla, B., Blanco, I., and Rojas- inhibitory activity from Cassia nomame, Phytochem-
Hidalgo, E., Carotenoid composition in raw and cooked istry, 1997; 46(5): 893–900.
Spanish vegetables, J. Agric. Food Chem., 1992; 40: 200. Haugan, J. A., Akermann, T., and Liaaen-Jensen, S.,
2135–2140. Isolation of fucoxanthin and peridinin, Method.
185. Gray, J. C., Control of isoprenoid biosynthesis, Adv. Enzymol. 1992; 213(part A): 231–245.
Bot. Res., 1987; 14: 25–91. 201. Haviv, E. A., Tausta, S. L., and Peterson, R. B.,
186. Green, B. R. and Durnford, D. G., The chlorophyll- Purification and properties of violaxanthin de-
carotenoid proteins of oxygenic photosynthesis, Ann. epoxydase from spinach, Plant Sci., 1997; 123: 65–
Rev. Plant Physiol. Plant Mol. Biol., 1996; 47: 685– 66.
714. 202. Havkin-Frenkel, D., Dorn, R., and Leustek, T., Plant
187. Guérin, E., Ludwig, M. G., Basset, P., and Anglard, Tissue Culture for production of secondary metabo-
P., Stromelysin-3 induction and interstitial collage- lites, Food Technol., 1997; 51: 56–61.
nase repression by retinoic acid, J. Biol. Chem., 1997; 203. Hawthorne, S. B., Analytical-scale supercritical fluid
272(17): 11088–11095. extraction, Anal. Chem., 1990; 62(11): 633A–642A.
277
204. Heinonen, M., Haila, K., Lampi, A. M., and 218. Hooijberg, J. H., Broxterman, H. J., Heijn, M., Fles,
Piironen, V., Inhibition of oxidation in 10% oil-in- D.L. A., Lankelma, J., and Pinedo, H. M., Modulation
water emulsions by β-carotene with α- and γ-toco- by (iso)flavonoids of the ATPase activity of the
pherols, J. Am. Oil. Chemists’ Soc., 1997; 74(9): multidrug resistance protein, FEBS Lett., 1997; 413:
1047–1052. 344–348.
205. Helariutta, Y., Elomaa, P., Kotilainen, M., 219. Hör, M., Heinrich, M., and Rimpler, H.,
Griesbach, R.J., Schröder, J., and Teeri, T.H., Chal- Proanthocyanidin polymers with antisecretory activ-
cone synthase-like genes active during corolla de- ity and proanthocyanidin oligomers from Guazuma
velopment are differentially expressed and encode ulmifolia bark, Phytochemistry, 1996; 42(1): 109–
enzymes with different catalytic properties in Ger- 119.
bera hybrida (Asteraceae), Plant Mol. Biol., 1995; 220. Hoshino, A., Abe, Y., Saito, N., Inagaki, Y., and
28: 47–60. Iida, S., The gene encoding flavanone 3-hydroxy-
206. Helariutta, Y., Kotilainen, M., Elomaa, P, Klakkinen, lase is expressed normally in the pale yellow flow-
N., Bremer, K., Teeri, T. H., and Albert, V. A., Dupli- ers of the Japanese morning glory carrying the
cation and functional divergence in the chalcone syn- speckled mutation which produce neither flavonol
thase gene family of Asteraceae: evolution with sub- nor anthocyanin but accumulate chalcone, aurone
strate change and catalytic simplification, Proc. Natl. and flavanone, Plant Cell Physiol., 1997; 36: 970–
Acad. Sci. USA, 1996; 93: 9033–9038. 974.
207. Hempel, J. and Böhm, H., Betaxanthin pattern of 221. Hoshino, T., Fujii, R., and Nakahara, T., Molecular
hairy roots from Beta vulgaris var Lutea and its alter- cloning and sequence analysis of the crtB gene of
ation by feeding of amino acids, Phytochemistry, 1997; Thermus thermophilus an extreme thermophile pro-
44(5): 847–852 ducing carotenoid pigments, Appl. Environ. Microbiol.,

208. Heuer, S. and Strack, D., Synthesis of betanin from 1993; 59(9): 3150–3153.
betanidin and UDP-glucose by protein preparation 222. Hosokawa, K., Fukunaga, Y., Fukushi, E., and
from cell suspension cultures of Dorotheanthus Kawabata, J., Seven acylated anthocyanins in the blue
bellidiformis (Burm, F.), Planta, 1992; 186: 626–628 flowers of Hyacinthus orientalis, Phytochemistry,
209. Heuer, S., Vogth, T., Böhm, H., and Strack, D., Partial 1995; 38: 1293–1298.
purification and characterization of UDP-glucose: 223. Hosokawa, K., Fukushi, E., Kawabata, J., Fujii, C.,
betanidin 5-O- and 6-O-glucosyltransferases from cell Ito, T., and Yamamura, S., Seven acylated anthocya-
suspension cultures of Dorotheanthus bellidiformis nins in blue flowers of Gentiana, Phytochemistry,
Burm. F.) N.E.Br., Planta, 1996; 199: 244–250. 1997; 45: 167–171.
210. Hill, S., Squeezing the death out of food, New Scien- 224. Hosokawa, K., Fukushi, E., Kawabata, J., Fujii, C.,
tist, 1997; 12 April: 28–32. Ito, T., and Yamamura, S., Three acylated cyanidin
211. Hinz, U. G., Fivaz, J., Girod, P. A., and Zyrd, J. P., glucosides in pink flowers of Gentiana, Phytochemis-
The gene coding for DOPA dioxygenase involved in try, 1995; 40: 941–944.
betalain biosynthesis in Amanita muscaria and its 225. Huang, A. S. and von Elbe, J. H., Effect of pH on the
regulation, Mol. Gen. Genet., 1997; 256: 1–6. degradation and regeneration of betanine, J. Food
212. Hirano, H. and Komamine, A., Correlation of Sci., 1987; 52: 1689–1693.
betacyanin synthesis with cell division in cell suspen- 226. Huang, A. S. and von Elbe, J. H., Kinetics of the
sion cultures of Phytolacca americana, Physiol. Plant., degradation and regeneration of betanine, J. Food
1994; 90: 239–245. Sci., 1985; 50: 1115–1120,1129.
213. Hirasuna, T. J., Shuler, M. L., Lackney, V. K., and 227. Huang, C., Ma, W. Y., Dawson, M. I., Rincon, M.,
Spanswick, R. M., Enhanced anthocyanin production Flavell, R. A., and Dong, Z., Blocking activator pro-
in grape cell cultures, Plant Sci., 1991; 78: 107–120. tein-1 activity, but not activating retinoic acid re-
214. Hirobe, C., Qiao, Z. S., Takeya, K., and Itokawa, H., sponse element, is required for the antitumor promo-
Cytotoxic flavonoids from Vitex agnus-castus, Phy- tion effect of retinoic acid, Proc. Natl. Acad. Sci.
tochemistry, 1997; 46(3): 521–524. USA, 1997; 94: 5826–5830.
215. Holton, T. A., Brugliera, F., Lester, D. R., Tanaka, Y., 228. Hulshof, P. J. M., Xu, C., van de Bovenkamp, P.,
Hyland, C. D., Menting, J. G. T., Lu, C. Y., Farcy, E., Muhilal, and West, C. E., Application of a validated
Stevenson, T. W., and Cornish, E. C., Cloning and method for the determination of provitamin A caro-
expression of cytochrome P450 genes controlling tenoids in Indonesian foods of different maturity and
flower colour, Nature, 1993; 366: 276–279. origin, J. Agric. Food Chem., 1997; 45(4): 1174–
216. Holton, T. A. and Cornish, E. C., Genetics and bio- 1179.
chemistry of anthocyanin biosynthesis, Plant Cell, 229. Hutt, P. B., Approval of food additives in the United
1995; 7: 1071–1083. States: a bankrupt system, Food Technol., 1996; 3:
217. Hong, W. K. and Sporn, M. B., Recent advances in 118–128.
chemoprevention of cancer. Science, 1997; 278: 1073– 230. Ihl, M., Shene, C., Schevermann, E., and Bifani, V.,
1077. Connection for pigment content through colour deter-
278
mination using tristimulus values in a green leafy 243. Jorgensen, R., Cluster, P. D., English, J., Que, Q., and
vegetable, Swiss chard, J. Sci. Food Agric., 1994; 66: Napoli, C. A., Chalcone synthase cosuppression phe-
527–531. notypes in petunia flowers: comparison of sense vs.
231. Im, J. S., Parkin, K. L., and von Elve, J. H., Endog- antisense constructs and single-copy vs. complex T-
enous polyphenoloxidase activity associated with the DNA, Plant Mol. Biol., 1996; 31: 957–973.
“Black ring” defect in canned beet (Beta vulgaris L) 244. Joy, R. W., Sugiyama, M., Fukuda, H., and Komamine,
root slices, J. Food Sci., 1990; 55(4): 1042–1059. A., Cloning and characterization of polyphenol oxi-
232. Inami, O., Tamura, I., Kikuzaki, H., and Nakatani, N., dase cDNAs of Phytolacca americana, Plant Physiol.,
Stability of anthocyanins of Sambucus canadensis 1995; 107(4): 1083–1089.
and Sambucus nigra, J. Agric. Food Chem., 1996; 44: 245. Ju, Z., Liu, C. and Yuan, Y., Activities of
3090–3096. chalcone synthase and UDPGal:flavonoid-3-O-
233. Inami, O., Tamura, I., Kikuzaki, H., and Nakatani, N., glycosyltransferase in relation to anthocyanin synthe-
Stability of anthocyanins of Sambucus canadensis sis in apple, Sci. Hortic., 1995; 63: 175–185.
and Sambucus nigra, J. Agric. Food Chem., 1996; 246. Kakegawa, K., Suda, J., Sugiyama, M., and
44(10): 3090–3096. Komamine, A., Regulation of anthocyanin bio-
234. Inuma, M., Okawa, Y., Tanaka, T., Ho, F. C., synthesis in cell suspension cultures of Vitis in
Kobayashi, Y., and Miyauchi, K. I. Anti-oral micro- relation to cell division, Physiol. Plant., 1995; 94:
bial activity of isoflavonoids in root bark of Ormosia 661–666.
monosperma, Phytochemistry, 1994; 17(3): 889–891. 247. Kamal-Eldin, A. and Appelqvist, L. A., The effects of
235. Isaksen, M. and Francis, G. W., Reversed-phase thin- extraction methods on sesame oil stability, J. Am. Oil.
layer chromatography of carotenoids, J. Chromatogr., Chem. Soc., 1995; 72(8): 967–969.
1986; 355: 356–362. 248. Kamei, H., Koide, T., Kojima, T., Hasegawa, M., and
236. Isaksen, M. and Francis, G. W., Silver ion spray re- Umeda, T., Suppression of growth of cultured malig-
agent for the discrimination of the β- and ε-end groups nant cells by allomelanins plant-produced melanins,
in carotenoids on thin-layer chromatograms, Canc. Biother. and Radiopharmacol., 1997; 12(1):
Chromatographia,1990; 29(7/8): 363–365. 47–49.
237. Ittah, Y., Kanner, J., and Granit, R., Hydrolysis study 249. Kanazawa, K., Kawasaki, H., Samejima, K., Ashida,
of carotenoid pigments of paprika Capsicum annuum H., and Danno, G.I., Specific demutagens
L. variety Lehava) by HPLC/photodiode array detec- (antimutagens) in oregano against a dietary carcino-
tion, J. Agric. Food Chem., 1993; 41: 899–901. gen, Trp-P-2, are galangin and quercetin, J. Agric.
238. Jeon, J. H., Kim, H. S., Choi, K. H., Joung, Y. H., Food Chem., 1995; 432): 404–409.
Joung, H., and Byun, S. M., Cloning and characteriza- 250. Kanner, J. and Budowski, P., Carotene oxidizing fac-
tion of one member of the chalcone synthase gene tors in red pepper fruits (Capsicum annuum L.): effect
family from Solanum tuberosum L., Biosci. Biotechnol. of ascorbic acid and cooper in a β-carotene-linoleic
Biochem., 1996; 60: 1907–1910. acid solid model, J. Food Sci., 1978; 43: 524–26.
239. Jiménez, A. A., Dávila, O. G., Villegas, G. T., and Del 251. Kaufmann, R., Wingerath, T., Kirsch, D., Stahl, W.,
Villar, M. A., Obtención de colorantes de interés and Sies, H., Analysis of carotenoids and carotenol
alimentario por cultivo de células de Opuntia fatty acid esters by matrix-assisted laser desorption
microdasys Lehm) Pfeiff., Rev. Latinoamer. Quím., ionization (MALDI) and MALDI-post source decay
1992; 23(1), 5–8. mass spectrometry, Anal. Chem., 1996; 238: 117–
240. Jiménez, A. A., Gutiérrez, L. G., and Rodríguez, M. 128.
M., Caracterización reológica e hidrodinámica del 252. Khachik, F., Beecher, G. R., Goli, M. B., and Lusby,
cultivo de células de betabel (B. vulgaris L.) W. R., Separation and quantitation of carotenoids in
productoras de betalaínas en un biorreactor tipo foods, Method. Enzymol., 1992; 213part A): 142–167.
tanque agitado. In: Hübinger, M., Murr, F. X., 253. Khachik, F., Goli, M. B., Beecher, G. R., Holden, J.,
Aguilera, J. M., Eds., Propiedades físicas de los Lusby, W. R., Tenorio, M. D., and Barrera, M. R.,
alimentos Vol. 1. Valencia: CYTED-UPV, 1996, 105– Effect of food preparation on qualitative and quanti-
112. tative distribution of major carotenoid constituents of
241. Jiménez, A. A. and Gutiérrez, L. G., Production of tomatoes and several green vegetables, J. Agric. Food
food related colorants by culture of plant cells. The Chem., 1992; 40(3): 390–398.
case of betalains. In: Shahidi, F., Kolodziejczyk, P., 254. Kim, S. W., Seo, W. T., and Park, Y. H., Enhanced
Whitaker, J., López-Munguía, A., and Fuller, G., Eds., production of β-carotene from Blakeslea trispora with
Chemicals via Higher Plant Bioengineering. Plenum span 20, Biotechnol. Lett., 1997; 19(6): 561–562.
Pu. Co., 1998, in press. 255. Kino-Oka, M. and Tone, S., Extracellular production
242. Johnston, T. V. and Morris, J. R., HPLC analysis of of pigment from red beet hairy roots accompanied by
cabernet sauvignon and noble wine pigment fractions, oxygen preservation, J. Chem. Eng. Jpn., 1996; 29(3):
J. Food Sci., 1997; 62: 684–687. 488–493
279
256. Kirmizigül, S., Gören, N., Yang, S. W., Cordell, G. vonoids to inhibit the procoagulant activity of adher-
A., and Bozok-Johansson, C., Spinonin, a novel gly- ent human monocytes, J. Nat. Prod., 1996; 593):
coside from Ononis spinosa subsp. Leiosperma, J. 273–276.
Nat. Prod., 1997; 60(4): 378–381. 271. Lashley, D. and Wile, R. C., A betacyanine decoloriz-
257. Kläui, H., Carotenoids and their applications. In: ing enzyme found in red beet tissue, J. Food. Sci.
Counsell, J. N. and Dunastable, J. A., Eds., Natural 1979; 44: 1568–1569
Colours for Food and Other Uses. Applied Science, 272. Learned, R. M. and Connolly, E. L., Light modulates
London, 1979, 91–122. the spatial patterns of 3-hydroxy-3-methylglutaryl co-
258. Klinman, J. P. and Mu, D., Quinoenzymes in biology, enzyme A reductase gene expression in Arabidopsis
Ann. Rev. Biochem., 1994; 63: 299–344. thaliana, Plant J., 1997; 11(3): 499–511.
259. Koes, R. E. K., Quattrocchio, F., and Mol, J. N. M., 273. Leathers, R., Davin, C., and Zrÿd, J., Betalain produc-
The flavonoid biosynthetic pathway in plants: func- ing cell cultures of Beta vulgaris L. red beet), In Vitro
tion and evolution, BioEssays, 1994; 16(2): 123–132. Cell Dev. Biol., 1992; 28: 39–45
260. Kolb, E., Haung, M., Janzowski, C., Vetter, A., and 274. Lee, S. H. and Min, D. B., Effects, quenching mecha-
Eisenbrand, G., Potential nitrosamine formation and nisms, and kinetics of carotenoids in chlorophyll-sen-
its prevention during biological denitrification of red sitized photooxidation of soybean oil, J. Agric. Food
beet juice, Food Chem. Toxicol, 1997; 35(2): 219– Chem., 1990; 38(8): 1630–1634.
224. 275. Lee, Y. N. and Wiley, R. C., Betalaine yield from a
261. Koo, M. H. and Park, Y. K., Investigation of fla- continuous solid-liquid extraction system as influ-
vonoid aglycones in Propolis collected by two differ- enced by raw product, post-harvest and processing
ent varieties of bees in the same region, Biosci. variables, J. Food Sci., 1981; 46: 421–424
Biotechnol. Biochem., 1997; 61(2): 367–369. 276. Lee, Y. N., Wiley, R. C., Sheu, M. J., and Schlimme,
262. Koplas-Lane, L. M. and Warthesen, J. J., Carotenoid D. V., Purification and concentration of betalaines by
photostability in raw spinach and carrots during cold ultrafiltration and reverse osmosis, J. Food Sci., 1982;
storage, J. Food Sci., 1995; 60(4): 773–776. 47(2): 465–471, 475.
263. Kragh, K. M., Nielsen, J. E., Nielsen, K. K., Dreboldt, 277. Lessin, W. J., Catigani, G. I., and Schwartz, S. J.,
S., and Mikkelsen, J. D., Characterization and local- Quantification of cis-trans isomers of provitamin A
ization of new antifungal cysteine-rich proteins from carotenoids in fresh and processed fruits and veg-
Beta vulgaris, Mol. Plant-Microbe Inter., 1995; 8(3): etables, J. Agric. Food Chem., 1997; 45(10): 3728–
424–434 3732.
264. Kull, D. and Pfander, H., Isolation and identification 278. Li, Z. H., Matthews, P. D., Burr, B., and Wurtzel, E.
of carotenoids from the petals of rape (Brassica napus), T., Cloning and characterization of a maize cDNA
J. Agric. Food Chem., 1995; 43(11): 2854–2857. encoding phytoene desaturase, an enzyme of the caro-
265. Kumon, K., Sasaki, J., Sejima, M., Takeuchi, Y., and tenoid biosynthetic pathway, Plant Mol. Biol., 1997;
Hayashi, Y., Betacyanin-decolorizing enzymes from 30: 269–279.
Phytolacca americana, Plant Cell Physiol., 1990; 279. Lichtenhaler, H. K., Chlorophylls and carotenoids:
31(2): 233–240 pigments of photosynthetic biomembranes, Method.
266. Kuntz, M., Römer, S., Suire, C., Hugueney, P., Weil, Enzymol., 1987; 148: 350–382.
J.H., Schantz, R., and Camara, B., Identification of a 280. Lichtenhaler, H. K., Schwender, J., Disch, A., and
cDNA for the plastid-located geranylgeranyl pyro- Rohmer, M., Biosynthesis of isoprenoids in higher
phosphate synthase from Capsicum annuum: cor- plant chloroplasts proceeds via a mevalonate path-
relative increase in enzyme activity and transcript way, FEBS Lett., 1997; 400: 271–274.
level during fruit ripening, Plant J., 1992; 2(1): 25– 281. Liebler, D. C. 1993. Antioxidant reactions of caro-
34. tenoids. In: Canfield, L. M., Krinsky, N. I., and
267. Kuwabara, K., Shudo, K., and Hori, Y., Synthetic Dunastable, J. A., Eds., Carotenoids in Human Health,
retinoic acid inhibits rat collagen arthritis and differ- Annals of the New York Academy of Sciences, New
entially affects serum immunoglobulin subclass lev- York, Vol. 691, 1993, 20–31.
els, FEBS Lett., 1996; 378: 153–156. 282. Lietz, G. and Henry, C. J. K., A modified method to
268. Lacomme, C. and Roby, D., Molecular cloning of a minimize losses of carotenoids and tocopherols dur-
sulfotransferase in Arabidopsis and regulation during ing HPLC analysis of red palm oil, Food Chem.,
development and in response to infection with patho- 1997; 60(1): 109–117.
genic bacteria, Plant Mol. Biol., 1996; 30: 995–1008. 283. Lin, C., Ahmad, M., and Cashmore, A. R.,
269. Lai, S. M., Gray, J. I., Partridge, J. A., and Flegal, C. Arabidopsis cryptochrome 1 is a soluble protein
J., Stability of cholesterol and paprika carotenoids in mediating blue light-dependent regulation of plant
egg powders as influenced by dietary and processing growth and development, Plant J., 1996; 10: 893–
treatments, J. Sci. Food Agric., 1996; 72: 171–178. 902.
270. Lale, A., Herbert, J. M., Augereau, J. M., Billon, M., 284. Lin, Y. M., Anderson, H., Flavin, M. T., Pai, Y. H. S.,
Leconte, M., and Gleye, J., Ability of different fla- Mata-Greenwood, E., Pengsuparp, T., Pezzuto, J. M.,
280
Schinazi, R. F., Hughes, S. H., and Chen, F. C., In 297. Marusich, W. L. and Bauernfeind, J. C. Oxicarotenoids
vitro anti-HIV activity of biflavonoids isolated from in poultry feeds. In: Bauernfeind, J. C., Ed., Caro-
Rhus succedanea and Garcinia multiflora, J. Nat. tenoids as Colorants and Vitamin A Precursors,
Prod., 1997; 60(9): 884–888. Academic Press, New York, 1981, 319–462.
285. Linden, H. and Macino, G., White collar 2, a partner 298. Massiot, P., Guiller, I., Baron, A., and Drilleau, J. F.,
in blue-light signal transduction, controlling expres- Cell wall polysaccharides modifications during heat
sion of light-regulated genes in Neurospora crassa, treatment and enzymatic degradation of carrot tissues,
EMBO J., 1997; 16(1): 98–109. J. Food Sci. Technol., 1992; 25: 559–563.
286. Lozovik, G., Nutraceutical properties of Anonia 299. Masson, G., Baumes, R., Puech, J. L., and Razungles,
colorant. In: International Congress and Symposium A., Demonstration of the presence of carotenoids in
on Natural Colorants. Puerto de Acapulco, México, wood: quantitative study of cooperage oak, J. Agric.
1996. Food Chem., 1997; 45(5): 1649–1652.
287. Mabry, T. J., Betalains. In: Bell, E. A. and Charlwood, 300. Mathews-Roth, M. M., Carotenoids in erythropoietic
B. V., Eds. Encyclopedia of Plant Physiology. Vol. 8, protoporphyria and other photosensitivity disease. In:
Secondary Plant Products. Springer-Verlag, Berlin, Canfield, L. M., Krinsky, N. I., and Dunastable, J. A.,
1980, 513–533. Eds., Carotenoids in Human Health. Annals of the
288. Mabry, T. J. and Dreiding, A. S., The betalaines. In: New York Academy of Sciences, New York, Vol.
Mabry, R., Alston, E., and Runeckles, V. C., Eds., 691, 1993, 127–138.
Recent Advances in Phytochemistry, Vol.1., Appleton 301. Matsuda, N., Tsuchiya, T., Kishitani, S., Tanaka, Y.,
Century Crofts, New York, 1968, 145–160. and Toriyama, K., Partial male sterility in transgenic
289. Mabry, T. J., Taylor, A., and Turner, B. L., The tobacco carrying antisense and sense PAL cDNA un-
betacyanins and their distribution, Phytochemistry, der the control of a tapetum-specific promoter, Plant
1963; 2: 61–64. Cell Physiol., 1996; 37(2): 215–222.
290. Macias, F. A., Molinillo, J. M. G., Torres, A., Varela, 302. Mattews, S., Mila, I., Scalbert, A., and Donnelly, M.
R. M., and Castellano, D., Bioactive flavonoids from X., Extractable and non-extractable proanthocyanidins
Helianthus annuus cultivars, Phytochemistry, 1997; in barks, Phytochemistry, 1997; 45(2): 405–410.
45(4): 683–687. 303. Mazza, G. and Gao, L., Malonylated anthocyanins in
291. Madhavi, D. L., Bomser, J., Smith, M. A. L., and purple sunflower seeds, Phytochemistry, 1994; 35(1):
Singeltary, K., Isolation of bioactive constituents from 237–239.
Vaccinium myrtillus (bilberry) fruits and cell cultures, 304. McCarty, D. R., Genetic control and integration of
Plant Sci., 1998; 131: 95–103. maturation and germination pathways in seed devel-
292. Madhavi, D. L., Juthangkoon, S., Lewen, K., Berber- opment, Ann. Rev. Plant Physiol. Plant Mol. Biol.,
Jimenez, M. D., and Smith, M. A. L., Characterization 1995; 46: 71–93.
of anthocyanins from Ajuga pyramidalis metallica 305. McGarvey, D. J. and Croteau, R., Terpenoid metabo-
crispa cell cultures, J. Agric. Food Chem., 1996; 44: lism, Plant Cell, 1995; 7: 1015–1026.
1170–1176. 306. Means, A. L. and Gudas, L. J., The roles of retinoids
293. Maier, V. P., Brewster, L. C., Norman, S. M., and in vertebrate development. Ann. Rev. Biochem., 1995;
Yokoyama, H., Bioregulation of citrus terpenoid con- 64: 201–233.
stituents and fruit quality. In: Inglett, G. E. and 307. Mehta, B. J,, Salgado, L. M., Bejarano, E. R., and
Charalambous, G., Eds., Tropical Foods: Chemistry Cerdá-Olmedo, E., New mutants of Phycomyces
and Nutrition. Vol. 1., Academic Press, New York, blakesleeanus for β-carotene production, Appl.
1979, 155–170. Environ. Microbiol., 1997; 63(9): 3657–3661.
294. Malhotra, B., Onyilagha, J. C., Bohm, B. A., Towers, 308. Mercadente, A. Z., Rodriguez-Amaya, D. B., and
G. H. N., James, D., Harborne, J. B., and French, C. Britton, G., HPLC and mass spectrometric analysis of
J., Inhibition of tomato ringspot virus by flavonoids, carotenoids from mango, J. Agric. Food Chem., 1997;
Phytochemistry, 1996; 43(6): 1271–1276. 45: 120–123.
295. Marin, E., Nussaume, L., Quesada, A., Gonneau, M. 309. Merlin, J. C., Statoua, A., Cornard, J. P., Saidi-Idrissi,
Sotta, B., Hugueney, P., Frey, A., and Marion-Poll, M., and Brouillard, R., Resonance RAMAN spectro-
A., Molecular identification of zeaxanthin epoxydase scopic studies of anthocyanins and anthocyanidins in
of Nicotiana plumbaginifolia, a gene involved in ab- aqueous solutions, Phytochemistry, 1994; 35: 227–
scisic acid biosynthesis and corresponding to the ABA 232.
locus of Arabidopsis thaliana, EMBO J., 1996; 15(10): 310. Merril, R.A., Food additive approval procedures dis-
2331–2342. courage innovation, Food Technol., 1996; 50(3): 110–
296. Marrs, K. A., Alfenito, M. R., Lloyd, A. M., and 113.
Walbot, V., A glutathione-S-transferase involved in 311. Meyer, H. J. and Van Staden, J., The in vitro produc-
vacuolar transfer encoded by the maize gene Bronze- tion of an anthocyanin from callus cultures of Oxalis
2, Nature, 1995; 375: 397–400. linearis, Plant Cell Tiss. Org. Cult., 1995; 40: 55–58.
281
312. Meyer, P., Heidmann, I., Forkmann, G., and Saeder, in petunia corollas, Plant Physiol., 1997; 113: 419–
H., A new petunia flower colour generated by trans- 424.
formation of a mutant with a maize gene, Nature, 326. Mol, J., Jenkins, G., Schäefer, E., and Weiss, D.,
1987; 330: 677–678. Signal perception, transduction, and gene expression
313. Middendorf, D. F., Childs, G. R., and Cravens, W. involved in anthocyanin biosynthesis, Crit. Rev. Plant
W., A rapid bioassay for the comparison of xantho- Sci., 1996; 15: 525–557.
phyll availability from various sources, Poultry Sci., 327. Mol, J., Jenkins, G., Schäfer, E., and Weiss, D., Signal
1980; 59: 1442–1450. perception, transduction and gene expression involved
314. Middleton, E. M. and Teramura, A. H., The role of in anthocyanin biosynthesis, Crit. Rev. Plant Sci.,
flavonol glycosides and carotenoids in protecting soy- 1996; (155 & 6): 525–557.
bean from ultraviolet-B damage, Plant Physiol., 1993; 328. Mol, J. N. M., Molecular biology of anthocyanin bio-
103: 741–752. synthesis. In: Scalbert, A., Ed., Polyphenolic Phe-
315. Miller, N. J., Sampson, J., Candeias, L. P., Bramley, nomena. Paris, 1993, 87–98.
P. M., and Rice-Evans, C. A., Antioxidant activities 329. Mooney, M., Desnos, T., Harrison, K., Jones, J., Car-
of carotenes and xanthophylls, FEBS Lett., 1996; 384: penter, R., and Coen, E., Altered regulation of tomato
240–242. and tobacco pigmentation genes caused by the delila
316. Minale, L., Piatelli, M., De Stefano, S., and Nicolaus, gene of Anthirrhinum, Plant J., 1995; 7: 333–339.
R. A., Pigments of Centrospermae VI. Acylated 330. Moore, P. P., Estimation of anthocyanin concentra-
betacyanins, Phytochemistry, 1966; 5: 1037–1052 tion from color meter measurements of red raspberry
317. Mínguez-Mosquera, M. I. and Gandul-Rojal, B., fruit, Hortscience, 1997; 32: 135.
Mechanism and kinetics of carotenoid degradation 331. Mori, T. and Sakurai, M., Effects of riboflavin and
during the processing of green table olives, J. Agric. increased sucrose on anthocyanin production in sus-
Food Chem., 1994; 42(7): 1501–1554. pended strawberry cell cultures, Plant Sci., 1995; 110:
318. Mínguez-Mosquera, M. I. and Hornero-Méndez, D., 147–153.
Comparative study of the effect of paprika processing 332. Mori, T. and Sakurai, M., Production of anthocyanin
on the carotenoids in peppers (Capsicum annuum) of from strawberry cell suspension cultures; effects of
the Bola and Agridulce varieties, J. Agric. Food Chem., sugar and nitrogen, J. Food Sci., 1994; 59: 588–593.
1994; 42: 1555–1560. 333. Mosha, T. C., Pace, R. D., Adeyeye, S., Laswai, H. S.,
319. Mínguez-Mosquera, M. I. and Jarén-Galán, M., Ki- and Mtebe, K., Effect of traditional processing practices
netics of the decolouring of carotenoid pigments, J. on the content of total carotenoid, β-carotene, α-carotene
Sci. Food Agric., 1995; 67: 153–161. and vitamin A activity of selected Tanzanian vegetables,
320. Mínguez-Mosquera, M.I., Jarén-Galán, M., and Plant Foods Hum. Nutr., 1997; 50: 189–201.
Garrido-Fernández, J., Competition between the pro- 334. Müeller, L. A., Hinz, U., Use, M., Sautter, C., and
cesses of biosynthesis and degradation of carotenoids Zryd, J. P., Biochemical complementation of the
during the drying of peppers, J. Agric. Food Chem., betalain biosynthetic pathway in Portulaca grandi-
1994; 42(3): 645–648. flora by a fungal 3,4-dihydroxyphenylalanine
321. Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, dioxygenase, Planta, 1997; 203(2): 260–263
S., Izawa, Y., Nakamura, K., and Harashima, K., Elu- 335. Müeller, L. A., Hinz, U., Uze, M., Sautter, C., and
cidation of the Erwinia uredovora carotenoid biosyn- Zrÿd, J. P., Complementation of betalain biosynthesis
thetic pathway by functional analysis of gene prod- in P. grandiflora by a fungal DOPA-dioxygenase,
ucts expressed in Escherichia coli, J. Bacteriol., 1990; Experientia, 1996; 522. : A20.
172(12): 6704–6712. 336. Müeller, L. A., Hinz, U., and Zrÿd, J. P., The forma-
322. Miséra, S., Müller, A. J., Weiland-Heidecker, U., and tion of betalamic acid and muscaflavin by recombi-
Jürgens, G., The FUSCA genes of Arabidopsis: nega- nant DOPA-dioxygenase from Amanita, Phytochem-
tive regulators of light responses, Mol. Gen. Genet., istry, 1997; 44(4): 567–569
1994; 244: 242–252. 337. Murakami, A., Ohigashi, H., and Koshimizu, K., Anti-
323. Misset-Smits, M., Oltshoorn, A. J. J., Dewanti, A., tumor promotion with food phytochemicals: A strat-
and Duine, J. A., Production, assay, and occurrence of egy for cancer chemoprevention, Biosci. Biotechnol.
pyrroloquinoline quinone, Method. Enzymol., 1997; Biochem., 1996; 60(1): 1–8.
280: 89–98. 338. Namiki, M., Antioxidants/antimutagens in food, Crit.
324. Miyake, Y., Yamamoto, K., and Osawa, T., Metabo- Rev. Food Sci. Nutr., 1990; 29(4): 273–300.
lism of antioxidant in lemon fruit (Citrus limon BURM. 339. Newsome, R. L., Food colors, Food Technol., 1986;
F.) by human intestinal bacteria, J. Agric. Food Chem., 40(7): 49–56.
1997; 4510): 3738–3742. 340. Nielsen, B. R., Mortensen, A., Jørgensen, K., and
325. Moalem-Beno, D., Tamari, G., Leitner-Dagan, Y., Skibsted, L. H., Singlet versus triplet reactivity in
Borochov, A., and Weiss, D., Sugar-dependent gib- photodegradation of C40 carotenoids, J. Agric. Food
berellin-induced chalcone synthase gene expression Chem., 1996; 44:2106–2113.
282
341. Nievelstein, V., Vandekerckhove, J., Tadros, M. H., growth inhibition by flavonoids, Phytochemistry, 1997;
Lintig, J. V., Nitschke, W., and Beyer, P., Carotene 46(3):499–502.
desaturation is linked to a respiratory redox pathway 355. Palozza, P. and Krinsky, N. I., Antioxidant effects of
in Narcissus pseudonarcissus chromoplast membranes. carotenoids in vivo and in vitro: an overview, Method.
Involvement of a 23-kDa oxygen-evolving-complex- Enzymol., 1992; 213(part A): 403–420.
like protein, Eur. J. Biochem., 1995; 233: 864–872. 356. Paquay, J. B. G., Haenen, G. R. M. M., Korthouwer,
342. Nilsson, T., Studies into pigments of beet root, R. E. M., and Bast, A., Peroxynitrite scavenging by
Lantbruk-shoegskolans Anal., 1970; 36: 179–219. wines, J. Agric. Food Chem., 1997; 45(9): 3357–
343. Norris, S. R., Barrette, T. R., and DellaPenna, D., 3358.
Genetic dissection of carotenoid synthesis in 357. Parajó, J. C., Vázquez, M., and Cruz, J. M., Produc-
Arabidopsis defines plastoquinone as an essential tion of carotenoids by Xanthophyllomyces dendrorhous
component of phytoene desaturation, Plant Cell, 1995; growing on enzymatic hydrolysates of prehydrolysed
7: 2139–2179. wood, Food Chem., 1997; 60(3): 347–355.
344. Norton, R. A., Effect of carotenoids on aflatoxin B1 358. Parellada, J. and Guinea, M., Flavonoid inhibitors of
synthesis by Aspergillus flavus, Phytopathology, 1997; trypsine and leucine aminopeptidase: a proposed math-
87: 814–821. ematical model for IC50 estimation, J. Nat. Prod.,
345. Nozue, M., Kubo, H., Nishimura, M., and Yasuda, H., 1995; 58(6): 823–829.
Detection and characterization of a vacuolar protein 359. Park, H., Flores, R. A., and Johnson, L. A., Prepara-
VP24) in anthocyanin-producing cells of sweet potato tion of fish feed ingredients: reduction of carotenoids
in suspension culture, Plant Cell Physiol., 1995; 36: in corn gluten meal, J. Agric. Food Chem., 1997;
883–889. 456): 2088–2092.
346. Nyengfack, A. E., Kouam, J., Vouffo, T. W., Meyer, 360. Parker, R. S., Absorption, metabolism, and transport
M., Tempesta, M. S., and Fomum, Z. T., An of carotenoids, FASEB J., 1996; 10: 542–551.
isoflavanone and a coumestan from Erythrina 361. Parra, J. M. and Muñoz, R., An approach to the char-
sigmoidea, Phytochemistry, 1994; 35(2): 521–526. acterization of betanine oxidation catalyzed by horse-
347. Ohba, R., Kitaoka, S., Ohda, S., and Ueda, S., Storage radish peroxidase, J. Agric. Food Chem., 1997; 458):
stability and thermal stability of hordeumin, an antho- 2984–2988
cyanin pigment from barley, Biosci. Biotechnol. 362. Pascal, N., Block, M. A., Pallett, K. E., Joyard, J., and
Biochem., 1995; 59: 746–748. Douce, R., Inhibition of carotenoid biosynthesis in
348. Olson, J. A., Molecular actions of carotenoids. In: sycamore cells deprived of iron, Plant Physiol.
Canfield, L. M., Krinsky, N. I., and Dunastable, J. A., Biochem., 1995; 33(1): 197–104.
Eds., Carotenoids in Human Health, Annals of the 363. Pasch, J. H. and von Elbe, J. H., Betanine stability in
New York Academy of Sciences, New York, Vol. buffered solutions containing organic acids, metal
691, 1993, 156–166. cations, antioxidants, or sequestrants, J. Food Sci.,
349. Olson, J. A. and Krinsky, N. I., Introduction. The 1979; 44: 72–74,81
colorful, fascinating world of the carotenoids: impor- 364. Pecker, I., Gabbay, R., Cunningham, F. X., and
tant physiologic modulators, FASEB J., 1995; 9: 1547– Hirschberg, J., Cloning and characterization of the
1550. cDNA for lycopene β-cyclase from tomato reveals
350. O’Neil, C. A. and Schwartz, S. J., Photoisomerization decrease in its expression during fruit ripening, Plant
β-carotene by photosensitization with chlorophyll de- Mol. Biol., 1996; 30: 807–819.
rivatives as sensitizers, J. Agric. Food Chem., 1995; 365. Pedersen, A. T., Andersen, O. M., Aksnes, D. W., and
43(3): 631–635. Nerdal, W., Anomeric sugar configuration of antho-
351. Ootaki, T., Yamazaki, Y., Noshita, T., and Takahashi, cyanin O-pyranosides determined from heteronuclear
S. 1996. Excess carotenoids disturb prospective cell- one-bond coupling constants, Phytochemical Anal.,
to-cell recognition system in mating responses of 1995; 6: 313–316.
Phycomyces blakesleanus. Mycoscience 37: 427–435. 366. Pelletier, M. K., Murrell, J. R., and Shirley, B. W.,
352. Packer, L., Antioxidant action of carotenoids in vitro Characterization of flavonol synthase and
and in vivo and protection against oxidation of human leucoanthocyanidin dioxygenase genes in Arabidopsis,
low density lipoproteins. In: Canfield, L. M., Krinsky, Plant Physiol., 1997; 113: 1437–1445.
N. I., and Dunastable, J. A., Eds, Carotenoids in 367. Pesek, C. A. and Warthesen, J. J., Kinetic model for
Human Health, Annals of the New York Academy of photoisomerization and concomitant photodegradation
Sciences, New York, Vol. 691, 1993, 48–60. of β-carotenes, J. Agric. Food Chem., 1990; 38(6):
353. Padmanabhan, S., Ramakrishna, M., Lonsane, B. K., 1313–1315.
and Krishnaiah, M. M., Enzymatic treatment of cas- 368. Pesek, C. A., Warthesen, J. J., and Taoukis, P. S., A
sava flour slurry for enhanced recovery of starch, kinetic model for equilibration of isomeric β-carotenes,
Food Biotechnol., 1993; 7(1): 1–10. J. Agric. Food Chem., 1990; 38(1): 41–45.
354. Padmavati, M., Sakthivel, N., Thara, K. V., and Reddy, 369. Phillips, L. G., Cowan, A. K., Rose, P. D., and Logie,
A. R., Differential sensitivity of rice pathogens to M. R. R., Operation of the xanthophyll cycle in non-
283
stressed and stressed cells of Dunaliella salina Teod. 386. Raina, B. L., Agrawal, S. G., Bhatia, A. K., and Gour,
in response to diurnal changes in incident irradiation: G. S., Changes in pigments and volatiles of saffron
a correlation with intracellular β-carotene content, J. (Crocus sativus L.) during processing and storage, J.
Plant Physiol., 1995; 146: 547–553. Sci. Food Agric., 1996; 71: 27–32.
370. Piatelli, M. and Imperato, F., Betacyanins of the fam- 387. Randolph, T. W., Supercritical fluid extractions in
ily cactaceae, Phytochemistry, 1969; 8: 1503–1507 biotechnology, Trends Biotechnol., 1990; 8: 78–82.
371. Piatelli, M., Betalains. In: Goodwin, T. W., Ed,. 388. Rao, M. N., Ghosh, P., and Lakshman, M. R., Purifi-
Chemistry and Biochemistry of Plant Pigments. cation and partial characterization of a cellular caro-
Vol., 1., Academic Press, New York, 1976, 560– tenoid-binding protein from ferret liver, J. Biol. Chem.,
596. 1997; 272(39): 24455–24460.
372. Piatelli, M. and Imperato, F., Pigments of Bougainvil- 389. Ray, J., Bird, C., Maunders, M., Grierson, D., and
lea glabra, Phytochemistry, 1970; 9: 2557–2560. Schuch, W., Sequence of pTOM5, a ripening related
373. Piatelli, M. and Minale, L., Pigments of cDNA from tomato, Nucleic Acids Res., 1987; 15:
Centrospermae. I. Betacyanins from Phyllocactus 10587.
hybridus Hort. and Opuntia ficus-indica, Phytochem- 390. Rayner, P. B., Food and drink colors from natural
istry, 1964; 3: 307–311. sources, Food Marketing Technol. 1993; 7(1): 9–10.
374. Piatelli, M. and Minale, L., Pigments of 391. Real, S. E. and Cerezal, M. P., Estudio de los
Centrospermae. III. Distribution of betacyanins, Phy- parámetros de operación en la ultrafiltración del jugo
tochemistry, 1964; 3: 547–557. de remolacha para colorante, Aliment. Latinoamer.,
375. Piatelli, M., The betalains: structure, biosynthesis and 1995; 206: 34–40.
chemical taxonomy. In: Conn, E. E., Ed., The Bio- 392. Real, S. E., Cerezal, M. P., and Ruiz, H. A., Empleo
chemistry of Plants: A Comprehensive Treatise. Vol. de la ultrafiltración en la purificación del jugo de

17. Secondary Plant Products, Academic Press, New remolacha para colorante, Alimentos, 1993; 18(4): 5–
York, 1981, 557–575. 10.
376. Pogson, B., McDonald, K. A., Truong, M., Britton, G., 393. Reay, P. F., Fletcher, R. H., and Thomas, V. J.,
and DellaPenna, D., Arabidopsis carotenoids mutants Chlorophylls, carotenoids and anthocyanin concen-
demonstrate that lutein is not essential for photosynthe- trations in the skin of ‘Gala’ apples during maturation
sis in higher plants, Plant Cell, 1996; 8: 1627–1639. and the influence of foliar applications of nitrogen and
377. Pommer, K. (Novo Nordisk A/S). 1994. Abstract: magnesium, J. Sci. Food Agric., 1998; 76: 63–71.
Method for isolation of vegetable oleoresins produc- 394. Reeves, M. J., Re-evaluation of Capsicum color data,
ible by hexane production. PCT Inf. Appl. WO94 13, J. Food Sci., 1987; 52(4): 1047–1049.
743 (CI.CO9B61/00) 23/Jun/1994, DK Appl. 92/1, 395. Reznik, H., Betalains. In: Pigments in plants. Czygan,
510, 17/Dec/1992. F. E., Ed., Akademie Verlag 1981; 370–392
378. Poole, C. F. and Poole, S. K., Instrumental thin layer 396. Rimington, C. and Kennedy, G. Y., Porphyrins: struc-
chromatography, Anal. Chem., 1994; 66(1): 27A-36A. ture, distribution, and metabolism. In: Florkin, M. and
379. Pourrat, A., Lejeune, B., Grand, A., and Pourrat, H., Mason, H.S., Eds., Comparative Biochemistry. A Com-
Betalains assay of fermented red beet root extract by prehensive Treatise. Vol. IV. Constituents of Life-Part
high performance liquid chromatography, J. Food Sci., B, Academic Press, New York, 1962, 558–614.
1988; 53(1): 294–295. 397. Rink, E. and Böhm, H., Changed betaxanthin pattern
380. Powrie, W. D. and Fennema, O., Electrophoretic sepa- in violet flowers of Portulaca grandiflora after the
ration of beet pigments, J. Food Sci., 1963; 28: 214– feeding of DOPA, Phytochemistry, 1984; 24(7): 1475–
216. 1477
381. Proctor, A. and Bowen, D. J., Ambient-temperature 398. Robards, K., Li, X., Antolvich, M., and Boyd, S.,
extraction of rice bran oil with hexane and isopro- Characterization of citrus by chromatographic analy-
panol, J. Am. Oil. Chem. Soc., 1996; 73(6): 811–813. sis of flavonoids, J. Sci. Food Agric., 1997; 75: 87–
382. Pszczola, D. E., Natural colors: pigments of imagina- 101.
tion, Food Technol., 1998; 52(6): 70–76. 399. Rockholm, D. C. and Yamamoto, H. Y., Violaxanthin-
383. Quatrano, R. S., Bartels, D., Ho, T. H. D., and Pagés, de-epoxydase, Plant Physiol., 1996; 110: 697–703.
M., New insights into ABA-mediated processes, Plant 400. Römer, S., Hugueney, P., Bouvier, F., Camara, B.,
Cell, 1997; 9: 470–475. and Kuntz, M., Expression of the genes encoding the
384. Quintanar-Hernández, J. A., Loarca-Piña, M. G. F., early carotenoid biosynthetic enzymes in Capsicum
and González-de-Mejía, E., Efecto de los carotenoides annuum, Biochem. Biophys. Res. Commun., 1993;
presentes en chile verde (Capsicum annuum spp.) de 196: 1414–1421.
mayor consumo contra tóxicos en alimentos, 401. Rose, A., Gläbgen, W. E., Hopp, W., and Seitz,
Tecnología Alimentaria, 1996; 31(6): 15–21. H. U., Purification and characterization of
385. Radlwimmer, F. B. and Yokoyama, S., Cloning and glycosyltransferases involved in anthocyanin biosyn-
expression of the red visual pigment of goat (Capra thesis in cell-suspension cultures of Daucus carota L.,
hircus), Gene, 1997; 198: 211–215. Planta, 1996; 198: 397–402.
284
402. Rosendal-Jensen, S., Juhl-Nielsen, B., and 417.Sarma, A. D., Sreelakshmi, Y., and Sharma, R., Antioxi-
Dahlgren, R., Use of chemistry in plant classifica- dant ability of anthocyanins against ascorbic acid
tion, Rev. Latinoamer. Quím., 1989; Suppl 1: 66– oxidation, Phytochemistry, 1997; 45: 671–674.
89. 418. Sato, K., Nakayama, M., and Shigeta, J. I., Culturing
403. Rosenthal, A., Pyle, D. L., and Niranjan, K., Aqueous conditions affecting the production of anthocyanin in
and enzymatic processes for edible oil extraction, Enz. suspended cell cultures of strawberry, Plant Sci., 1996;
Microb. Technol., 1996; 19: 400–420. 113: 91–98.
404. Rüdiger, W. and Schoch, S., Chlorophylls. In: 419. Scita, G., Stability of ß-carotene under different labo-
Goodwin, T. W., Ed., Plant Pigments, Academic Press, ratory conditions, Method. Enzymol., 1992; 213(part
London, 1988, 1–60. A): 175–185.
405. Rwabahizi, S. and Wrolstad, R. E., Effects of mold 420. Scolnik, P. A. and Bartley, G. E., Phytoene desaturase
contamination and ultrafiltration on the color stability from Arabidopsis, Plant Physiol., 1993; 103: 1475.
of strawberry juice and concentrate, J. Food Sci., 421. Scolnik, P. A. and Bartley, G. E., Nucleotide se-
1988; 53: 857–861, 872. quence of an Arabidopsis cDNA for geranylgeranyl
406. Sacchettini, J. C. and Poulter, C. D., Creating iso- pyrophosphate synthase, Plant Physiol., 1994; 104:
prenoid diversity, Science, 1997; 277: 1788–1789. 1469–1470.
407. Saguy, I., Goldman, M., Bord, A., and Cohen, E., 422. Scolnik, P. A. and Bartley, G. E., A table of some
Effect of oxygen retained on beet powder on the sta- cloned plant genes involved in isoprenoid biosynthe-
bility of betanine and vulgaxanthine-I, J. Food Sci., sis, Plant Mol. Biol. Rep., 1996; 14(4): 305–319.
1984; 49: 99–101. 423. Schedz, M., Al-Babili, S., Lintig, J. V., Haubruck, H.,
408. Saguy, I., Kopelman, I. J., and Mizrahi, S., Computer- Rabbani, S., Kleining, H., and Beyer, P., Phytoene
aided determination of beet pigments, J. Food Sci., synthase from Narcissus pseudonarcissus: Functional
1978; 43: 124–127. expression, galactolipid requirement, topological dis-
409. Sainz, M. B., Grotewold, E., and Chandler, V. L., tribution in chromoplasts and induction during flow-
Evidence for direct activation of an anthocyanin pro- ering, Plant J., 1996; 10(5): 781–792.
moter by the maize C1 protein and comparison of 424. Scheller, H. V., Naver, H., and Møller, B. L., Molecu-
DNA binding by related Myb domain proteins, Plant lar aspects of photosystem I, Physiol. Plant., 1997;
Cell, 1997; 9: 611–625. 100: 842–851.
410. Saito, N., Ku, M., Tatsuzawa, F., Lu, T. S., Yokoi, M., 425. Schmitz, H.H., Emenhiser, C., and Schwartz, S. J.,
Shigihara, A., and Honda, T., Acylated cyanidin gly- HPLC separation of geometric carotene isomers using
cosides in the purple-red flowers of Bletilla striata, a calcium hydroxide stationary phase, J. Agric. Food
Phytochemistry, 1995; 40: 1521–1529. Chem., 1995; 43(5):1212–1218.
411. Sakamoto, K., Iida, K., Sawamura, K., Hajiro, K., 426. Schmitz, H. H., Van Breemen, B., and Schwartz, S. J.,
Asada, Y., Yoshikawa, T., and Furuya, T., Effects of Fast-atom bombardment and continuous-flow fast-
nutrients on anthocyanin production in cultured cells atom bombardment mass spectrometry in carotenoid
of Aralia cordata, Phytochemistry, 1993; 33: 357– analysis, Method. Enzymol., 1992; 213(part A): 322–
360. 336.
412. Sakurai, M., Ozeki, Y., and Mori, T., Induction of 427. Schoefs, B., Bertrand, M., and Lemoine, Y., Separa-
anthocyanin accumulation in rose suspension-cultured tion of photosynthetic pigments and their precursors
cells by conditioned medium of strawberry suspen- by reversed-phase high-performance liquid chroma-
sion cultures, Plant Cell Tiss. Org. Cult., 1997; 50: tography using a photodiode-array detector, J.
211–214. Chromatogr. A, 1995; 692: 239–245.
413. Samejima, K., Kanazawa, K., Ashida, H., and Danno, 428. Schroeder, W. A., Calo, P., DeClercq, M. L., and
G.I., Luetolin: a strong antimutagen against dietary Johnson, E. A., Selection for carotenogenesis in the
carcinogen, Trp-P-2, in peppermint, sage, and thyme, yeast Phaffia rhodozyma by dark-generated singlet
J. Agric. Food Chem., 1995; 43(2): 410–414. oxygen, Microbiology, 1996; 142: 2923–2929.
414. Sánchez-Torres, P., González-Candelas, L., and 429. Schwartz, S. J., Chemical alterations of betacyanin
Ramón, D., Heterologous expression of a Candida and chlorophyll pigments during processing and a
molischiana anthocyanin-β-glucosidase in a wine yeast toxicological investigation of betalain pigments, Diss.
strain, J. Agric. Food Chem., 1998; 46: 354–360. Abst. Intern., 1983; 43(11): 3524–3525
415. Sander, L. C., Sharpless, K. E., Kraft, N. E., and Wise, 430. Schwartz, S. J., Hildenbrand, B. E., and von Elbe, J.
S. A., Development of engineered stationary phases H., Comparison of spectrophotometric and HPLC
for the separation of carotenoid isomers, Anal. Chem., methods to quantify betacyanins, J. Food Sci., 1981;
1994; 66(10): 1667–1674. 46: 286–297.
416. Sapers, G. M. and Hornstein, J. S., Varietal differ- 431. Schwartz, S. J., von Elbe, J. H., Pariza, M. W.,
ences in colorant properties and stability of red beet Goldsworthy, T., and Pitot, H. C., Inability of red beet
pigments, J. Food Sci., 1979; 44: 1245–1248. betalain pigments to initiate or promote
285
hepatocarcinogenesis, Food Chem. Toxicol., 1983; 445. Spanos, G. A., Chen, H., and Schwartz, S. J.,
21(5): 531–535 Supercritical CO2 extraction of ß-carotene from sweet
432. Schwartz, S. J. and von Elbe, J. H., Quantitative de- potatoes, J. Food Sci., 1993; 58(4): 817–820.
termination of individual betacyanin pigments by high- 446. Stafford, A., The manufacture of food ingredients
performance liquid chromatography, J. Agric. Food using plant cell and tissue cultures, Trends Food Sci.
Chem., 1980; 28: 540–543 Technol., 1991; 5(2): 116–122
433. Schwinn, K. E., Davies, K. M., Deroles, S. C., 447. Stahl, W. and Sies, H., Physical quenching of singlet
Markham, K. R., Miller, R. M., Bradley, J. M., oxygen and cis-trans isomerization of carotenoids. In:
Manson, D. G., and Given, N. K., Expression of Canfield, L. M., Krinsky, N. I., and Dunastable, J. A.,
an Antirrhinum majus UDP-glucose:flavonoid-3- Eds., Carotenoids in Human Health, Annals of the
O-glucosyltransferase transgene alters flavonoid New York Academy of Sciences, New York, Vol.
glycosylation and acylation in lisianthus (Eustoma 691, 1993, 10–19.
grandiflorum Grise.), Plant Sci., 1997; 125: 53– 448. Stahl, W., Nicolai, S., Briviba, K., Hanusch, M.,
61. Broszeit, G., Peters, M., Martin, H. D., and Sies, H.,
434. Schwitzguébel, J. P., Zrÿd, J. P., and Leathers, R., Biological activities of natural and synthetic caro-
From plant cells to biotechnology, Swiss Biotechnol., tenoids: induction of gap junctional communication
1991; 9(1): 17–24 and singlet oxygen quenching, Carcinogenesis, 1997;
435. Seibert, M., Picorel, R., Kim, J. H., and Cotton, T. M., 181): 89–92.
Surface enhanced Raman scattering of photosynthetic 449. Steglich, W. and Strack, D., Betalains. In: Brossi, A.,
membranes and complexes, Method. Enzymol., 1992; Ed., The Alkaloids. Vol. 39,Academic Press, Orlando,
213(part A): 31–42. 1991, 1–62.
436. Shapira, M., Lers, A., Heifetz, P. B., Irihimovitz, V., 450. Stevens, J. F., Hart, H. T., and Wollenweber, E., The
Osmond, C. B., Gillham, N. W., and Boynton, J. E., systematic and evolutionary significance of exudate
Differential regulation of chloroplast gene expression flavonoids in Aeonium, Phytochemistry, 1995; 394):
in Chlamydomonas reinhardtii during 805–813.
photoacclimation: light stress transiently suppresses 451. Strack, D., Steglich, W., and Wray, V., Betalains. In:
synthesis of the Rubisco LSU protein while enhanc- Methods in Plant Biochemistry Vol. 8, Academic
ing synthesis of the PSII D1 protein, Plant Mol. Biol., Press, Orlando, 1993, 421–450.
1997; 33: 1001–1011. 452. Stuppner, H. and Egger, R., Application of capillary
437. Shih, C. C. and Wiley, R. C., Betacyanine and zone electrophoresis to the analysis of betalains from
betaxanthine decolorizing enzymes in the beet (Beta Beta vulgaris, J. Chromatogr. A., 1996; 735: 409–
vulgaris L.) root, J. Food Sci., 1981; 47: 164–166,172 413.
438. Siefer, E., Rettura, G., and Levenson, S. H., Caro- 453. Sun, N. K., Huang, S. L., Lin-Chao, S., and Chao, C.
tenoids and cell-mediated immune responses. In: C. K., Induction, not associated with host-cell re-
Charalambous, G. and Inglett, G., Eds., The Quality of activation of damaged plasmid. DNA, of damaged-
Foods and Beverages. Vol. 2: Chemistry and Tech- DNA-recognition proteins by retinoic acid and
nology, Academic Press, New York, 1981, 335–347. dibutyryl cyclic AMP in mammalian cells, Biochem.
439. Simon, P., Drdák, M., and Altamirano, R., Influence J., 1996; B13: 441–445.
of water activity on the stability of betanin in various 454. Suvarnalatha, G., Rajendran, L., Ravishankar, G. A.,
water/alcohol model systems, Food Chem., 1993; 46: and Venkataraman, L. V., Elicitation of anthocyanin
155–158. production in cell cultures of carrot (Daucus carota
440. Singer, J. W. and von Elbe, J. H., Degradation rates of L.) by using elicitors and abiotic stress, Biotechnol.
vulgaxanthine-I, J. Food Sci., 1980; 45: 489–491 Lett., 1994; 16: 1275–1280.
441. Singh, G., Reactor design for plant cell culture of food 455. Swain, T. and Bate-Smith, E. C., Flavonoid Com-
ingredients and additives, Food Technol., 1997; 51: pounds. In: Florkin, M. and Mason, H. S., Eds., Com-
62–66. parative Biochemistry Vol. III: Constituents of Life.
442. Slivarts, M., Borochov, A., and Weiss, D., Low tem- Part A, Academic Press, New York, 1962, 755–809.
perature enhances petunia flower pigmentation and 456. Takano, Y., Kubo, Y., Kawamura, C., Tsuge, T., and
induces chalcone synthase gene expression, Physiol. Furusawa, I., The Alternaria alternata melanin bio-
Plant., 1997; 99: 67–72. synthesis gene restores appresorial melanization and
443. Snyder, B. A. and Nicholson, R. L., Synthesis of penetration of cellulose membranes in the melanin-
phytoalexins in sorghum as a site-specific response to deficient albino mutant of Colletotrichum lagenarium,
fungal ingress, Science, 1990; 248: 1637–1639. Fungal Genet. Biol., 1997; 21: 131–140.
444. Soleas, G. J., Dam, J., Carey, M., and Goldberg, D. 457. Tanaka, T., Urade, Y., Kimura, H., Eguchi, N.,
M., Toward the fingerprinting of wines: cultivar-re- Nishikawa, A., and Hayaishi, O., Lipocalin-type pros-
lated patterns of polyphenolic constituents in Ontario taglandin D synthase β-trace) is a newly recognized
wines, J. Agric. Food Chem., 1997; 45(10): 3871– type of retinoid transporter, J. Biol. Chem., 1997;
3880. 272(25): 15789–15795.
286
458. Tatsuzawa, F., Saito, N., Yokoi, M., Shigihara, A., 472. Trezzini, G. F. and Zrÿd, J. P., Characterization of
and Honda, T., Acylated cyanidin 3,7,3'-triglucosides some natural and semi-synthetic betaxanthins, Phy-
in flowers of × Laeliocattley a cv. mini purple and its tochemistry, 1991; 30(6): 1901–1903.
relatives, Phytochemistry, 1996; 41: 635–642. 473. Trezzini, G. F. and Zrÿd, J. P., Two betalains from
459. Taya, M., Mine, K., Kino-Oka, M., Tone, S., and Ichi, Portulaca grandiflora, Phytochemistry, 1991; 30(6):
T., Production and release of pigments by culture of 1897–1899
transformed hairy root of red beet, J. Ferm. Bioeng., 474. Tsimidou, M. and Biliaderis, C. G., Kinetic studies of
1992; 73(1): 31–36 saffron (Crocus sativus L.) Quality deterioration, J.
460. Taylor-Mayne, S., Beta-carotene, carotenoids, and dis- Agric. Food Chem., 1997; 458): 2890–2898.
ease prevention in humans, FASEB J., 1996; 10: 609– 475. Tsimidou, M. and Tsatsaroni, E., Stability of saffron
701. pigments in aqueous extracts, J. Food Sci., 1993;
461. Terahara, N., Callebaut, A., Ohba, R., Nagata, T., 58(5): 1073–1075.
Ohnishi-Kameyama, M., and Suzuki, M., Triacylated 476. Tsuda, T., Shiga, K., Ohshima, K., Kawakishi, S.,
anthocyanins from Ajuga reptans flowers and cell and Osawa, T., Inhibition of lipid peroxidatin and
cultures, Phytochemistry, 1996; 42: 199–203. the active oxygen radical scavenging effect of an-
462. Terahara, N., Oda, M., Matsui, T., Osajima, Y., thocyanin pigments isolated from Phaseolus vul-
Saito, N., Toki, K, and Honda, T., Five new antho- garis L., Biochem. Pharmacol., 1996; 52: 1033–
cyanins, ternatins, A3, B4, B3, B2, and D2, from 1039.
Clitoria ternata flowers, J. Nat. Prod., 1996; 59: 477. Tsushima, M., Fujiwara, Y., and Matsuno, T., Novel
139–144. marine di-Z-carotenoids: cucumariaxanthins A, B and
463. Terradas, F. and Willer, H., 2,3- and 4,5-secodopa, C from the sea cucumber Cucumaria japonica, J. Nat.
the biosynthesis intermediates generated from L-DOPA Prod., 1996; 59: 30–34.
by an enzyme system extracted from the fly agaric, 478. Tyczkowski, J. K. and Hamilton, P. B., Research
Amanita muscaria L., and their spontaneous conver- note: preparation of purified lutein and its diesters
sion to muscaflavin and betalamic acid, respectively, from extracts of marigold (Tagetes erecta), Poultry
and betalains, Helv. Chim. Acta, 1991; 74: 124–140. Sci., 1991; 70: 651–654.
464. Thomson, R. H., Quinones: structure and distribution. 479. Unten, L., Koketsu, M., and Kim, M., Antidiscoloring
In: Florkin, M. and Mason, H. S., Eds., Comparative activity of green tea polyphenols on β-carotene, J.
Biochemistry Vol. III: Constituents of Life. Part A, Agric. Food Chem., 1997; 456): 2009–2012.
Academic Press, New York, 1962, 631–725. 480. van Breemen, R. B., Innovations in carotenoid analy-
465. Thomson, R. H., Melanins. In: Florkin, M. and Ma- sis using LC/MS, Anal. Chem., 1996; 68(9): 299A-
son, H. S., Eds., Comparative Biochemistry Vol. III: 304A.
Constituents of Life. Part A, Academic Press, New 481. van der Biezen, E. A., Brandwagt, B. F., van Leeuwen,
York, 1962, 727–753. W., Nijkamp, H. J. J., and Hille, J., Identification and
466. Tobe, H., Komiyama, O., Komiyama, Y., and isolation of the FEEBLY gene from tomato by
Maruyama, H. B., Daidzein stimulation of bone re- transposon tagging, Mol. Gen. Genet., 1996; 251: 267–
sorption in pit formation assay, Biosci. Biotechnol. 280.
Biochem., 1997; 61(2): 370–371. 482. van der Krol, A. R., Lenting, P. E., Veenstra, J., van
467. Todd, J. J. and Vodkin, L. O., Duplications that sup- der Meer, I. M., Koes, R. E., Gerats, A. G. M., Mol,
press and deletions that restore expression from chal- J. N. M., and Stuitje, A. R., An anti-sense chalcone
cone synthase multigene family, Plant Cell, 1996; 8: synthase gene in transgenic plants inhibits flower pig-
687–699. mentation, Nature, 1988; 333: 866–869.
468. Toguri, T., Umemoto, N., Kobayashi, O., and Ohtani, 483. van Eldik, G. J., Ruiter, R. K., Colla, P. H. W. N., van
T., Activation of anthocyanin synthesis genes by white Herpen, M. M. A., Schrauwen, J. A. M., and Wullems
light in eggplant hypocotyl tissues, and identification G. J., Expression of an isoflavone reductase-like gene
of an inducible P-450 cDNA, Plant Mol. Biol., 1993; enhanced by pollen tube growth in pistils of Solanum
23: 933–946. tuberosum, Plant Mol. Biol., 1997; 33: 923–929.
469. Toki, K., Saito, N., Kuwano, H., Terahara, N., and 484. van Gadow, A., Joubert, E., and Hansmann, C. F.,
Honda, T., Acylated anthocyanins in verbena flowers, Comparison of the antioxidant activity of aspalathin
Phytochemistry, 1995; 38: 515–518. with that of other plant phenols of rooibos tea
470. Tomás-Barberan, F.A., Tomás-Lorente, F., Ferreres, Aspalathus linearis), α-tocopherol, BHT, and BHA,
F., and García-Viguera, C., Flavonoids as biochemi- J. Agric. Food Chem., 1997; 45(3): 632–638.
cal markers of the plant origin of bee pollen, J. Sci. 485. van Gorsel, H., Li, C., Kerbel, E. L., Smits, M., and
Food Agric., 1989; 47(3): 337–340. Kader, A. A., Compositional characterization of prune
471. Trebst, A. and Depka, B., Role of carotene in the rapid juice, J. Agric. Food Chem., 1992; 40: 784–789.
turnover and assembly of photosystem II in Chlamy- 486. Vernon-Carter, E. J., Gómez, S. A., Beristaín, C. I.,
domonas reinhardtii, FEBS Lett., 1997; 400: 359– Mosqueira, G., Pedroza-Islas, R., and Moreno-
362. Terrazas, R. C., Color degradation and coalescence
287
kinetics of Aztec marigold oleoresin-in-water emul- 499. Wang, H., Cao, G., and Prior, R. L., Oxygen radical
sions stabilized by mesquite or arabic gums and their absorbing capacity of anthocyanins, J. Agric. Food
blends, J. Text. Stud., 1996; 27: 625–641. Chem., 1997; 45: 304–309.
487. Vernon-Carter, E. J., Ponce-Palafox, J. T., and Pedroza- 500. Watson, J. F. and Goldman, I. L., Inheritance of a
Islas, R. Pigmentation of Pacific white shrimp Penaeus gene conditioning blotchy root color in table beet
vannamei) using Aztec marigold (Tagetes erecta) ex- (Beta vulgaris L.), J. Heredity, 1997; 886. : 540–543.
tracts as the carotenoid source, Arch. Lat. Nutr., 1996; 501. Wegener, C., Bartling, S., Olsen, O., Weber, J., and
46(3): 243–246. von Wettstein, D., Pectate lyase in transgenic potatoes
488. Vicent, K. R. and Scholz, R. G., Separation and quan- confers preactivation of defense against Erwinia
tification of red beet betacyanins and betaxanthins by carotovora, Physiol. Mol. Plant Pathol., 1996; 49:
high-performance liquid chromatography, J. Agric. 359–376.
Food Chem., 1978; 264): 812–816. 502. Weiss, D., van Blokland, R., Kooter, J. M., Mol, J. N.
489. Vinha, C. A. and Hass, U., Qualitative and M., and van Tunen, A. J., Gibberellic acid regulates
semiquantitative analysis of dried fruits and season- chalcone synthase gene transcription in the corolla of
ing products of paprika using photoacoustic spectros- Petunia hybrida, Plant Physiol., 1991; 98: 191–197.
copy, J. Agric. Food Chem., 1997; 45(4): 1273–1277. 503. Weiss, M. R., Floral color change: a widespread func-
490. Vishnevetsky, M., Oradis, M., Itzhaki, H., Levy, M., tional convergence, Am. J. Bot., 1995; 82(2): 167–
Libal-Weksler, Y., Adam, Z., and Vainstein, A., 185.
Molecular cloning of a carotenoid associated protein 504. Wiley, R. C. and Lee, Y. N., Recovery of betalaines
from Cucumis sativus corollas: homologous genes from red beets by a diffusion-extraction procedure, J.
involved in carotenoid sequestration in chromoplasts, Food Sci., 1978; 43: 1056–1058.
Plant J., 1996; 10(6): 1111–1118. 505. Wiley, R. C., Lee, Y. N., Saladini, J. J., Wyss, R. C.,
491. Vishnevetsky, M., Ovadis, M., Itzhaki, H., and and Topalian, H. H., Efficiency studies of a continu-
Vainstein, A., CHRC, encoding a chromoplast-spe- ous diffusion apparatus for the recovery of betalaines
cific carotenoid-associated protein, is an early gibber- from red table beet, J. Food Sci., 1979; 44(1): 208–
ellic acid-responsive gene, J. Biol. Chem., 1997; 211.
272(40): 24747–24750. 506. Williams, C. A., Goldstone, F., and Greenham, J.,
492. Vogelien, D. L., Hrazdina, G., Reeves, S., and Dougall, Flavonoids, cinnamic acids and coumarins from the
D. K., Phenotypic differences in anthocyanin accu- different tissues and medicinal preparations of
mulation among clonally related cultured cells of car- Taraxcum officinale, Phytochemistry, 1996; 42(1):
rot, Plant Cell Tiss. Org. Cult., 1990; 22: 213–222. 121–127.
493. Vogt, T., Zimmermann, E., Grimm, R., Meyer, M., 507. Wissgot, U. and Bortlik, K., Prospects for new food
and Strack, K., Are the characteristics of betanidin colorants, Trends Food Sci. Technol., 1996; 7: 298–
glucosyltransferases from cell-suspension cultures 302.
of Dorotheanthus bellidiformis indicative of their 508. Wodicka, V. O., Regulation of food: where have we
phylogenetic relationship with flavonoid been?, Food Technol., 1996; 50(3): 106–109.
glucosyltransferases, Planta, 1997; 203(3): 349– 509. Wollenweber, E., Occurrence of flavonoid aglycones
361. in medicinal plants. In: Cody, V., Middleton E., Jr.,
494. von Elbe, J. H., Influence of water activity on pigment Harborne, J. B., and Beretz, A., Eds., Progress in
stability in food products. In: Rockland, L. B. and Chemical and Biological Research Vol. 280. Alan R.
Belichat, L. R., Eds., Water Activity: Theory and Liss Inc., New York, 1988, 45–55.
Applications to Food, IFT Basic Sym. Ser. Marcel 510. Wong, D. W. S., Colorants. In: Mechanism and Theory
Dekker, New York, 1987, 55–83 in Food Chemistry, AVI, Westport, CT, 1989, 147–
495. von Elbe, J. H., Maing, I., and Amundson, C. H., 187.
Color stability of betanin, J. Food Sci., 1974; 39: 511. Wrolstad, R. E., Skrede, G., Lea, P., and Enersen, G.,
334–337. Influence of sugar on anthocyanin pigment stability in
496. von Elbe, J. H., Sy, S. H., Maing, I. Y., and Gabelman, frozen strawberries, J. Food Sci., 1990; 55: 1064–
W. H., Quantitative analysis of betacyanins in red 1065, 1072.
table beets (Beta vulgaris), J. Food Sci., 1972; 37: 512. Yadav, S. K. and Sehgal, S., Effect of home process-
932–934. ing and storage on ascorbic acid and β-carotene con-
497. von Elbe, J. H., The betalaines. In: Furia, E. T., Ed., tent of bathua (Chenopodium album) and fenugreek
Current Aspects of Food Colorants, CRC Press Inc., (Trigonella foenum graecum) leaves, Plant Foods
Boca Raton, 1977, 29–39. Hum. Nutr., 1997; 50: 239–247.
498. von Lintig, J., Welsch, R., Bonk, M., Giuliano, G., 513. Yamaguchi, M. A., Maki, T., Ohishi, T., and Ino, I.,
Batschauer, A., and Kleinig, H., Light-dependent regu- Succinyl-coenzyme A: anthocyanidin 3-glucoside
lation of carotenoid biosynthesis occurs at the level of succinyltransferase in flowers of Centaurea cyanus,
phytoene synthase expression and is mediated by 1995; Phytochemistry, 39: 311–313.
phytochrome in Sinapis alba and Arabidopsis thaliana 514. Yamakawa, T., Ohssuka, H., Onomichi, K., Kodama,
seedlings, Plant J., 1997; 12(3): 625–634. T., and Minoda, Y. Production of anthocyanin pig-
288
ments by grape cell culture. In: Proc. 5th. Int. Congr. 523. Zhang, D. and Quantick, P. C., Effects of chitosan
Plant Tissue and Cell Culture, Japan: Japanese Asso- coating on enzymatic browning and decay during
ciation for Plant Tissue Culture, 1982. postharvest storage of litchi (Litchi chinensis Sonn.)
515. Yamamoto, H. and Oritani, T., Isomeric ratio and fruit, Postharvest Biol. Technol., 1997; 12: 195–
level of xanthoxin in tomato plants measured by a 202.
new analytical method, Biosci. Biotechnol. Biochem., 524. Zhang, W., Seki, M., and Furusaki, S., Effect of tem-
1997; 61(7): 1142–1145. perature and its shift on growth and anthocyanin pro-
516. Yamamoto, Y. and Mizuguchi, R., Selection of a high duction in suspension cultures of strawberry cells,
stable pigment-producing strain in cultured Euphorbia Plant Sci., 1997; 127: 207–214.
millii cells, Theor. Appl. Genet., 1982; 61: 113–116. 525. Zhang, W. and Furusaki, S., Regulation of anthocya-
517. Yamane, Y., Higashida, K., Nakashimada, Y., nin synthesis in suspension cultures of strawberry cell
Kakizono, T., and Nishio, N., Astaxanthin production by pH, Biotechnol. Lett., 1997; 19: 1057–1061.
by Phaffia rhodozyma enhanced in fed-batch culture 526. Zhong, J. J., Seki, T., Kinoshita, S. I., and Yoshida, T.,
with glucose and ethanol feeding, Biotechnol. Lett., Effect of light irradiation on Anthocyanin production
1997; 19(11): 1109–1111. by suspended culture of Perilla frutescens, Biotechnol.
518. Yamane, Y. I., Higashida, K., Nakashimada, Y., Bioeng., 1991; 38: 653–658.
Kakizono, T., and Nishio, N., Influence of oxygen and 527. Zhu, B. T. and Liehr, J. G., Inhibition of catechol O-
glucose on primary metabolism and astaxanthin pro- methyltransferase-catalyzed O-methylation of 2- and
duction by Phaffia rhodozyma in batch and fed-batch 4–hydroxyestradiol by quercetin, J. Biol. Chem., 1996;
cultures: kinetic and stoichiometric analysis, Appl. 271(3): 1357–1363.
Environ. Microbiol., 1997; 63(11): 4471–4478. 528. Zhu, X., Suzuki, K., Saito, T., Okada, K., Tanaka, K.,
519. Yamauchi, R., Miyake, N., Inoue, H., and Kato, K., Nakagawa, T., Matsuda, H., and Kawamukai, M.,
Products formed by peroxyl radical oxidation of ß- Geranylgeranyl pyrophosphate synthase encoded by
carotene, J. Agric. Food Chem., 1993; 41(5): 708–713. the newly isolated gene GGPPS6 from Arabidopsis
520. Yuan, J. P., Gong, X. D., and Chen, F., Separation and thaliana is localized in mithocondria, Plant Mol. Biol.,
analysis of carotenoids and chlorophylls in 1997; 35: 331–341.
Haematococcus lacustris by high-performance liquid 529. Ziegler, R. G., Carotenoids, cancer, and clinical trials.
chromatography photodiode array detection, J. Agric. In: Canfield, L. M., Krinsky, N. I., and Dunastable,
Food Chem., 1997; 45(5): 1952–1956. J.A., Eds., Carotenoids in Human Health, Vol. 691, ,
521. Zakharova, N. S., Petrova, T. A., Shcherbukhin, V. Annals of the New York Academy of Sciences, New
D., and Gins, V. K., Betacyanin and betalain oxidase York 1993, 110–119.
in different Amaranthus species, Appl. Biochem. 530. Zwayyed, S. K., Frazier, G. C., and Dougall, D. K.,
Microbiol., 1995; 31(2): 202–205. Growth and anthocyanin accumulation in carrot cell
522. Zallocchi, E. M. and Pomilio, A. B., Evolution of suspension cultures growing on fructose, glucose,
flavonoids in the Phaseolinae, Phytochemistry, 1994; or their mixtures, Biotechnol. Prog., 1991; 7: 288–
37(2): 449–453. 290.
289
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Natural Pigments: Carotenoids, Anthocyanins, and Betalains —

Characteristics, Biosynthesis, Processing, and Stability
F. Delgado-Vargas a; A. R. Jiménez b; O. Paredes-López c
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Natural Pigments: Carotenoids, Anthocyanins, and Betalains — Characteristics,

Article in Critical Reviews in Food Science and Nutrition · June 2000

Francisco Delgado-Vargas Antonio Ruperto Jiménez-Aparicio

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Natural Pigments: Carotenoids, Anthocyanins, and

II. PIGMENTS IN GENERAL ..............................................................................................................176

III. NATURAL PIGMENTS ................................................................................................................177

IV. SOME IMPORTANT PLANT PIGMENTS: CAROTENOIDS,

a. Higher Plants .............................................................................................................197

V. FUTURE TRENDS .........................................................................................................................270

KEY WORDS: color, carotene, xanthophyll, betacyanin, betaxanthin, food.

synthesis of flavonoids; the quinones are very impor-

phenoxazines, and betalains. prochloron present only two chlorophylls (“a”

Isoprenoids, also called terpenoids, repre-

3. N-Heterocyclic Compounds Different b. Pterins

FIGURE 2. N-heterocyclic compounds.

Flavones and flavanones are found free

FIGURE 3. Benzopyran derivatives.

are found in plants: plastoquinones are found in

ity, possibly by the inhibition of uncoating. Fla-

Rutin, silibin Against disorders of the respiratory system

Adapted from Refs. 10, 198, 509.

enzymes have a quinone as cofactor. Methanol

1. Reasons to Use Color Additives

additives, and this attitude was converted in a

Color CEE FDA

Annato extract Yes Yes

indirect additives to group those additives used in

FIGURE 6. Carotenoid structure. (Adapted from Ref. 179.)

FIGURE 7. Structures of common carotenoids.

class; the exceptions are Chlorophyta caro-

Enzyme that is coded by the geneb

a Adapted from Refs. 422, 498.

of the PDS gene that shows a cis-activation of the

mals that disperse pollen, seeds, or spores. In

activity was reduced in aged rats and recovered

chromatography in alumina followed by thin layer speed of analysis; additionally, it is possible to

cially canned. Fresh and frozen fruits showed

Substituted with a characteristic hydroxyl group

Namea Position of substitution Some of the produced colors

Apigeninidin 5,7,4’ Orange

Substituted with a characteristic methyl ether group

Capensinidin 5,3’,5’ Bluish red

Europenidin 5,3’ Bluish red

a Underlined names represent the most common anthocyanidins.

characterized, but involves an oxidation and a and pelargonidin, but not to

densed tannins (CT) in alfalfa has supported a

has been considered as important agents in reduc-

cyanin acylation is also observed by this method-

Reaction conditions Structural characteristic Observed characteristic

AlCl3 (5% in methanol) Flavonoids with o-hydroxyl Bathochromic change of

maticity coordinates give an appreciation of

Plant model Remarks Ref.

Verbena flowers First report of a diacylated anthocyanin: 469

Blue flowers of Identification of anthocyanins with a cis-p-coumaric 222

Geranium flowers First structure elucidation of a monoacetylated 7

Gentiana flowers Identification of anthocyanins in which the 5-O- 224

Stem of Allium First report on acylation of the 3”-position in the sugar 8

Water lily First report involving a diacylation, which involves 154

Model Conditions Effects Ref.