natural pigments carotenoids, anthocyanins, and betalains

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173 1040-8398/00/$.50 © 2000 by CRC Press LLC 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-López 3* 1 Fac. de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sin.; 2 Centro de Productos Bióticos del Instituto Politécnico Nacional, Yautepec, Mor.; 3 Centro 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 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. Downloaded By: [Cinvestav del Ipn] At: 15:20 24 November 2009

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1040-8398/00/$.50© 2000 by CRC Press LLC

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

Natural Pigments: Carotenoids, Anthocyanins, andBetalains — 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 ProductosBióticos del Instituto Politécnico Nacional, Yautepec, Mor.; 3Centro de Investigación y de Estudios Avanzadosdel 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

II. PIGMENTS IN GENERAL ..............................................................................................................176A. Distribution ................................................................................................................................ 176B. Classification ..............................................................................................................................176

1. By Their Origin .....................................................................................................................1762. By the Chemcial Structure of the Chromophore ...................................................................1763. By the Structural Characteristics of The Natural Pigments .................................................1764. As Food Additives .................................................................................................................177

III. NATURAL PIGMENTS ................................................................................................................177A. Distribution ..............................................................................................................................177

1. Tetrapyrrole Derivatives .......................................................................................................1772. Isoprenoid Derivatives .........................................................................................................1773. N-Heterocyclic Compounds Different from Tetrapyrroles ..................................................178

a. Purines .....................................................................................................................178b. Pterins .....................................................................................................................178c. Flavins ......................................................................................................................178d. Phenazins .................................................................................................................180e. Phenoxazines ...........................................................................................................180f. Betalains ..................................................................................................................180

4. Benzopyran Derivatives .......................................................................................................1805. Quinones ..............................................................................................................................1826. Melanins ...............................................................................................................................182

B. Functions ..................................................................................................................................1821. Tetrapyrrole Derivatives .......................................................................................................1822. N-Heterocyclic Compounds Different from Tetrapyrroles ..................................................1853. Benzopyran Derivatives .........................................................................................................185

a. Antioxidant ..............................................................................................................185b. Color and Sexual Processes in Plants .....................................................................186c. Photoprotection ..........................................................................................................186

*Corresponding author.

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d. Defense Mechanism in plants ...................................................................................187e. Other Ecological Functions .....................................................................................188f. Pharmacological Effects .............................................................................................189

4. Quinones ................................................................................................................................1925. Iridoids ...................................................................................................................................1936. Melanins ..............................................................................................................................193

C. Importance As Food Colorants ................................................................................................1931. Reasons to Use Color Additives .........................................................................................1932. 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 ...............................................................................................................................1971. Definition ...............................................................................................................................1972. Classification ..........................................................................................................................1973. Distribution ...........................................................................................................................197

a. Higher Plants .............................................................................................................197b. Algae .........................................................................................................................200c. Bacteria .....................................................................................................................200d. Fungi ........................................................................................................................200

4. Biosynthesis: Biochemistry and Molecular Biology ............................................................200a. Biochemistry .............................................................................................................200b. Biosynthesis Regulation .............................................................................................205c. Molecular Biology of Carotenogenesis .........................................................................207

5. Functions ................................................................................................................................214a. Color ........................................................................................................................214b. Photosynthesis ...........................................................................................................214c. Xanthophyll Cycle ...................................................................................................214d. Antioxidant ..............................................................................................................214e. Pharmacological Effects .........................................................................................217

6. Methodological Aspects ......................................................................................................220a. Extraction ................................................................................................................221b. Saponification .........................................................................................................222c. Separation ...............................................................................................................222d. Charaterization ........................................................................................................224

7. Importance as Food Colors — Stability, Processing, andProduction ...............................................................................................................................................225

a. Stability in Model Systems ......................................................................................225b. Processing and Stability in Foods ............................................................................227c. Production of Carotenoids in Bioreactors ................................................................230

B. Anthocyanins .............................................................................................................................2311. Definition ...............................................................................................................................2312. Classification .........................................................................................................................2313. Distribution ...........................................................................................................................2314. Biosynthesis: Biochemistry and Molecular Biology ...........................................................231

a. Biochemistry ............................................................................................................231b. Biosynthesis Regulation ............................................................................................236c. Molecular Biology of Anthocyanin Biosynthesis ....................................................237

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5. Functions ...............................................................................................................................240a. Color and Ecological Functioins ............................................................................240b. Marker for Good Manufacturing Practices in Food ................................................240c. Pharmacological Effects ..........................................................................................240

6. Methodological Aspects ........................................................................................................241a. Extraction .................................................................................................................241b. Separation ................................................................................................................242c. Characterization .......................................................................................................242

7. Importance as Food Colors — Stability, Processing, and Production ................................244a. Stability in Model Systems .......................................................................................244b. Processing and Stability in Foods ..........................................................................247c. Production of Anthocyanins by Plant Tissue Culture...............................................249

C. Betalains ..................................................................................................................................2511. Definition ................................................................................................................................2512. Classification .........................................................................................................................2533. Distribution ...........................................................................................................................2534. Biosynthesis: Biochemistry and Molecular Biology ............................................................254

a. Biochemistry .............................................................................................................254b. Biosynthesis Regulation ...........................................................................................259c. Molecular Biology of Betalain Biosynthesis .................................................................259

5. Functions ...............................................................................................................................260a. Taxonomic Markers................... .............................................................................260b. Ecological and Physiological Aspects ...................................................................260c. Pharmacological Effects ..........................................................................................261

6. Methodological Aspects .......................................................................................................261a. Extraction .................................................................................................................261b. Separation .................................................................................................................262c. Characterization ........................................................................................................263

7. Importance as Food Colors — Stability, Processing, and Production ...............................266a. Stability in Model Systems .....................................................................................266b. Processing and Stability in Foods ..........................................................................267c. Production of Betalains by Plant Tissue Culture ...................................................269

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

ACKNOWLEDGMENTS ......................................................................................................................271REFERENCES .......................................................................................................................................271

ABSTRACT: Pigments are present in all living matter and provide attractive colors and play basic roles in thedevelopment of organisms. Human beings, like most animals, come in contact with their surroundings throughcolor, and things can or cannot be acceptable based on their color characteristics. This review presents the basicinformation 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 theirbiosynthesis, 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.

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I. INTRODUCTION

Pigments produce the colors that we observe ateach step of our lives, because pigments are presentin each one of the organisms in the world, and plantsare the principal producers. They are in leaves, fruits,vegetables, and flowers; also, they are present in skin,eyes, and other animal structures; and in bacteria andfungi. Natural and synthetic pigments are used inmedicines, foods, clothes, furniture, cosmetics, and inother products. However, natural pigments have im-portant functions other than the imparted beauty, suchas the following: we could not have photosynthesis orprobably life all over the world without chlorophyllsand carotenoids. In animals how could oxygen andcarbon dioxide be transported without hemoglobin ormyoglobin? Under stress conditions plants show thesynthesis 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 andother vertebrates, and in some fungi melanins areessential for their vital cycle; last but not least, a lot ofpigments have a well-known pharmacological activ-ity in sickness such as cancer and cardiovasculardiseases, and this has stressed pigment importance forhuman beings.162,196,259,327

Additionally, since time immemorial humanbeings have associated product qualities with theircolors, this is especially true for meals. Historically,at the beginning of the food industry consumers didnot take care about the kind of pigments used in foodcoloring (natural or synthetic), but recently peoplehave shown their phobia to synthetic pigments whenthe concepts “synthetic pigments” and “illness” wereassociated, and when the attributed pharmacologicalbenefits of natural pigments came into consider-ation. However, the natural pigments that are per-mitted for human foods are very limited, and theapproval of new sources is difficult because the U.S.Food and Drug Administration (FDA) considers thepigments as additives, and consequently pigmentsare under strict regulations.80,144,507,508

Thus, an adequate understanding of the actualsources of pigments will contribute to their better use.In this review we present the basic information aboutpigments focusing our attention on the natural pig-ments. At this time, it must be emphasized the ubiq-uity of pigments in living organisms (plants, fungi,bacteria, among others), the variety of chemical struc-tures, and the large quantity of information generated

for each pigment group. Consequently, it is not ourmajor goal to present a thorough review of all pig-ment groups. This would be impossible in a singledocument, and although we present here a globaloverview about natural pigments our work focuses onthe more representative pigments in the main naturalproducers, namely, plants, which in addition are themore highly consumed as food products. In the fol-lowing pages, we emphasize their salient characteris-tics; their biosynthesis, taking into account the bio-chemical and molecular biology information generatedfor their elucidation, and the processing and stabilityproperties of these pigments as food colorants.

II. PIGMENTS IN GENERAL

A. Definition

Pigments are chemical compounds that ab-sorb light in the wavelength range of the visibleregion. Produced color is due to a molecule-spe-cific structure (chromophore); this structure cap-tures the energy and the excitation of an electronfrom an external orbital to a higher orbital isproduced; the nonabsorbed energy is reflectedand/or refracted to be captured by the eye, andgenerated neural impulses are transmitted to thebrain where they could be interpreted as a color.196

B. Classification

1. By Their Origin

Pigments can be classified by their origin asnatural, synthetic, or inorganic. Natural pigments areproduced by living organisms such as plants, animals,fungi, and microorganisms. Synthetic pigments areobtained from laboratories. Natural and synthetic pig-ments are organic compounds. Inorganic pigmentscan be found in nature or reproduced by synthesis.29

2. By the Chemical Structure of theChromophore

Also, pigments can be classified by takinginto account the chromophore chemical structureas:510

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Chromophores with conjugated systems: caro-tenoids, anthocyanins, betalains, caramel, syn-thetic pigments, and lakes.

Metal-coordinated porphyrins: myoglobin,chlorophyll, and their derivatives.

3. By the Structural Characteristics ofthe Natural Pigments

Moreover, natural pigments can be classifiedby their structural characteristics as:29,196

Tetrapyrrole derivatives: chlorophylls and hemecolors.Isoprenoid derivatives: carotenoids and iridoids.N-heterocyclic compounds different from tetrapy-rroles: purines, pterins, flavins, phenazines,phenoxazines, and betalains.Benzopyran derivatives (oxygenated heterocycliccompounds): anthocyanins and other flavonoidpigments.Quinones: benzoquinone, naphthoquinone, an-thraquinone.Melanins.

4. As Food Additives

By considering the pigments as food addi-tives, their classification by the FDA is150,162,510

Certifiable. These are manmade and subdi-vided as synthetic pigments and lakes.

Exempt from certification. This group in-cludes pigments derived from natural sources suchas vegetables, minerals, or animals, and man-made counterparts of natural derivatives.

III. NATURAL PIGMENTS

A. Distribution

1. Tetrapyrrole Derivatives

These compounds have a structure with pyr-role rings in linear or cyclic arrays. Figure 1shows some common structures of tetrapyrrolederivatives. Phytochrome is very common in al-gae (Rhodophyta, Cryptophyta), and bilin is this

basic structure (lineal array). In the cyclic com-pounds, we can mention the heme group (theporphyrin ring is bonded to an iron atom); thisgroup is present in hemoglobin and myoglobin,present in animals, and also in cytochromes,peroxidases, catalases, and vitamin B12 as a pros-thetic group, all of them with a wide distribu-tion. However, chlorophylls constitute the mostimportant subgroup of pigments within the tet-rapyrrole derivatives. Chlorophyll is mainlypresent in the chloroplasts of higher plants andmost algae. The number of known chlorophyllstructures have shown increments through theyears: up until 1960 only three structures hadbeen described, 20 up until 1970, and more than100 up until 1980. Higher plants, ferns, mosses,green algae, and the prokaryotic organismprochloron present only two chlorophylls (“a”and “b”), and the rest of them have been foundin other groups such as algae and bacte-ria.60,87,196,279,404

2. Isoprenoid Derivatives

Isoprenoids, also called terpenoids, repre-sent a big family of natural compounds; they arefound in all kingdoms where they carry out mul-tiple functions (hormones, pigments, phytoalex-ins). Over 23,000 individual isoprenoid com-pounds have been identified and many newstructures are reported each year. By their abun-dance and structure, two subgroups of compoundsare considered pigments: quinones and caro-tenoids. However, in addition, and only recently,iridoids is a third group of plant isoprenoid com-pounds that have acquired some relevance.Quinones are considered another group becausenot all of them are produced by this biosyntheticpathway. On the other hand, carotenoids as amajor point of our review are described below.In relation to iridoids, these are found inabout 70 families (Capriofilaceae, Rubiaceae,Cornaceae, among others) grouped in some 13orders. Saffron (Crocus sativus L.) and capejasmine fruit (Gardenia jasminoids Ellis) are thebest-known iridoid-containing plants, but theircolors are more importantly influenced by caro-tenoids.406

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3. N-Heterocyclic Compounds Differentfrom Tetrapyrroles

Figure 2 shows some structures for compoundsof this group.

a. Purines

As most of the nucleotides, purines are foundin two macromolecules: deoxyribonucleic acid(DNA) and ribonucleic acid (RNA). These mol-ecules are an essential component of life, thusthey are present in each living organism. Freepurines have been found in animals (golden andsilvery fish).196

b. Pterins

The pteridin ring system is probably present inevery form of life. Most of the natural pteridins havean amino group at C-2 and an hydroxyl group at C-4. Also, 2,4-dihydroxipteridins have been describedas important components in the flavin biosynthesis.Pterins are responsible for color in some insects, invertebrate eyes, human urine, and bacteria (Lacto-bacillus casei and Streptomyces faecalis R.).152,196

c. Flavins

In these compounds a pteridin and a ben-zene ring are condensed. Riboflavin is the main

FIGURE 1. Basic structure of porphyrinic pigments (pyrrol) and of some porphyrinic pigments with biologicalimportance.

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FIGURE 2. N-heterocyclic compounds.

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compound of this group, and it is synthesized inall live cells of microorganisms and plants. Ri-boflavin is found in milk. Other sources are awide range of leafy vegetables, meat, andfish.87,152,196

d. Phenazines

They are found in bacteria.196

e. Phenoxazines

They are found in fungi and insects.196

f. Betalains

As a main component of our review, thissubgroup of compounds are discussed below.

4. Benzopyran Derivatives

The most studied secondary metabolites arethe flavonoids. These are phenolic compoundswith two aromatic rings bonded by a C3 unit(central pyran ring) and divided in 13 classesbased on the oxidation state of the pyran ringand on the characteristic color: anthocyanins,aurons, chalcones, yellow flavonols,flavones, uncolored flavonols, flavanones,dihydroflavonols, dihydrochalcones, leuco-anthocyanidins, catechins, flavans, andisoflavonoids. Figure 3 shows some flavonoidstructures. Each type of flavonoid can be modi-fied by hydroxylation, methylation, acylation,and glycosylation to obtain a great natural diver-sity of compounds. Flavonoids are water solubleand show a wide distribution in vascular plants.They are present in each part of the plant. Morethan 5000 flavonoids have been chemically char-acterized, and new structures are described con-tinuously.194,259

In the flavonoids, the anthocyanins are themost important pigments; they produce colorsfrom orange to blue in petals, fruits, leaves,and roots and are discussed below. Flavonoids

also contribute to the yellow color of flowers,where they are present with carotenoids oralone in 15% of the plant species.259

The aurones are present in flowers (Bidenssp., Cosmos sp., Dahlia sp.), wood (Rhussp., Schinopsis sp.), bryophytes (Funariahygrometrica), and in Cypreceae plants. Theyare more common in inflorescences, seeds, andleaves. The chalcones are common in mixtureswith aurones to generate the anthclor pigment inthe Compositae). In nature, it is uncommon tofind them without substitutions, but hydroxy-lated have been described in wood and peelsof trees in the genus Acacia, Rhus, Macherium,and Adenanthera. Flavonols are ubiquitous inwoody angiosperms, common in shrubby an-giosperms, and exceptionally in inferior plants.Flavones and flavanones are found freeor glycosylated in leaves of angiosperms.Flavanones are especially common inRosaceae, Rutaceae, legumes and Compositae.Dihydrochalcones are found mainly hydroxy-lated in apple and in some species of Rosaceae,Ericaceae, Fagaceae, legumes, and Salicaceae.Leucoanthocyanidins (flavan 3,4-diols) arewidely distributed in plants, and they have beenisolated from wood and peel of trees (particu-larly Acacia) and methylated, and C-alkylatedleucoanthocyanidins have been identified in avariety of sources (e.g., Neuroautaneniaamboensis, Marshallia sp.). Catechins flavan 3-ols) and flavans are found mainly in leaves. Cat-echins and epicatechins are among the common-est flavonoids known, sharing a distributionalmost as widespread as quercetin in theDicotyledoneae. The 3,4,5-trihydroxy B-ringflavan 3-ols gallocatechin and epigallocatechinhave also a wide distribution (parallelingmyricetin), especially in more primitive plants(the Coniferae being outstanding). Many flavansare lipid soluble and appear to be leaf-surfaceconstituents. Isoflavonoids have the B ring in C-3 instead of C-2, and many natural products arein this group: isoflavones, rotenoids, pterocarps,and coumestans. Isoflavones are the most com-mon in legumes, especially in Lotoideae, al-though they have also been reported inAmaranthaceae, Iridicaceae, Miristicaceae, andRosaceae.29,157,193,259

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5. Quinones

Quinones have a great number of coloringcompounds (Figure 4). This group is the biggestone in number and structural variation. Also, theyare more widely distributed than other naturalpigments (with the exception of carotenoids andmelanins). The basic structure consists of adesaturated cyclic ketone that is derived from anaromatic monocyclic or polycyclic compound.Quinones can be divided by their structure asbenzoquinones, naphthoquinones, anthraquinones,and miscellaneous quinones; moreover,dibenzoquinones, dianthraquinones, anddinaphthoquinones have been reported. The vari-ability in the kind and structure of substituentsconduce to large number of quinones. Quinonesare found in plants: plastoquinones are found inchloroplasts of higher plants and algae; ubiquino-nes are ubiquitous in living organisms;menaquinones are found in bacteria;naphthoquinones in animals; and anthraquinonesin fungi, lichens, flowering plants, and insects.Many quinones are byproducts of the metabolicpathways and a few organisms (fungi) producelarge quantities. In general, quinones produceyellow, red, or brown colorations, but quinonesalts show purple, blue, or green colors.196,464

6. Melanins

Melanins are nitrogenous polymeric compoundswhose monomer is the indole ring (Figure 5). Ingeneral, melanins are not homopolymers but presenta mixture of macromolecules. Melanins are respon-sible of many of the black, gray, and brown colora-tions of animals, plants, and microorganisms.Eumelanins are widely distributed in vertebrate andinvertebrate animals. Phaemelanins are macromol-ecules of mammals and birds. Allomelanins havebeen described in seeds, spores, and fungi, andesclorotins in arthropods.64,196,465

B. Functions

Up to 1898 the increased interest on color inorganisms was due to three main reasons: (1) the

color phenomena is conspicuous for the survivalof animals and plants; (2) the relation betweencolor and evolution theories; and (3) their impor-tance in comparative physiology. Thus, the stud-ies on pigments were greatly impulsed by theirmultiple functions.396

In agreement with the distribution and abun-dance, the most important pigments in higherplants were chlorophylls, carotenoids, and antho-cyanins when considering their capacity to impartcolors. By the same criteria, algae phycobilins areconsidered important pigments. Other pigmentshave less importance as colorant substances be-cause they are present in low quantities.465 In thefollowing paragraphs we describe other functionsof this colorant substance.

1. Tetrapyrrole Derivatives

The heme groups show chemical combina-tion with metals (Fe, V, Cu, Mg) to form metalporphyrins. The metal atom is bonded to the ni-trogen atoms of the heme group by a coordinationbond. The most important metal porphyrins areformed with Fe. In these compounds, nature hasexploited the change of the valence states of ironfrom ferric (Fe+3) to ferrous (Fe+2) and vice versato establish a system for electron transport. Thissystem gives the connection between the intracel-lular dehydrogenases with atmospheric oxygenand in consequence the support for the transportand storage of oxygen, an essential componentfor life. Additionally, the bonding of metal tocytochrome permitted joining the air oxygen withthe metabolic substrates in the cell, contributingwith electron transportation, and consequentlyproviding the energy for vital processes.396 Cata-lase and peroxidase are enzymes with an hemecofactor and have a participation in reduction-oxidation (redox) reactions of living organisms.Vitamin B12 with its heme group participates inchemical-biological reactions that involve rear-rangements.196

The most important function of chlorophyllsis in the photosynthetic process. Chlorophyllsare porphyrins and have a phytyl group confer-ring on them hydrophobic characteristics. Themetal bonded in chlorophylls is magnesium in-

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FIGURE 4. Basic structures of Quinones (A, B, and C) and some of their most known compounds.

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FIGURE 5. Melanin-related structures and some properites. (A) Basic structure (indolic ring). (B) Resonancestructures that are probaly involved in the process of color. (C) Some of the suggested forms of indolic polymerizationto form melanins. The arrows show the points and sense of polymerization.

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stead of the iron in the heme. These compoundsabsorb light in the visible region, red (peak at670 to 680 nm) and blue (peak at 435 to 455nm). The reflection and/or transmittance of thenonabsorbed green light (intermediate wave-length) gives the characteristic green color ofplants and chlorophyll solutions. Light absorp-tion is carried out by the porphyrin ring and inthe absorption process a π-electron is excited tothe higher energy π*-orbital. Part of the lumi-nosity energy is absorbed by the chlorophyllmolecules, where it is transformed into biochemi-cal energy. Normally, in this process an electronis lost and transferred to an acceptor molecule.This process needs highly specialized chloro-phyll molecules that are associated with mem-branes (reaction centers P680 and P700). A smallportion of chlorophyll molecules shows this func-tion, but the reaction centers are the startingpoint of the chain of electron transportation thatwill finally produce the energy in the form ofATP and NADPH2, and these molecules supportall the plant metabolism. Photosynthesis asknown nowadays cannot be possible withoutchlorophyll. Thus, chlorophyll is a very impor-tant molecule for life all over the world.404

Among the linear tetrapyrroles, bilins pigmentthe integuments of many invertebrates (worms andinsects). In algae, the function of phycobilin is simi-lar to chlorophyll as accessory pigments in lightharvesting, transmitting this energy to chlorophyllmolecules in the photosynthetic membrane. It hasbeen described that pigment content in cyanobacteriaand some algae is regulated by light.196

Phytochrome is a blue-green photochromicpigment and controls a wide variety of metabolicand developmental processes in green plants. Thechromophore of phytochrome is a lineal tetrapyr-role and has been shown that the activity of manyenzymes is regulated by phytochrome at biochemi-cal and transcriptional level (mRNA species). Phy-tochrome is involved in the germination, flower-ing, ripeness, anthocyanin, and protein synthesis.196

2. N-Heterocyclic Compounds Differentfrom Tetrapyrroles

The main function of purines is to serve asstructural components in DNA and RNA, where

all the information for the survival of organismsis described. Some pterins are growth factors:rhizopterin (Streptomyces faecalis), pteroyl-glutamic acid (Lactobacillus casei and chickens),other growth factors are teropterin, vitamin Bc,folinic acid, and probably the most important pterinis folic acid, which is an essential vitamin in-volved in the transference of methyl groups andin the insertion of two carbon atoms in the pyri-midine ring.152,196 Flavins are in the molecule offlavinadenin mono- (FMN) and di-nucleotide(FAD), important molecules in the redox reac-tions of organisms, where they are found. FMNand FAD are coenzymes of many enzymes (e.g.,nitrate reductase, pyruvate decarboxylase). Ribo-flavin is an essential vitamin in animals.152 Phena-zines are in bacteria and have bacteriostatic prop-erties. Phenoxazines are in fungi and insects; forexample, Streptomyces sp. produces actinomycin(antibiotic).196

3. Benzopyran Derivatives

a. Antioxidant

Several reports have shown the antioxidantactivity of flavonoids, which is important in thedevelopment of their functions such as scavengingof radicals and disease treatment and prevention.Rutin inhibited malonaldehyde formation from ethylarachidonate by 70% at the level of 0.125 µmol.With ethyl linoleate, naringin, galangin, and rutinexhibited dose-related activities and showed inhi-bitions of 30% at 0.5 µmol. It has been pointed outthat the major mode of flavonoids as antioxidantsis in their ability to scavenge free radicals, and thathydroxylation of the B-ring is an important con-tributor to such activity; in particular, hydroxylgroups at the 3′- and 4′- positions of the B-ringexhibited the highest antioxidant activity, but itwas lower than the observed with BHT.484 Itwas shown that luteolin has good antioxidantactivity evaluated by the β-carotenebleaching method, and it was suggested that C2-C3double bond and C4 keto group seem to be essen-tial for high antioxidant activity (quercetin > (+)-catechin). Also, it was shown that aglycone fla-vonoids are more potent antioxidants. However,luteolin did not exhibit quality antioxidant activity

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with the α,α-diphenyl-β-picrylhydrazyl (DPPH)method, and quercetin and other flavonoids werebetter, and a similar trend was obtainedwith the Rancimat method.484 Anotherstudy used 18 different intestinal bacteria to me-tabolize eriocitrin (eriodictyol 7-rutinoside),which is an antioxidant in lemon fruit. It was shownthat in a first step eriocitrin is transformed toeriodictyol by different bacteria except Clostridiumspp., but from eriodictyol Clostridium spp. pro-duced 3,4-dihydroxyhydrocinnamic acid and phlo-roglucinol. Interestingly, eriodictyol showed higheractivity than eriocitrin or α-tocopherol.324 The an-tioxidant activity of wines has also been evaluated,and it has been reported that almost 96% of theactivity can be explained by the content of cat-echin, m-coumaric acid, epicatechin, cis-polydatin,and vanillic acid, showing higher correlations withseveral flavonoids (catechin, myricetin, quercetin,rutin, epicatechin, cyanidin, and malvidin 3-gluco-side). Wine flavonoids also show a goodperoxynitrite scavenging activity; this was demon-strated by removing the activity with the elimina-tion of these compounds, an interesting aspect ofthe antioxidant profile of wine.356

b. Color and Sexual Processes in Plants

Most of the pigments in flowers are flavonoids,and they impart colors in the range red or purpleassociated with anthocyanins to yellow associatedwith aurones and chalcones. Also, flowers containflavonols and flavanones that by themselves do nothave colors, but modify the coloration by complex-ation with anthocyanins and/or metallic ions (co-pigmentation). The coloration patterns of flowersattract insects to anthers (masculine organ) andpistils (feminine organs) and contribute to the pol-linating process, and as a matter of fact it is wellknown that many species accumulate flavonoids inthese organs. The most common flavonoids inanthers are anthocyanins, flavonols, and chalcones.Also, flavonoids have been involved in the sexualprocess: in the cross- pollination of two varieties offorsythia, rutin, and quercetin are required. An-other important aspect: animals are attracted byfruit color, and after the consumption the seeds aredispersed through the excrement.193,259,455 It has been

established that flavonols are required to inducepollen germination in vivo and in vitro. Some ofthe structural requirements of compounds to de-velop this function are a double bond betweencarbons two and three, a keto group at carbon four,and a hydroxyl group at carbon three of the hetero-cycle, and only the flavonol class has all thesefeatures. It has been established that phenylalanineammonia-lyase (PAL) might play an essential rolein the development of microspores to mature grains.The introduction of the sweet potato PAL intotobacco generated transgenic plants with reducedpollen fertility (10 to 45%). It was suggested thatpollen reduction in PAL activity could reduce thelevels of flavonols and a reduction of levels ofsporollenin of the pollen cell walls, and conse-quently affect the development, germination, andgrowth of pollen tubes in tobacco. Thus, PAL ac-tivity in the anther tapetum is a significant factor inthe development of pollen.301 p100 cDNA ofSolanum tuberosum is up-regulated in pistils afterpollen tube growth and, much more transiently, bytouching the stigma. This cDNA clone shows ho-mology with various isoflavone reductase-like se-quences that have been involved in the response topathogen attack and stress conditions. In potatopistils was shown that not only pollination but alsothe physical presence of the pollen tubes in thepistil is a constant stimulus for expression. Anantioxidant protective activity has been proposedfor p100 because oxygen radicals are naturalbyproducts of metabolism, and a high metabolismis required for the fast-growing pollen tubes.483

c. Photoprotection

Severe illumination produces and oxidative stressin plant tissues and flavonoids protect them fromdamage. One of the responses of UV-illuminatedseedlings is the transcriptional activation of flavonoidbiosynthetic genes. Also, it must be considered thatUV-B radiation itself modify the pigment composi-tion and consequently induces a reduction of theradiation levels over plant tissues. Moreover, it hasbeen established that better UV-B photoprotectors arethe noncolored flavonoids (flavones, flavonols, andisoflavonoids). Additionally, it is known that the pro-duction of pyrimidine dimers and 6,4-photoproducts

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is higher in plants without flavonoids.196,259,327

Middleton and Teramura314 showed that flavonoidsprotect soybean but without an effect on the photosys-tems, and they suggested that carotenoids developthis function. In 1992, it was shown that quercetin andits glycoside (rutin), acting as antioxidants, reducedthe rate of formation of malondialdehyde and lipidperoxidation in chloroplast thylakoids of wheat undersevere illumination; it was suggested that flavonoidsacted by trapping the singlet oxygen produced in thebiological reactions.96

d. Defense Mechanism in Plants

When attacked by pathogens, some plants shoottheir flavonoid biosynthesis (phytoalexins, as thosefrom Broussonetia papyrifera and Narcissuspseudonarcissus); interestingly, these flavonoids aredifferent to those induced under severe illumination.Sorghum (Sorghum bicolor) synthesizes thephytoalexins apigeninidin and luteolinidin(deoxyanthocyanidin flavonoids) as a response toattempted infection by the fungus Colletotrichumgraminicola that causes the anthracnose disease. Itwas reported that susceptible plants lose the ability torespond rapidly to fungal infection. It has been ob-served that after synthesis of phytoalexins in inclu-sions within the cell under attack, the pigments re-leased were observed to accumulate in the fungus.443

The major rice pathogens are Xanthomonasoryzae pv. Oryzae (the bacterial blight pathogen),Pyricularia oryzae (the fungal blast pathogen),and Rhizoctonia solani (the fungal sheath blightpathogen); several flavonoids with variable activ-ity have been reported. Naringenin inhibited thegrowth of X. Oryzae, naringenin, and kaempferol,the spore germination of P. oryzae. It was estab-lished that nonpolar flavonoids showed higherinhibition than their polar counterpart, but noneof the tested compounds showed inhibition toRhizoctonia. Also, cyanidin and peonidin glyco-sides (anthocyanins) showed growth inhibition ofXanthomonas.354 Downy mildew caused byPlasmopara viticola (Berk et Curt.) is an impor-tant disease threatening the world’s viticulturalareas. Three Vitis sp.(V. vinifera cv. Grenache,susceptible; V. rupestiris cv. du Lot, intermediateresistant; and V. rotundifolia cv. Carlos, resistant)

were studied. It was determined that the flavonoidcompounds were induced following infection byP. viticola in both the resistant and in the interme-diate resistant species. However, the kind of fla-vonoids was different. In the resistant species,necrosis of the stomatal tissues occurred afterinfection, resulting in the cessation of fungalgrowth of the tissue. This response was also ob-served in the species with intermediate resistance,but response time was longer in the last one.Thus, it is clear that flavonoids play a key role inthe high resistance of V. rotundifolia and thatprecocity in the resistance of Vitis sp. to P. viticolais very important.111 Flavonoids of sour cherrieswere extracted and assayed against the ascomyceteLeucostoma persoonii that causes the perennialcanker on the bark of peach, plum, and sweetcherry. In general, all tested flavonoids sloweddown the mycelial growth. It was shown to havesignificant differences in the toxicity of aglyconesand glucosides, showing higher toxicity the agly-cones; therefore, suggesting glucoside hydrolysisas a defense reaction. Naringenin and chrysinshowed the highest fungistatic effects.167 Erwiniacarotovora causes blackleg and soft-rot of pota-toes by macerating activity in the host tissue. Themain enzymes of this bacterium are pectate lyases(PL), and the transformation of potato with PLwas used as a protection mechanism. It was deter-mined that transgene was expressed in differenttissues and tuber disks of transformants inhibitedthe Erwinia growth. Interestingly, a strong induc-tion of PAL was observed in the transformantwith the high inhibitory effect that was a markerfor the activation of defense-related genes. Thus,the resistance might be based on the liberation ofphenolic compounds and phenol oxidases.501

Isolated in Arabidopsis thaliana was asulfotransferase cDNA (RaR047) that regulationis developmentally regulated and associated withstages of active plant growth. RaR047 was in-duced under stress or pathogen attack. RaR047showed a maximal expression in the incompatibleinteraction between 3 and 7 days after inoculationfor Xanthomonas campestris pv. campestris andbetween 7 and 24 h after inoculation withPseudomonas syringae pv. maculicola. It was sug-gested that sulfotransferase could be involved inthe molecular communication between the inter-

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acting organisms; in particular, it was proposedthat the RaR047 product participates in the syn-thesis of toxic compounds directed against thepathogen during the resistance reaction.268

Several studies have been carried out to inducevirus resistance, and flavonoids have shown antivi-ral activity against tomato ringspot virus(TomRSV). The most effective compounds werequercetin, quercetin 7,4′-dimethyl ether, quercetin3,7,4′-trimethyl ether, and fisetin 4′-methyl ether,causing a 67 to 76% inhibition of TomRSV infec-tivity when applied at 10 µg ml-1. Quercetin did notaffect viral replication. When quercetin was usedin combination with RNA, an increment in theinfectivity was observed. Thus, it was concludedthat inhibitory effect of quercetin is dependent onthe presence of the coat protein for antiviral activ-ity, possibly by the inhibition of uncoating. Fla-vonoids are proposed as a class of compounds withpotent antiviral activity that can be used to reduceor eliminate viruses from plant material.294 On theother hand, leucoanthocyanins are accumulated insome seeds and make them less attractive for theconsumption by animals or the attack of otherpests. Additionally, these leucoanthocyanins pro-vide a seed protection against digestive enzymes,permitting that after the excretion the seed cangerminate. The flavan from Lycoris radiata bulbswas found to be antifeedant for the larvae of theyellow butterfly Euroma hecabe mandarina.193,259,455

Studying the root bark and stem wood of Erythrinasigmoidea, the methylene chloride extract was foundto be active against the Gram-positive bacteriaStaphylococcus aureus. After successive purifica-tions, it was isolated with the bioactive flavonoidneobavaisoflavone, which showed a good inhibi-tory action compared with streptomycin sulfate.346

Also, it has been established that proanthocyanidinsare the main components of tannin. In particular,bark procyanidins are dominant (Monterrey pine50%, Scots pine 75%, and birch 65%), beingprodelphinidins in a higher proportion. It has beensuggested that proanthocyanidins contribute to pro-tecting internal plant tissues (phloem, cambium,and xylem) against invasion by pathogens becausethey are particularly abundant in the more exposedouter bark.302

It was proposed that flavonoids isolatedfrom Helianthus annuus possibly play roles inthe allelopathic activity of sunflower. They prin-

cipally influence, the shoot growth of seedling,but germination and radical length can be af-fected by chalcones. Tambulin did not showany effect against germination and radical lengthof tomato and barley but inhibited shoot growthof tomato (approximately 25%) and barley(22%). Kukulkanin B and heliannone A differin the presence of an additional methyl group inheliannone A; however, their activities are quitedifferent. Heliannone A inhibited germinationof tomato (20%) and barley (35%) and slightlyaffected barley shoot growth length. On theother hand, kukulkanin B only showed inhibi-tory activity on the shoot growth of tomato.290

e. Other Ecological Functions

Studying the loblolly pine (Pinus taeda) needles,it was shown that increments in catechin andproanthocyanidin concentration are generated by theeffect of O3 exposure. It was suggested that theseincrements are an adaptive response by acting as anantioxidant, and it was proposed that elevated cat-echin levels might also be a suitable biochemicalindicator of forest damage.51

Flavonoids are important compounds used asecological indicators.169 It has been reported con-siderable quantitative and qualitative differencesof flavonoids in propolis and has been concludedthat such differences were dependent mainly onplant ecology and the variety of the bee.261 More-over, it has been established that bee pollen canbe used to identify their plant origin, for example,the major compound in almond bee pollen is8-methoxykaempferol 3-glycoside, while jarabee pollen contained mainly quercetinand isorhamnetin 3-glycosides. Thus, flavonoidscan be used as a markers of pollens.470 The fla-vonoid profile has been used to evaluate the evo-lution in the Phaseolinae: it was determined thatkaempferol and quercetin glycosides are the mainflavonoids, whereas the glycosidic pattern was3,7-O-diglycoside and 3-O-glycoside. These fea-tures represent intermediate to advanced evolu-tionary characters among the Phaseolinae fla-vonoids. The flavone frequency was higher thanin other subtribes, thus indicating a highly ad-vanced position in the subtribe Phaseolinae withinthe subfamily Papilionoideae, which was sup-

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ported by the C-glycosylation and C-acylationprofiles, and it was concluded that flavonoids ofthese Phaseolinae species correspond to an inter-mediate evolutionary level.522

On the other hand, leaf waxes of 32 Aeoniumspecies were obtained, and 32 flavonoids wereidentified as methyl ethers of kaempferol, 6-hydroxykempferol, quercetin, myricetin, andscutellarein (6-hydroxyapigenin). It was suggestedthat waxes of different plants of the same speciescontained the same principal flavonols, with in-traspecific variation limited to the minor fla-vonoids. It was determined that in the Aeoniumgenus the occurrence of myricetin methyl ethersand 6-hydroxykaempferol methyl ethers is taxo-nomically most significant.450 In addition, fla-vonoids have been used to characterize wines, forexample, in Pinot Noir wine, the highest concen-trations of catechin and epicatechin were found,while Cabernet Sauvignon shows quercetin (typi-cal of red wines).444 Citrus has been studied byconsidering their flavonoid composition, whichare quantitative fingerprints of the juices: the pre-dominant flavonoids in lemon were hesperidinand eriocitrin, which were also present in additionto neoponcirin in lime, while pummelo containsalmost exclusively naringin. In grapefruit, theprofile was more complex with neohesperidosideas the main component. Based on this informa-tion, naringin is used as a chemotaxonomic markerto distinguish sweet orange from other citrus va-

rieties, and is especially used as indicator of grape-fruit addition to orange juice.398

f. Pharmacological Effects

It has been clearly established that flavonoidsin natural products (e.g., grape, soybean, peanut,wine, tea) have a good antioxidant activity. Insome instances a better activity than in the com-mercial antioxidants has been determined.159 Ad-ditionally, flavonoids contribute to the protectionand/or regeneration of antioxidants. It has beensuggested that the flavonoid action is by trappingfree radicals and that flavonoids are antimutagenicthat can reduce the atherogenesis and the risk forstrokes, and that are modulators of arachidonicacid metabolism. This last point involves fla-vonoids in many metabolic processes.159,198,338

Interestingly, flavonoids are ubiquitous of plantsand a person can consume up to 1 g of them. Thisquantity implies that flavonoids can reach phar-macological concentrations in the organism. Thus,it is interesting to point out that many flavonoidshave assigned pharmacological activities (Table1), and that many medicinal plants are known andused by their flavonoid content. Moreover, it hasbeen determined that flavonoid structure has theresponsibility of a large part of the activity. Also,it has been shown that different substitutes give amolecule with an specific polarity that permits

TABLE 1Pharmacological Activities of Some Flavonoids

Flavonoid Activity

Rutin, silibin Against disorders of the respiratory systemNaringenin Antimicrobial and antifungic on skinQuercetin, morin, procyanidin, pelargonidin Antiviral(+)-Catechin, 3-O-Metil-(+)-Catechin, Against ulcers

naringeninButrin, isobutrin Hepatic disorders and viral hepatitisChisin, floretin, apigenin, quercetin, Antiinflammatory

kaempferol, baicaelinEpicatechin Against diabetesGenistein, kaempferol, sophoricoside AntifertilityGalangin Activity against Staphylococcus epidermisProanthocyanidins Astringent for digestive system, diuretic, cardiac

tonic, in the treatment of high blood pressure

Adapted from Refs. 10, 198, 509.

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the presence of privileged bonds in the activesites.10 This characteristic could have a relationwith the antioxidant activity, because it is knownthat variations in glycosylation and/or acylationinduce differences in the activity and stability offlavonoids.233 From an acetone extract of the rootbark of Ormosia monosperma several flavonoidswere isolated, and it was observed that2,3-dihydroauriculatin showed a moderate activ-ity against oral microbial organisms such as Strep-tococcus mutants, Porphyromonas gingivalis, andActinobacillus actinomycetemcomitans, each at6.3 µg ml-1.234 From Ononis spinosa subsp.Leeiosperma, two flavonoids with biological ac-tivity were isolated: spinonin showed a moderateactivity against P. aeruginosa and higher activitywas obtained with ononin against β-hemolyticStreptococcus (minimal inhibitory concentrationor MIC = 25 µg/ mL).256 It was shown that someflavonoids (isorhamnetin, rhamnetin, and querce-tin) diminished the total serum cholesterol levelswhen rats were fed with these compounds; athero-genic index, bile acids, serum triacylglycerols,and thiobarbituric acid-reactive substances(TBARS) tended to low as well. Flavonoids in-hibited the generation of superoxide anion (higherwith rhamnetin) and the oxidation of linoleic acid(up to 84% with quercetin). The activities of su-peroxide dismutase and xanthine oxidase did notvary, thus the flavonoid abilities could be ascrib-able in part to the scavenging of free radicals andin particular to their antioxidative activities.198

Studies on oregano extracts showed thatgalangin and quercetin are active desmutagensagainst the mutagenicity of Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole), a liver-specificcarcinogen, and other mutagens and it was sug-gested that flavonoid desmutagenicity plays animportant role in cancer prevention; however, it isconvenient to mention that under certain condi-tions these flavonoids did not inhibit mutagenic-ity, that is, in the presence of a buffered mixtureof polychlorinated biphenyls, essential cofactors,NADP, and glucose-6-phosphate to form the S9mix.249 It has been reported that quercetin or itsanalogs could be interesting substitutes ofcamptothecin, which is used in the treatment ofcancer and produces undesirable secondary reac-tions. It was proposed that all of these substances

act at the level of the union of topoisomeraseenzymes with DNA. Flavones can either stabilizethe catalytic topoisomerase I-DNA intermediateor inhibit the DNA binding of the free enzyme.Thus, in view of the relatively low toxicity ofquercetin and related antitumor activity, its use asan anti-cancer drug seems feasible.43

It is well known that iron could induce thegeneration of harmful reactive species and thatspecial proteins are required to capture iron (trans-ferrin, ferritin, heme proteins) and to prevent itsreaction with oxygen species. Released iron in-duces the peroxidation of membrane lipids andhemolysis, and it has been established that quer-cetin inhibits the harmful effect of free iron prob-ably by forming a complex with it (in assays witherythrocytes). Interestingly, it has been suggestedthat the release of iron in a redox-active formcorrelated with the generation of senescent anti-gen (the antigen appearing on aged erythrocytes),and quercetin induces the chelation of iron atintracellular level and delays the formation ofsenescent antigen, prolonging the storage time ofblood in blood stores.145 Also, it was shown thatan hydroxyl group at C-3 and carbonyl at C-4 ofthe C-ring are necessary to bind iron. From ethylacetate extracts of peppermint, sage, and thyme,luteolin was identified as the main active com-pound, showing desmutagenicity againstTrp-P-2. It was suggested that mutagenicity gen-erated from consumption of 1 g of broiled meatcould be mitigated with very small amounts ofthese herbs: 2.8 mg of peppermint, 13 mg of sage,or 0.9 mg of thyme. A correlation was identifiedbetween a desmutagenic mechanism of flavonoidsand their antioxidant potency.413 Also, it was sug-gested that the choleretic, antirheumatic, and di-uretic activities of dandelions could be relatedwith the content of luteolin glycosides (7-gluco-side and 7-diglucosides).506 Also, luteolin glyco-sides have been isolated from Vitex agnus-castus(e.g., luteolin 6-C-[4′′ -methyl-6 ′′ -O-trans-caffeoylglucoside]; luteolin 6-C-[6′′ -O-trans-caffeoylglucoside]) and these compounds showedcytotoxic activity against P388 lymphocytic leu-kemia cells IC50 (values were 0.1 for 4′ to5-dihydroxy-3,3′,6,7-tetramethoxy-flavone and0.31 for luteolin).214 Trypsin and leucine ami-nopeptidase have been implicated in a variety of

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pathological process such as acute pancretitis, in-flammatory process, various cancers, etc. Inter-estingly, most flavonoids showed inhibitory prop-erties against trypsin aminopeptidase (strongestinhibition with quercetin and myricetin), but only3′,4′-dihydroxyflavone and quercetin inhibitedleucine aminopeptidase. From the studies withthe trypsin aminopeptidase inhibition, it was es-tablished that hydroxyl groups at C-5 and C-7 inring A are essential, while a double bond at posi-tion C-2, C-3 in ring C and hydroxylation at boththe C-3′ and C-4′ enhance the inhibitory activity.On the other hand, inhibition of leucine ami-nopeptidase by flavonoids requires of a doublebond at C-2, C-3.358 L868276 is a synthetic ana-log of flavopiridol that is obtained from Dysoxylumbinectariferum. These substances are used to in-hibit the growth of breast and lung carcinoma celllines by its effect on the cyclin-dependent kinaseactivities. Interestingly, it has been shown thatflavopiridol do not inhibit other protein kinases.113

Brysonima crassifolia (known in México asnanche) extracts have been used in traditionalmedicine by Mixe Indians in the treatment of gas-trointestinal disorders and skin infections, and invitro assays have shown that ethanolic extractsinhibited the nematode multiplication (IC50 = 175ppm). In this extract the main components wereproanthocyanidins.168 In Brazil, decoctions or infu-sions of Stryphnodendron adstringens are used tra-ditionally in the treatment of leukorrhoea, diar-rhoea, and as antiinflammatory. An acetone-H2Oextract was analyzed and prodelphinidins withbioactive pyrogallol units (epigallocatechin deriva-tives) were identified and proposed as the pharma-cological active compounds.122 In Guazumaulmifolia, which is used in the treatment of diar-rhoea by the Mixe Indians were identifiedprocyanidins (epicatechins). These compounds in-activate the cholera toxin, and toxin-binding activ-ity increased with their molecular weight.219 Also,flavonoids have been involved in the inhibition ofseveral individual steps related to the thrombosisprocess, and one of the most active compoundswas the biflavonoid hinokiflavone. The activity ofthis flavonoid has been suggested that is mediatedby the inhibition of induction of tissue factorexpression by interleukin-1.270 Other biflavonoids(e.g., amentoflavone, agathisflavone, robusta- fla-

vone) showed activity against the human immuno-deficiency virus (HIV) reverse transcriptase (HIV-1 RT). It was indicated that biflavones with twoapigenin units linked either with C-C or C-O-Cbonds (robustaflavone, hinokiflavone) exhibited sig-nificant inhibitory activity. However, in a wholecell assay (human PBM cells infected with HIV-1strain (LAV-1) morelloflavone exhibited potentinhibitory activity (promising anti-HIV activity),while it possessed moderate activity in the HIV-1RT, emphasizing the importance of cellular mecha-nisms in flavonoid action. It was concluded that thepresence of an unsaturated double bond at C-2, C-3 and three hydroxyl groups at C-5, C-6, and C-7positions were prerequisites for inhibition of RT.284

From the ethanolic extract of the stem bark ofMitrella hentii, it was isolated linderatin. This com-pound was tested in vitro on a non-small-cell bron-chopulmonary lung carcinoma type of cancer thatrepresents 80% of all human bronchopulmonarycancers that is highly chemoresistant to medicaltreatment. It was reported that only (-)-linderatinexhibited a significant activity toward this type ofcarcinoma (IC50 = 3.8 µg mL-1).34 It is interestingto mention that quercetin or its structural analogfisetin) enhanced the estradiol-induced tumorigen-esis in hamsters. The effect of quercetin was medi-ated by increased levels of S-adenosyl-L-homocys-teine (SAH) which inhibited the methylation of2- and 4-hydroxyestradiol by catecholO-methyltransferase. Also, it was shown that lowrenal pools of S-adenosyl-L-methionine contrib-uted to this phenotype.527 The estrogenic activity ofisoflavonoids has been reported, and7-isopropoxyisoflavone is now sold on the market(‘Osten’ from Takeda Chemical Industries, Ltd.)as a therapeutic drug for osteoporosis. Also, it hasbeen reported that daidzein increased the cell num-ber of mouse osteoclasts at a very low concentra-tions.466 One of the main flavonoids in teas isscutellarein, which has been used as a diuretic,antiinflammatory, and antiasthmatic drug.218 Thewide spectrum of resistance of cancer cells to natu-ral agents has been assigned in part to theoverexpression of proteins that belongs to the ATPbinding cassette transporter proteins (ABC).Multidrug resistance protein (MRP), pertains toABC proteins, has been cloned from a drug-resis-tant small cell lung cancer line. Several flavonoids

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(genistein, kaempferol and flavopiridol) have showninhibition of MRP-mediated transport of anti-can-cer drugs by a direct interaction of flavonoids onthe active site of MRP. Additionally, it has beensuggested that a glucose moiety in the flavonoidstructure plays an important role for its activity.218

Flavonoids have also been proposed in thetreatment of obesity. Extracts of Cassia nomamehave lipase-inhibitory activity, and it was deter-mined that (2S)-3′,4′,7-trihydroxyflavan-(4α→ 8)-catechin has a significant inhibition of the porcinepancreatic lipase (IC50 = 5.5 µM). However, inter-estingly, an oligomeric fraction showed the mostpotent inhibitory effect (IC50 = 0.20 µM). Thisoligomer was mainly composed of 3′,4′ ,7-trihydroxyflavan and catechin.199

4. Quinones

As colorant substances, quinones are not veryimportant. They only provide coloration in somehigher organisms and microorganisms: in mem-bers of the Echinodermata family, quinones con-tribute to the pigmentation of spines, shell, ova-ries, and eggs. In microorganisms, Polyporusrutilans is a fungus and accumulates up to 23%dry weight (d.w.) of polyporic acid, a terpenylquinone of bronze color. Helminthosporiumgramineum reaches up to 20% d.w. of quinones(islandicin, crisophanol, emodin). Some bacteriaproduce good quantities of quinones; Streptomy-ces coelicor accumulates up to 15% d.w.464

On the other hand, some quinones have beenused as food colorants at the industrial level. Rubiatinctoreum Linn. is obtained from an extract com-posed of quinones (alizarin, xanthopurpurin,rubiadin, purpurin, etc.). The peel of Coprosomaacerosa A. contained 3-hydroxi-2-methyl-an-thraquinone, methyl-ether of rubiadin, and lucidin,etc. From the root of Rumex chinensis is obtaineda denticulate, which in China is used as a substi-tute of rhubarb. However, the most importantquinones at the industrial level are the anthraquino-nes: carminic acid is obtained from the Mexicanspecies of Dactylopuis coccus; carmesic acid isobtained from Laccifer lacca Kerr.464

Quinones are very reactive compounds duetheir structural characteristics. Between all the

quinone reactions only one is common to all ofthem, a reversible reduction. By this reaction,quinones participate in the redox reactions of theorganisms where they are present. Ubiquinoneand plastoquinone are essential components ofthe electron transport in mitochondrial membranes(ubiquinone) and chloroplasts (plastoquinone).Ubiquinone is an hydrophobic compound of lowmolecular weight, and it can be found in its re-duced or oxidized state: ubiquinone is lipid solubleand can diffuse through the membranes to act asan electron carrier between the components of therespiratory chain. Plastoquinone has similar char-acteristics to ubiquinone, and it is involved in theelectron transport between PSI and PSII in thephotosynthetic process.196,464

Moreover, it has been reported that severalenzymes have a quinone as cofactor. Methanoldehydrogenase has a pyrroloquinoline quinone(PQQ) as cofactor, and also other bacterial dehy-drogenases are quinone dependent (quinoproteins).These enzymes are involved in the oxidation ofsubstrates such as alcohols, amines, and sugars.Additionally, it has been found that some copper-dependent amino oxidases (AO) have an alternatecofactor called topaquinone (TPQ). Several me-thyl-amine dehydrogenases have the tryptophantryptoquinone (TTQ) as a cofactor. TPQ is a cofac-tor ubiquitous in bacteria, yeasts, plants, and mam-mals. Bacterial and yeast TPQ enzymes permitthem to grow in different amines as a nitrogensource, and in some instances as source of carbon.In plants, the main function of the AO is in theproduction of hydrogen peroxide to heal woundsby the induction of new cell wall. Moreover, it hasbeen suggested that plant AO participate in thegrowth process by the regulation of polyaminelevels. The AO functions in animals are more elu-sive and diverse. The well-defined enzyme is themammal lysyl-oxidase that catalyzes the connec-tive tissue maturation by the crossover of elastinand collagen. On the other hand, some evidenceindicates that free PQQ has other functions in eu-karyotes: in the production of superoxides inneutrophiles (neuroprotector); prevents cataractsby exposure to cortisone; edemas induced by car-rageenans; liver injury by hepatotoxins (carbontetrachloride). In plants, it has been shown thatPQQ stimulates 5 to 10 times the conversion of

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acetyl-CoA to hydroxymethylglutaryl-CoA (HMG-CoA).258 However, new methodologies for the as-say of PQQ (bioassay and GC-MS) have showntheir presence in Gram-negative bacterium, and ithas been reported in only one Gram-positive bac-teria (Amycolatopsis methanolica). However, assuch, it was concluded that PQQ is not present inmaterials obtained from plants and animals, conse-quently, previous reports about their presence inthese organisms must come from contamination.323

Defense mechanisms. Streptomyces sp. pro-duces the antibiotic tetracycline. Many insects(Dyctiopters, Diplopodes, and Opiplions) producealkylbenzenequinones as a defense mechanism. TheAfrican tree Mansonia altissima excretes mansononsthat protect it from fungi and insect attack.465 Inaddition to the pharmacological contributions men-tioned above, it has been reported that several natu-ral laxatives rhubarb, senna) have mixtures of an-thraquinones and anthrones of the emodine type,and vitamin K functions as a cofactor of theprothrombines (coagulation factor). At the indus-trial level, skikonin is the most important quinonewith pharmacological activity, and it is produced bytissue cultures of Lithospermum erythrorhizon.196,464

5. Iridoids

These compounds are not particularly impor-tant as colorants and their relevance, until a fewyears ago, had been restricted to be taxonomicmarkers. Iridoid glucosides are only found in moreadvanced dicotyledonous plants. Remarkably, seco-iridoids are biosynthetic precursors of alkaloids.406

6. Melanins

In general, melanins are not essential for thegrowth and development but induce an incrementin the possibilities of organism survival by actingas a defense mechanism. Melanins have been alsoassociated with the immune response. It has beenobserved that rice resistance to Piricularia oryzeis associated with the induction of phenol oxi-dase/polyphenol oxidase and the synthesis ofmelanin. In fungi, melanins provide resistance tostress conditions.32,64,456,465

By considering the pharmacological effect, ithas been found that allomelanins (free of pro-teins) from plants, black bean, soybean, andsesame suppress the growth of tumorigenic cellsof animals and mammals. Also, it has been men-tioned that melanin inhibits the in vitro infectionof the lymphocyte cell line by the HIV-1 virus.However, the free radicals in melanin moleculescould produce a toxic effect in cells or the growthinhibition by melanin association with minor el-ements such as iron and copper. It has been re-ported that soluble melanins and their pre-cursors (catecholamines, 3-hydroxyquinurenine,3-hydroxyanthranilic acid, catechol) can inducedegenerative processes in connective tissues, bloodvessels, heart valves, liver, and other tissues, simi-larly to that observed in the illness called alcapto-nuria. Parkinson’s disease and the normal agingprocess are characterized by the loss of pigmentedneurons in nigra substance, which correlates withthe accumulation of neuromelanin and with theoxidative conditions in that brain region, and ithas been hypothesized that neuromelanin acts asa protector under oxidative stress in normal indi-viduals, but in those with Parkinson’s this mol-ecule has the potential to exacerbate the oxidativeconditions through the generation of hydrogenperoxide or through the liberation of active met-als for the redox process. This information em-phasizes the importance of protection by antioxi-dants in the normal human metabolism, in diseases,or in the aging process.32,248

C. Importance as Food Colorants

1. Reasons to Use Color Additives

Color is associated with many aspects of ourlife. For example, the main factors to evaluatefood quality are color, flavor, and texture, butcolor can be considered the most important ofthem, because if it is not appealing consumerswill not enjoy the flavor and texture of any givenfood. Color may as well give the key to catalogea food as safe, with good aesthetic and sensorialcharacteristics: the undesirable colors in meat,fruits, and vegetables warn us about a potentialdanger or at least of the presence of undesirable

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flavors, among other reactions. Many studies haveemphasized the relation of color with the flavordetection threshold, with the sweetness or salinitysensations, with the susceptibility and preferencefor products.80,162,339 Nowadays, people know thatfoods have their own natural colors, but mostpeople live in big cities, far from the places offood production, and processing and transporta-tion of foods are required. During such events,foods may lose some of their properties and someadditives must be added to permit such productshaving desirable appearance and are safe for con-sumption. Thus, additives are used to emulatecolors that are found in natural products such aspepper, red beet, grapes, saffron, meat, andshrimp.58,144 Moreover, it is possible to enumeratethe reasons to use food colorants:150

1. To restore the original food appearancebecause of the occurrence of changesduring processing and storage.

2. To assure the color uniformity to avoidvariations in tone color by seasonal variations.

3. To intensify colors that are normally foundin food and the consumer will associatethis improved color with food quality.

4. To protect the flavor and light susceptiblevitamins.

5. To give to food an attractive appearance,whereas without the additive food willnot be an appetizing item.

6. To preserve the identity or character bywhich food is recognized.

7. To help in the visual assignation of thefood quality.

2. Importance of Natural Colorants

Since the early civilizations, natural prod-ucts were used to give an attractive presentationto man-made products. Saffron and other spe-cies were used frequently to provide yellow colorin a variety of foods and evidence exists thatbutter was pigmented with these products. Thefirst reports regarding the use of derived colorsof minerals dated from the ninteenth century;however, some of them caused serious health

problems. Lead chromate and copper sulfate wereused to pigment candies and sauerkraut, but inthe pigmentation process arsenic and other ven-omous impurities were added frequently. Also,in that epoch began the use of tar colorants andother petroleum derivatives in the processing offoods, medicines, and cosmetics.144 Syntheticcolorants have been used for many years, in1938 it was recognized the use of approximately200, in 1970 to 1965, and nowadays only sevencan be used in food pigmentation.144,150 How-ever, in the last 30 years synthetic additives havebeen severely criticized, and consumers show aphobia toward these products and consequentlypeople prefer the natural colorants.157,161 In theperiod 1960 to 1970 in the U.S., the environ-mental activist movements attacked the foodadditives, and this attitude was converted in aworld-wide phenomenon. In general, this move-ment was against the technologies that have aharmful effect on the environment, and they useda very particular association between food andnutrition. The criticism of these groups was fo-cused on the fast food and was difficult for themto attack these products as a group. However,colorants were an easy target; they indicated thatpigments only have a cosmetic value, and, on theother hand, they could cause damage. The activ-ist consumers reviewed the concepts health andfound it harmful to many foods and diets, andcompanies began with the aim of producinghealthier foods. They started using the nutri-tional characteristics as a sale tool, a strategythat has previously failed, but after the socialmovement was converted into a total success;thus, a world-wide tendency to use naturalcolorants was generated. At the present time,most people interpret the content of chemicalproducts as a contaminant and the tendency hasbeen reinforced, and all seems to indicate that itwill continue to in the future.158 To emphasizethis situation, it should be pointed out that up to1986 356 patents on natural colorants have beenregistered and for the synthetics only 71.156 Nowa-days, the number of advantages of natural oversynthetic colorants have been increased becauseof the pharmacological properties of the naturalpigments. In 1994, the market of natural colorantshad an estimated value of $250 million U.S.

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dollars, and 65% of this corresponded to coloradditives for food, with an annual growth rate of5 to 10% in relation to 3 to 5% for the syntheticpigments. However, it is necessary to note thatsynthetic colorants have well-known advantagesover the natural ones based on the higher pig-menting power, stability, storage, facility in theprocessing, and they are cheaper and availablein unlimited quantities.507 On the other hand,some products have a good market value only ifthey are colored with natural products: in themanufacture of Cheddar cheese, only the annatopigment is used;161 in the pigmentation of poul-try products, synthetic colorants are not ad-equate.297 With the information mentioned aboveand by considering the large number of papersabout natural colorants, one tends to believe thatthe area of these food color additives is veryactive, but this is a mistake because most ofthem are not applicable at the industrial levelbecause they have not covered the economical,legal, and safety requirements that most govern-ments have established for food additives. Nowa-days, the number of approved colorants for foodindustry is very limited, and this phenomenon isobserved practically all over the world (Table2), probably with the exception of Japan.Colorants produced by the Monascus fungi arewell-known pigments. Traditionally, Monascuspigments are obtained from fermented productsof rice and bread and have been used as foodcolorants and medicinal agents. Interestingly,iridoids and Monascus pigments have remark-able properties, such as a good range of colorsand stability, that can be used as food colorants;however, none of these groups are approved inneither the U.S. nor in the European Union.58

D. Some Regulatory Aspects AboutColor Additives

Color additive regulation has an older historythan any other additive regulation, with the excep-tion of that of preservatives. These two kinds ofadditives were the subject of a special legislationby the U.S. Congress in the act of 1900 on colorsand preservatives. In this act it was pointed out thatthe government had the responsibility to investi-

gate the safety of these products before issuing anyregulation. With the 1906 act, the control on thecolor additives was greater than on the other addi-tives. In 1938, the Food, Drug & Cosmetic Act(FD&C) was established and published a list of tarcolorants that could be used as food additives; also,it was decreed that the FDA had the authority tocertify the color lots. With this, color additiveswere the first substances that must be revised be-fore their commercialization. In the first years,synthetic colors were prohibited as health-harmfulproducts. The next important change came in 1958;additives were redefined and a new classificationwith three categories appeared: (1) substances ap-proved by the FDA or the USDA (United StatesDepartment of Agriculture) during 1938 to 1958(prior-sanctioned substances); (2) substances thatare Generally Recognized as Safe (GRAS sub-stances), which did not require of a previous FDAevaluation to be commercialized; and (3) all theremaining added substances in the food supplyfood additives), which must be evaluated by theFDA before commercialization.

All new and color additives were included inthe last category. Moreover, in 1958 included wasthe Delaney clause “no additive shall be deemed tobe safe if it is found to induce cancer when ingestedby man or animal, or if it is found after tests whichare appropriated, for the evaluation of the safety offood additives, to induce cancer in man or animal”.In 1960 and with the FD&C amendment, Congressestablished that all color additives required theprior FDA approbation for their commercializa-tion. It may appear that it did not difference existedbetween this and the regulation before 1958. How-ever, the new regulation also included prior-sanc-tioned colors (before 1958). When the 1900 actwas published in 1912, 80 tar-derived colors hadbeen used in food industry, the number was re-duced to 12 after the FD&C act, and nowadaysonly 7 are approved. Additionally, color additiveshave another disadvantage, that is, all must beconsidered as additives and not as GRAS sub-stances, thus the regulations for color additives arestronger than for other additives, and certainly upto date there exists no scientific basis to establishdifferences between color and other additives.507,508

On November 9 1990, an act was approved inwhich the producers are obliged to label all products

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under certain regulation. With respect to color, theact established that all certified colors used in foodsmust be indicated in the label, but the exempt ofcertification (this includes the accepted natural pig-ments) must be generically grouped as colorants.This act gave a clear advantage to the use of naturalover synthetic colorants, because consumers are

suspicious about synthetics. On the other hand, theexemption of certification additives are from com-mon natural products: red beet, carrot, fruits, pepper,among others, and regulation could be expected tobe less severe for these products; however, it is clearthat FDA policies do not share this idea, and theresearch for natural colorant sources is uncertain

TABLE 2Approved Colors for Food Industry in the European Union and in the Food and DrugAdministration (FDA) of USA

Color CEE FDA

CertifiableRed allure AC No Yes (red #40)Brilliant blue No Yes (blue #1)Carmosine Yes (E122) NoEritrosin Yes (E127) Yes (red # 3)Fast green FCF No Yes (green # 3)Indigotine Yes (E132) Yes (blue # 2)Ponceau 4R Yes (E124) NoSunset yellow FCF Yes (E110) Yes (yellow # 6)Tartrazine Yes (E110) Yes (yellow # 5)Polymeric colors No NoCitric red # 2 No No

Exempt of certificationAnnato extract Yes YesDehydrated red beet Yes YesUltramarine blue YesCanthaxanthin No YesCaramel Yes Yesβ-apo-8'-carotenal No Yesβ-carotene Yes YesDactylopuis coccus extract Yes YesMeal of cotton seeds Yes YesIron glutamate No YesSkin grape extract Yes YesIron oxide No YesFruit juices Yes YesVegetable juices Yes YesAlgae meals No NoTagetes and extracts Yes? NoCarrot oil Yes YesOil of corn endosperm Yes YesPaprika Yes YesPaprika oleoresin Yes YesRiboflavin Yes YesSaffron Yes YesTitanium dioxide No YesTurmeric Yes YesTurmeric oleoresin Yes YesChlorophyll Yes NoXanthophylls, flavoxanthins, rubiaxanthin, Not all Not all

zeaxanthin and other natural products withsome of these carotenoids

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because of the difficulty in getting FDA approval. Inthis respect, the history of color additive amendmentis disastrous; red #40 is a colorant that was devel-oped in the 1960s and approved by FDA in 1974,the only synthetic colorant approved in the last 75years that it is still used). In general, the approbationprocess is expensive, a synthetic colorant requires$2.5 millions dollars only for the FDA evaluations,and in the European Union about $1 million dollars.Nevertheless, the regulations for the GRAS sub-stances are very successful: olestra lipid substitute)was invented in 1968, this product was cataloged asan additive and it was approved by the end of 1996,28 years after its invention; on the other hand, theinventors of the high fructose corn syrup (HFCS)decided that their product was a GRAS substance,thus HFCS was immediately commercial-ized.144,229,310,507,508 Additionally, FDA uses the termindirect additives to group those additives used inthe pigmentation of animal meals, and finally ani-mals will be used as human food. Something that itis not easy to understand.161

Undoubtedly, it is technologically feasible toobtain new colorants from plants and microorgan-isms, but the greater obstacles for the introductionof new colorants are (1) the current legislation; (2)cost of manufacture, which includes that of thebiotechnological culture or production; and (3) theacceptance of unknown materials by consumers.507

IV. SOME IMPORTANT PLANTPIGMENTS: CAROTENOIDS,ANTHOCYANINS, AND BETALAINS

A. Carotenoids

1. Definition

In general, carotenoids are compounds comprisedof eight isoprenoid units (ip) whose order is invertedat the molecule center (Figure 6). All carotenoids canbe considered as lycopene (C40H56) derivatives byreactions involving: (1) hydrogenation, (2) dehydro-genation, (3) cyclization, (4) oxygen insertion, (5)double bond migration, (6) methyl migration, (7)chain elongation, (8) chain shortening.179

2. Classification

Carotenoids are classified by their chemical struc-ture as: (1) carotenes that are constituted by carbon andhydrogen; (2) oxycarotenoids or xanthophylls that havecarbon, hydrogen, and, additionally, oxygen.

Also, carotenoids have been classified as pri-mary or secondary. Primary carotenoids groupthose compounds required by plants in photosyn-thesis (β-carotene, violaxanthin, and neoxanthin),whereas secondary carotenoids are localized infruits and flowers (α-carotene, β-cryptoxanthin,zeaxanthin, antheraxanthin, capsanthin,capsorubin).279 Some carotenoid structures arepresented in Figure 7.

3. Distribution

Carotenoids are the widest distributed groupof pigments. They have been identified in photo-synthetic and nonphotosynthetic organisms: inhigher plants, algae, fungi, bacteria, and at least inone species of each form of animal life. Caro-tenoids are responsible for many of the brilliantred, orange, and yellow colors of fruits, vegetables,fungi, flowers, and also of birds, insects, crusta-ceans, and trout.179,180,182,196,510 Only microorgan-isms and plants can synthesize carotenoids denovo; carotenoids in animals come from thesetwo sources, although they can be modified dur-ing their metabolism to be accumulated in tis-sues.179 More than 300 carotenoids have beenidentified up to 1972, and around 600 up to 1992.Actually, this number has been exceeded by con-sidering that many carotenoids have been isolatedfrom marine organisms.477 Total production ofcarotenoids in nature has been estimated at 108

ton/year, most of which is concentrated in fourcarotenoids: fucoxanthin, in marine algae; andlutein, violaxanthin, and neoxanthin, in greenleaves.200

a. Higher Plants

Carotenoids are accumulated in chloroplastsof all green plants as a mixture of α- andβ-carotene, β-cryptoxanthin, lutein, zeaxanthin,

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violaxanthin, and neoxanthin. These pigments arefound as complexes formed by a noncovalent bond-ing with proteins. In green leaves, carotenoids arefree, nonesterified, and the composition dependson the plant and developmental conditions. Someleaves of gymnosperms accumulate not very com-mon carotenoids in oily droplets, which areextraplastidial: rhodoxanthin in some members ofthe families Cupressaceae and Taxaceae, and semi-β-carotenone in young leaves of cycads. In repro-ductive tissues the following have been found:liliaxanthin in white lily and crocetin in Crocus sp.stigmas; in flowers more than 40 pigments exclu-sive of petals have been identified. Flowers havebeen identified that synthesize: (1) highly oxygen-

ated carotenoids, frequently 5,8-epoxydes; (2) prin-cipally β-carotenes; and (3) carotenoids that arespecies specific (e.g., eschscholzxanthin in pop-pies). Fruits are yet more prodigious in their syn-thetic ability than flowers. More than 70 character-istic carotenoids have been described and havebeen classified as those with minimal quantities,higher quantities, and specific carotenoids, for ex-ample, capsanthin and capsorubin in pepperfruits.180,181,279 Interestingly, carotenoids have beenidentified in wood: samples of oak (Quercus roburL., Quercus petrae Liebl., and Quercus alba L.),chestnut (Castanea sativa Mill.), and beech (Fagussilvatica L.) were studied at different ages andsections. Lutein and β-carotene were identified in

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

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oak wood and also in others deciduous species.These carotenoids could be the origin of β-iononeand more than 30 other norisoprenoid substancesidentified in oak wood. Considering that carotenoidsare hydrophobic and not soluble in sap, it is sug-gested the in situ formation of carotenoids in theliving cells is in the sapwood. It was reported thatsapwood was richer in β-carotene than lutein, andthe ratio was reversed in the heartwood. Also, itwas found that lutein could be used as a marker todistinguish between the wood samples.299

b. Algae

Carotenoids are in chloroplasts as com-plex mixtures that are characteristic of eachclass; the exceptions are Chlorophyta caro-tenoids, which have a tendency to accumulatethe pigments characteristics of higher plants.The red algae Rhodophyta have α - andβ-carotene and their hydroxylated derivatives.In the Pyrrophyta, the main pigments areperidinin, dinoxanthin and fucoxanthin.Chrysophyta accumulates epoxy-, allenic-, andacetylenic-carotenoids, and between them fu-coxanthin and diadinoxanthin. Eutreptielanonehas been found in Euglenophyta. The princi-pal carotenoids in Chloromonadophytaare diadinoxanthin, heteroxanthin, andvaucheriaxanthin. Chryptophyta is character-ized by their acetylenic carotenoids, for ex-ample, alloxanthin, monadoxanthin, andcrocoxanthin. While the Phaeophyta is char-acterized by its main pigment, fucoxan-thin.180,181

c. Bacteria

Approximately 80 different carotenoids aresynthesized by photosynthetic bacteria. Usually,the characteristics of the accumulated carotenoidsare (1) most of carotenoids are aliphatic, but inChlorobiaceae and Chloroflexaceae some caro-tenoids have aromatic or β-rings; (2) aldehydeswith crossover conjugations and tertiary methoxygroups; (3) various classes of carotenoids in eachspecies; (4) all carotenoids are bound to the light-

harvesting complexes or reaction centers in mem-branal systems of bacterial cells; and (5) usuallystructural elements are not found, that is, allenicor acetylenic bonds, epoxydes, furanoxides C45 orC50 carotenoids. In vivo, one of the main groups ofcarotenoids are the sulfates of eritoxanthin sulfateand of caloxanthin sulfates. The sulfates of caro-tenoids are not associated with pigment-proteincomplexes, for example, they are neither part ofthe light harvesting complexes nor of the reactioncenters. In non-photosynthetic bacteria, caro-tenoids appear sporadically and when present,they have unique characteristics, for example,some Staphylococcus accumulate C30 carotenoids,flavobacteria C45 and C50, while some mycobacte-ria accumulate C40 carotenoid glycosides.180

d. Fungi

Carotenoid distribution in fungi, non- pho-tosynthetic organisms, are apparently capricious, butthey usually accumulate carotenes, mono-and bi-cyclic carotenoids, and withoutcarotenoids with ε-rings. For example,plectaniaxanthin in Ascomycetes and canth-axanthinin Cantharellus cinnabarinus has been found.180

4. Biosynthesis: Biochemistry andMolecular Biology

a. Biochemistry

Carotenoids as terpenoids are synthesized bythe isoprenoid pathway (Figure 8).185 Most of thepathways have been elucidated, but more infor-mation is required about the involved en-zymes.179,180,181 Isopentenyl pyrophosphate (IPP)is the common precursor of many of the iso-prenoid compounds (Figure 8), thus sophisticatedmechanisms of control must exist to assure theproduction of the appropriate levels of these com-pounds in the context of the metabolic pathway,developmental stage, and environmental condi-tions.305 Initial steps of the pathway involve thefusion of three molecules of acetyl-CoA to pro-

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FIGURE 8. (A) Pathway of biosynthesis of isoprenoid compounds. (B) Proposed stages for the carotenogenesispathway. (Adapted from Ref. 185.)

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duce 3-hydroxi-3-methylglutaryl-coenzyme A(HMG-CoA). In animals and yeasts these reac-tions are catalyzed by two enzymes, the acetyl-CoA transferase and the HMG-CoA synthase.However, these reactions have not been studiedextensively in plants, but apparently the reactionmust be similar, a cabocationic mechanism. Evi-dence exists that only one enzyme catalyzes bothreactions with Fe+2 and quinone as cofactors.305

The conversion of HMG-CoA to mevalonateis catalyzed by the HMG-CoA reductase. Thisenzyme is very important in animals, limiting thestage of the reaction, thus this enzyme is highlyregulated. The HMG-CoA reductase activity hasbeen detected in endoplasmic reticulum, mito-chondria, and plastids, and catalyzes two steps ofreduction and uses NADPH as a cofactor.305

IPP synthesis from mevalonate is well charac-terized in plants (Figure 9A). Mevalonateis phosphorylated sequentially by the en-zymes mevalonate kinase and after bymevalonate-5-pyrophosphate kinase to form5-pyrophosphomevalonate. This compound suffersa decarboxylating elimination catalyzed bypyrophosphomevalonate decarboxylase that re-quires ATP and a divalent cation.185,305 These enzy-matic activities have been characterizedin plant extracts or in vitro. It seems that mevalonatekinase is feedback regulated by phosphomevalonateand other allylic-diphosphate intermediates. It hasbeen shown in potato that mevalonate diphosphatedecarboxylase is induced by pathogen attack or bytreatment with chemical products. It could be pos-sible that the main controversy is in the subcellularlocalization of these enzymes.185

In 1997 Lichtenhaler et al.280 proposed analternative pathway for the IPP synthesis in thechloroplasts of higher plants. These authors usedlabeled glucose (1-13C-glucose) to grow L. gibba,D. carota, and H. vulgare. They then analyzed thelabeling in sterols produced in cytoplasm andfound that they were synthesized from acetatethrough the mevalonate pathway as it had beendescribed previously. However, the labeling pat-tern of carotenoids, phytyl, and plastoquinone weredifferent (produced in plastids). The pattern sug-gested an alternative pathway that uses pyruvateand 3-phosphoglycerate (from the glycolysis path-way) to obtain D-1-deoxy-xylulose-5-phosphate,

which is rearranged to IPP. Thus, they suggestedthe existence of an alternative pathway for iso-prenoid synthesis in higher plants.

Isopentenyl pyrophosphate is the basic unitfor constructing terpenoids of longer chains.IPP itself is not reactive enough to start thecondensation reactions. Thus, the first step isits isomerization to dimethyl-allyl pyrophos-phate (DMAPP), the reaction is catalyzed bythe IPP isomerase with a divalent metallic ionas cofactor. The next step is the condensationof IPP and DMAPP to form geranyl pyrophos-phate (GPP). After this, two molecules ofIPP are condensed to GPP to obtain thegeranylgeranyl pyrophosphate (GGPP) by ca-talysis with GGPP synthase. The GGPP syn-thase has been purified to homogeneity, and theactivity has been corroborated by in vitro reac-tions.123 The enzyme has requirements for twoions, Mg+2 or Mn+2, per catalytic site. The ion isbonded to the pyrophosphate group of GPP fora better salient group. The ionization of theallylic pyrophosphate leads to the formation ofan allylic carbocation with a stabilized charge.The electrophilic addition of the IPP moleculegenerates the intermediate carbocation of 15carbons that is attacked by another IPP mol-ecule to obtain the GGPP (Figure 9B). BecauseGGPP is a precursor of many other compounds(phytyls, phylloquinones, plastoquinones, taxol,tocopherol, gibberellic acid, diterpenoid phy-toalexins, and diterpenes), it is possible that thetransformation of GGPP, in this branching ofthe pathway, is highly regulated by complexmechanisms and is possible to be compartmen-talized. These assumptions are supported bythe existence of multiple genes for the GGPPsynthase of Arabidopsis.24,93,185,266,305

The first specific step for the synthesis of caro-tenoids is the condensation of two molecules ofGGPP to cis-phytoene (C40). The reaction has theprephytoene pyrophosphate PPPP) as an interme-diate (Figure 10A).185 In tomato and pepper it hasbeen shown that one enzyme phytoene synthase)catalyzes both steps.172 Complexes with IPPisomerase, GGPP synthase, and phytoene synthaseactivities have been isolated.70 In the mechanism,two GGPP molecules are condensed, initially hy-drogen and one of the pyrophosphate groups are

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FIGURE 10. (A) Phytoene biosynthesis and (B) desaturation reactions to form lycopene. (Adapted from Refs.179,185.)

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produced to obtain PPPP. Afterward, the PPPPmolecule is rearranged to form phytoene. Most ofthe organisms (especially higher plants, algae, andfungi) synthesize 15,15′-cis-phytoene. The 15,15′-cis-phytoene has the basic structure of the caro-tenoids, and subsequent reactions involve chemi-cal transformations of this structure. However, mostof natural carotenoids have a trans configuration,and it seems that the cis-trans transformation doesnot require any specific isomerase.14,24,93

Later, phytoene suffers four desaturation re-actions to give phytofluene, ζ-carotene,neurosporene, and finally lycopene (Figure10B).24,179 It has been established that iron couldplay an important role in the desaturation elec-tron-transport chain involved in phytoenedesaturation. This conclusion was reached bygrowing sycamore (Acer pseudoplatanus L.) cellsin an iron-deprived medium, producing a largeaccumulation of phytoene and corresponding dimi-nution of carotenes and xanthophylls.362 Addi-tionally, studies on chromoplasts of Narcissuspseudonarcissus have been establishedthat quinone compounds (plastoquinone/plastohydroquinone) are involved in the redoxreactions in membranes. Membrane oxidationleads to a loss of desaturase activity and the mainredox active components in chromoplast mem-branes, quinones and tocopherols, become oxi-dized. Then, NADPH is able to reactivatedesaturase reactions by the reduction of thesecomponents. By considering that chromoplastsshowed a pronounced NADPH-dependent respi-ratory activity, it is thus considered that phytoenedesaturation and chromorespiration are overlap-ping biochemical phenomena by using in the re-dox process the same quinone pool.2,341

Lycopene is converted to β- or ε-carotene bylycopene cyclases (Figures 11A and 11B); theseenzymes accept other acyclic substrates, such asneurosporene. In Capsicum annuum the direct con-version of lycopene to β-carotene catalyzed bylycopene cyclase has been detected.71 This reactionrequires FAD as a cofactor, and the proposedmechanism is shown in Figure 11A. However, thisreaction is not yet perfectly defined.14,24,179

The hydroxylation reactions occur in an earlystage, but different structural and functional modi-fications (epoxy, furanoxy, and oxy derivatives)

are believed to happen in the last stages of thebiosynthetic pathway (Figure 11C).68,72 The firstxanthophylls are formed from cyclic carotenoids(e.g., β-carotene) by hydroxylation in the 3 and 3′positions of the ionone ring, followed by theepoxydation in the 5,6 and 5′,6′ positions. Byconsidering the high diversity of xanthophylls innature, nowadays a lot of information is requiredto completely define this route (enzymes, reac-tion mechanisms). In pepper, the capsanthincapsorubin synthase that catalyzed the conver-sion of antheraxanthin and violaxanthin to theketocarotenoids capsanthin and capsorubin, re-spectively, has been purified to homogeneity.14,24,69

In 1996, a protein of 43 kDa was purified fromlettuce thylakoids that corresponded to aviolaxanthin deepoxydase. This was the first re-ported enzyme that catalyzes the conversion ofviolaxanthin and antheraxanthin into zeaxanthin.Also, it was established that acidic conditions andascorbate in lumen are required for the reaction.399

Afterward, the violaxanthin deepoxydase fromspinach was isolated and shown that it is a mono-mer of 46 to 50 kDa molecular weight.201

By using Arabidopsis thaliana mutants, it wasshown that plants with deficiencies in carotenoidsynthesis also had low levels of abscisic acid(ABA), and it was suggested that ABA was formedby the oxidative breakdown of epoxyxanthophylls,specifically it was related with violaxanthin andantheraxanthin.24,293 This was supported by thevery recent isolation of genes of the biosyntheticpathway of ABA from Nicotiana plumbaginifolia,Arabidopsis, and maize.383 The fast conversion ofcis-xanthoxin (obtained from epoxyxanthophylls)to cis-ABA was reported in tomato plants, and itwas observed that cis-xanthoxin levels were sig-nificantly induced by water stress in older plants.515

b. Biosynthesis Regulation

Regulation of the carotenoid biosynthesis iscomplex, as been observed for most of the terpe-noids because it seems that regulation must occurat several levels. Evidence shows that carotenoidbiosynthesis is restricted to specific tissues, wherethey are used. Carotenoids are produced in fruitchromoplasts during the maturation process (or-

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FIGURE 11. (A) Proposed mechanism for ring formation of carotenoids. Examples of (B) cyclization reactions oflycopene, 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.)

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gan-specific regulation). Also, it has been deter-mined that GGPP synthase activity is in chro-moplast stroma (tissue-specific regulation).24 En-zymes of carotenoid biosynthesis, and other relatedproteins, are functional in chloroplast/chromoplastbut are codified by nuclear genes; then, the corre-spondent precursors are posttranscriptionally im-ported toward plastids. This could suggest theexistence of transcriptional or posttranscriptionalpathway control. However, although some ge-netic mutants in fruit ripening and carotenoid ac-cumulation have been identified (from maize, to-mato, and Arabidopsis), none of the regulatorygenes have been isolated.24,93

Studies on carotenogenesis provided signifi-cant advances thanks to use of mutants of oxy-genic photosynthetic organisms. However, theidentification of mutants block in the initial stepsof the biosynthesis pathway has proven to bedifficult because of cells with chlorophyll that aresimultaneously exposed to illumination and oxy-gen, in the absence of carotenoids, suffer photo-oxidative damage and then die. Additionally, mu-tations in the final stages are masked byendogenous chlorophylls; thus, few mutants incarotenoid synthesis have been isolated. In plants,the situation is not so discouraging: potential le-thal mutants affecting green photosynthetictissues can survive in heterozygosis, andcarotenogenic mutants in flower and fruits do notnecessarily affect the viability. In particular, acollection of mutants affected in carotenoid bio-synthesis in leaves and endosperm of maize havebeen used.14

Interestingly, exogenous regulatory agents havebeen used to direct the carotenoid synthesis or blockcatabolism; they are attractive approaches for produc-ing fruits or vegetables with improved characteristics.2-(4-chlorophenylthio)-triethylamine hydrochloride(CPTA) and N,N-diethyl nonylamine cause an accu-mulation of the red carotenoid lycopene. 2-(4-ethyl-phenoxy)-triethylamine hydrochloride has been usedto improve orange color by postharvest applicationinduce the accumulation of xanthophylls in endocarp).Esters of 2-diethylaminoethanol induce the accumula-tion of β-carotene, while p-bromobenzylfurfurylamineapplied on grapefruit produces poly-cis lycopeneprolycopene), which has a characteristic orange colorcontrasting with the red color of lycopene.293

c. Molecular Biology of Carotenogenesis

Starting models. Biochemical studies haveoutlined the carotenoid biosynthetic pathway;however, the biochemical approach for studyingthe involved enzymes have shown to be ineffi-cient, because most of them are in membranesand are difficult to isolate. On the other hand,carotenoids are very important for human beings,and scientists work to control the carotenogenesispathway (overproduce or change). It is clear thatpathway control requires a well-defined pathwayand that molecular biology has permitted advancesin this area.

The current knowledge on the molecular bi-ology of the carotenogenesis pathway is derivedfrom studies of specific organisms. Completesequences of clusters of genes for carotenogenesishave been reached in the photosynthetic prokary-otes Rhodobacter capsulatus and Rhodobactersphaeroides. Much data on the carotenogenesispathway have been reported for Synechococcus,and all the genes of some nonphotosyntheticbacteria, for example, Myxococcus xanthus, My-cobacterium aurum, Thermus thermophilus, andsome bacteria Erwinia sp. Also, the pathway iswell defined in the fungi Neurospora andPhycomyces.13,25,160,221,321 It was isolated and char-acterized the Neurospora wc-2 gene that codesfor a second central regulator of blue light re-sponses. The coded proteins represent the firsttwo putative GATA transcription factors thathave been characterized in any organism beinginvolved in light-activated gene regulation ratherthan in a general transcriptional regulation.285

Gene clusters have been observed in somemicroorganisms but not in eukaryotesor cyanobacteria; however, generated informa-tion from microorganisms and fromthe carotenogenesis genetics of higher plantshelped to identify carotenogenic genesof cyanobacteria and of higher plants (Zeamays, Lycopersicon esculentum, Narcissuspseudornarcissus, and Capsicum annuum).14,23,420

Altogether, these models provided the essentialtools to obtain the plant carotenogenesis genesreported up to date (Table 3).422 In general,carotenogenesis eukaryotic genes seem to be inonly one copy with the exemption of phytoene

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TABLE 3Some Genes (genomic or cDNA Clones) Isolated from Plants That Are Directly Related to theCarotenogenesis 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 ● ● ● ● ●

esculentumZea mays ● ●Narcissus ● ● ●

pseudonarcissusNicotiana ● ● ●

tabacumGlycine max ● ●Cucumis melo ●Oryza sativa ● ● ●Brassica ● ●

campestrisCatharanthus ●

roseusLupinus albus ●Nicotiana ●

benthamianaSinapsis alba ● ● ●Haematococcus ●

pluvialisClarkia breweri ●Clarkia xantiana ●Lactuca sativa ●

aAdapted 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-carotenedesaturase; (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-per.90,278,423

Initial stages (Figure 9). Mevalonate syn-thesis is catalyzed by HMG-CoA reductase(HMGR). In animal systems isoprenoid biosyn-thesis is strongly regulated at HMGR level, and itwas suggested that in plants mevalonate-synthe-sis catalyzed by HMGR is also the first major

rate-limiting step. However, plants obtain moreproducts from the mevalonate pathway than ani-mals, thus some tissues and organelles have mul-tiple HMGR genes (Arabidopsis, Hevea, andSolanum). These genes are differentially regu-lated: different genes are expressed in differentparts and developmental stages.272 On the otherhand, one cDNA clone of mevalonate kinase has

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been isolated from Arabidopsis and IPP isomerasefrom some plants and yeasts. All the informationindicates that reactions catalyzed by HMGR,mevalonate kinase, and IPP isomerase are notlimiting steps of carotenoid biosynthesis.93,185,305

The number of plant prenyltransferase genesisolated is very limited, for example, GGPP syn-thase cDNAs from C. annuum266 and fromArabidopsis.421 The pepper cDNA was obtained byusing antibodies against the purified GGPP syn-thase in a cDNA expression library;266 this cDNAwas used as a probe to obtain the Arabidopsisgene.421 However, most of the information aboutprenyltransferases come from animal and micro-bial systems. Prenyltransferases have high homol-ogy between different kingdoms. They are struc-turally and functionally conserved, suggesting thepresence of a common ancestor for prokaryotesand eukaryotes.13 However, GGPP synthase ofpepper chromoplasts segregates with eubacterialprenyltransferases, thus this gene could be trans-ferred from a bacterial symbiont. Also, a consensusregion has been observed, DDXX(XX)D, whichmust have an important role in substrate bond-ing.305 Recently, a new GGPP synthase (GGPS6)gene from Arabidopsis thaliana was isolated. GGPPsynthase showed a strong amino acid homologywith other GGPP synthases. This is the first GGPPsynthase localized in mitochondria; thus, it wassuggested that this isozyme produces the precur-sors of the side chain of isoprenoid quinones, and,importantly, that GGPP synthase could be pro-duced in different organelles rather than organelle-specific production and posterior distribution.528

Phytoene synthesis and desaturation reactions(Figure 10). Tomato phytoene synthase was thefirst cDNA clone obtained for a carotenogenicenzyme that was isolated by using the correspon-dent cyanobacterial cDNA.389 Later on, the exist-ence of two phytoene synthase genes was discov-ered: one leaf-specific (PSY1) and another fruitspecific(PSY2). In contrast to tomato, pepper PSYexpression is not induced by fruit ripening.400 Itwas suggested that differences between these ma-terials could be assigned to differences betweenclimacteric and non-climacteric fruits.24 Schedz etal.423 isolated the PSY cDNA clone from daffodilby using an heterologous tomato PSY1 probe; thegene was in one copy (Southern blot). They re-

ported two enzyme forms: one soluble and inactivein stroma and another active, which are membranebound. The carotenoid content was increased dur-ing flower development; however, the mRNA levelwas constant during the process. Also, there was anincrement of the PSY protein level that was notrelated with activity, because the soluble PSY pro-tein was only increased. It was reported that mem-brane galactolipids are necessary for PSY activity,in particular an aldopyranose is necessary to acti-vate and lipids are necessary to membrane enzymebounding. Additionally, sequence analysis showedthat PSY is membrane integral protein, and it wassuggested the existence of postranscriptional regu-lation mechanisms that involve the membrane re-dox state.423 In 1996,65 phytoene synthase y1 genewas identified from maize and evidence of mul-tiple copies was presented. Interestingly, y1 is re-sponsible of leave and endosperm carotenogenesis.

When tobacco plants were transformed withPSY1 cDNA, an increment was observed in caro-tenoid content but with a reduction in gibberellicacid and chlorophyll levels. This information sug-gested the existence of a common GGPP pool forthese compounds. On the other hand, the cDNAsclones for GGPS, PSY, and PDS were isolatedfrom white mustard (Sinapis alba). It was estab-lished that PSY transcript is up-regulated by lightduring etioplast/chloroplast conversion, but nei-ther GGPS nor PDS. Also, it was shown that thisresponse is mediated by phytochrome in mutantsthe PSY mRNA level was not induced). Thus,enhanced carotenoid biosynthesis should occuronly after the light-dependent development ofstructured chloroplast membranes.498

Phytoene desaturases (PDS) of anoxygenic pho-tosynthetic microbes catalyzes the conversion ofphytoene to lycopene or neurosporene; in contrast,PDS cDNAs of Arabidopsis, tomato, and soybeancatalyzes the transformation phytoene to ζ-carotene.420

In both models, a dinucleotide binding site are iden-tified in their coding sequence.26 Norris et al.343 usedArabidopsis mutants that accumulate phytoene to mapthree loci (Figure 12). Two of these loci do notcorrespond to the PDS gene locus. Thus, at least threegenes are required in the phytoene desaturation; itwas showen that mutants showed deficiencies in plas-toquinone and α-tocopherol accumulation. Then acomplementation assay with quinone biosynthesis

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intermediates (homogenistic acid or plastoquinone)was used and carotenoid production was recovered. Itwas concluded that plastoquinones and/or tocopherolsare essential components for phytoene the desaturationreaction in higher plants. It was proposed that plasto-quinone is an important component in electron trans-portation and ubiquinone was proposed as the trans-porter of anoxygenic photosynthetic organisms, PDSexpressed in E. coli is active, where plastoquinone isabsent. In general, it was concluded that quinones areuniversal adapter molecules in the carotenoiddesaturation reactions.343

Corona et al.86 studied carotenogenesis regula-tion in tomato fruits. During tomato development,carotenoid content was increased (approximately10 times), which corresponded to PDS mRNAincrement. A 2-kb region was identified upstreamof the PDS gene that shows a cis-activation of thechromoplast-specific PDS expression. Also, it wasreported that chromoplast differentiation and PDSexpression are coordinately regulated in petals andtomato fruits, and it suggested the presence of a“chromoplast factor” that activates the PDS pro-moter. This relation was not found in tobacco,which does not have chromoplasts in its leaf struc-ture but accumulates good carotenoid levels. It wasproposed that PDS could be regulated by the endproduct (probably β-carotene, xanthophylls, or ab-scisic acid) that the effect of specific inhibitorsactivates the PDS promoter, and that etioplasts alsoaccumulate carotenoids.86 Recently, maize PDScDNA clone was isolated. This enzyme catalyzesthe transformation of phytoene to ζ-carotene, anheterologous complementation assay was used inthe demonstration. With these results the require-ment of two enzymes for the desaturation ofphytoene to lycopene in higher plants were con-firmed and observed both in mono- and dicotyle-donous plants. Southern blot analysis showed onlyone gene copy. Three possible regulatory locusvp2, and especially vp9, were reported becauserecessive alleles accumulate ζ-carotene. Northernanalysis showed that mRNA level was constantdespite the carotenoid content increments. It waspointed out that maize PDS regulation contrastswith that observed in the temporal control of PSYand PDS of tomato fruits.

Cyclization (Figures 11A and 11B). The ini-tial step to clone a plant β-lycopene cyclase was

the isolation by a complementation analysis ofthe gene of the cyanobacteria Synechococcus.91

By using this sequence, β-lycopene cyclasecDNAs were obtained from tobacco and tomato.364

Protein sequences are conserved between plantsand cyanobacteria but different in bacteria. Theenzyme showed activity in the cyclization ofneurosporene (neu) to β-zeacarotene and of lyco-pene to β-carotene, suggesting that the enzymecould perform two cyclizations. However, β-ly-copene cyclase was not capable of cyclizing ζ-carotene, and it was proposed that the presence ofa double bond in the 7 to 8 or 7′ to 8′ is requiredfor the cyclization process. Sequence analysisshowed FAD or NAD dependence. Also, the en-zyme was expressed in an active form in E. coli,showing that membrane special elements are notrequired. β-lycopene cyclase mRNA decreasedduring fruit ripening, contrary to the observationwith PSY and PDS, and a transcriptional regula-tion mechanism was proposed. Up to date, infor-mation of regulatory mechanisms of fruitcarotenogenesis does not exist, but it is clear thatregulation must be different in relation to leaves.Also, a locus B that seems to be a β-lycopenecyclase regulatory element was identified, al-though its mode of action is unknown.364

As could be expected from the evidence men-tioned above, β- and ε-lycopene cyclases cDNAswere obtained from Arabidopsis thaliana (Figure13).90 The importance of 7 to 8 or 7′ to 8′ insaturationfor the cyclization process was reiterated. It wasfound that ε-lycopene cyclase is a monocyclase thatcannot introduce a second ε-ring where previouslyanother exists. It was concluded that lycopene cy-clization is a key process in plant and algae caro-tenoid biosynthesis, because the relation betweenthese two enzymes gives the balance between cycliccarotenoids with or without an ε-ring. Also, it wasreported that these genes are in one copy.90

Hydroxylation and other modifications(Figure 11C). A. thaliana β-carotene hydroxy-lase cDNA was isolated. Hydroxylation activityof the ε-ring was low, and by considering thedifferences in chirality of β- and ε-rings, the pres-ence of an ε-carotene hydroxylase was sug-gested.376 In addition, the Zeaxanthin epoxydasegene was identified, and it was shown thatepoxyxanthophylls are the precursors of abscisic

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FIGURE 12. (A) Pathway for α-tocopherol and (B) model for the participation of plastoquinone in carotenoiddesaturations. 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 withstars were analyzed. (Adaped from Ref. 343.)

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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 arerepresented by solid arrows. Broken arrows are proposed activities. Parallel lines over the arrow indicate that theenzymatic activity was observed but no product was detected. (Adapted from Refs. 90, 364.)

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acid (ABA). The enzyme catalyzes the conver-sion of zeaxanthin to antheraxanthin andviolaxanthin.295

Carotenogenesis of pepper (Capsicumannuum) fruit is one of the better studied models,and molecular biology has permitted the elucida-tion at the enzymatic level the last biosyntheticsteps for its major carotenoids, capsanthin, andcapsorubin (Figure 14).24

In the elucidation of carotenogenesis regula-tory mechanisms, some interesting genes havebeen isolated. A cDNA that codes for a protein

similar to pepper fibrillin was isolated.490 Thisprotein does not contain cysteines and has severalaspartic and/or glutamic residues arranged in tan-dem; it is peripheral in thylakoid membranes; themRNA level have a temporal and tissue specificregulation and shows increments during flowerdevelopment up to anthesis, then decreases; andmRNA level is higher in corolla. Similar resultswere observed in melon and watermelon (fibrilarchloroplasts), tomato (crystalline chromoplasts),and orange (globulous chromoplasts). It was con-cluded that some apoproteins act as chromoplast

FIGURE 14. Hydroxylation and other modifications in the carotenoid production by pepper fruits. Enzymes involvedin the latter steps are (1) zeaxanthin epoxidase and (2) capsanthin capsorubin synthase. (Adapted from Ref. 54.)

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sequestrate agents of carotenoids, and altogethergene expression of these proteins and those ofPSY1 and PDS genes are strongly regulated at thetranscriptional level. Something similar has beenobserved with chloroplastidial proteins. Interest-ingly, corolla-specific carotenoid-associated pro-tein (CHRC) is highly unstable and shows a fastdisappearance when it is not bounded to caro-tenoids.490 It was established that the CHRC ex-pression is up-regulated in cucumber (Cucumissativus) corollas by GA3. Moreover, carotenoid-associated genes were not affected, and it wassuggested that modulation of carotenogenesis byGA3 is at the level of carotenoid sequestration.Thus, CHRC up-regulation is proposed to be cru-cial for enhanced carotenoid accumulation whilepreserving the structural organization of the orga-nization of chromoplast.491

Nowadays, molecular biology has prospectednew horizons to produce materials with a highcontent of biological active carotenoids (e.g., withvitamin A activity) or to introduce new caro-tenoids.13 The crtI gene of Erwinia uredovoracodes for phytoene desaturase. Nicotiana tabacumwas transformed with crtI via Agrobacteriumtumefaciens. Transformed plants were herbicideresistant: norfluorazon, fluridone, diflufenican,flurtamone, and fluorochloridone were inhibitorsof phytoene desaturase, and KM143-958, J852,and LS80707 interfere with ζ-carotene desaturase.Interestingly, higher levels of β-carotene and itsxanthophyll derivatives (e.g., violaxanthin) andlower levels of lutein were observed in trans-formed tobacco, while total carotenoid contentwas conserved between transformed and controlplants. The meaning of the change in the xantho-phyll pattern have not yet been explained.13 An-other important goal of biotechnology is the pro-duction of important crops of high humanconsumption with enhanced carotenoid content.Rice endosperm does not contain carotenoids, andto obtain plants that produce carotenoids it wasnecessary to introduce carotenogenesis genes. Riceplants were transformed via biolistic withthe phytoene desaturase gene of Narcissuspseudonarcissus; this rice formed phytoene, theprecursor of colored carotenoids. The highphytoene production with transformants was0.74 µg phytoene/g dry seed weight. It was men-

tioned that these results were attractive becausethe goal was 2 µg β-carotene/g dry seed weight tocover the minimal requirements of young chil-dren (100 µg retinol equivalents). Thus, the intro-duction of the next carotenogenesis enzymes (ζ-carotene desaturase and lycopene cyclase) couldgenerate rice transformants with good levels of β-carotene.13,66

5. Functions

a. Color

Carotenoids provide colors to flowers, seeds,fruit, and to some fungi, and color has an impor-tant role in reproduction: coloration attracts ani-mals that disperse pollen, seeds, or spores. InPhycomyces blakesleanus it was observed thatintracellular accumulation of excess carotenoidsdisturb the mating recognition system, which ap-pears to be involved in the later stages of matingby inhibiting the cell-to-cell recognition systems.351

b. Photosynthesis

Main pigments involved in photosynthesisare chlorophylls and carotenoids. Carotenoidshave two well-known functions in photosynthe-sis: (1) accessory pigments in light harvesting,and (2) as photoprotectors against oxidative dam-ages. It has been proposed that carotenoids aslight harvesting compounds evolved from anaero-bic organisms, then generalized to all of theaerobic photosynthetic organisms. One of thecarotenoid structural characteristics is their abil-ity to absorb visible light: p delocalized elec-trons suffer a photoinduced transformation inwhich a singlet state (s2) is produced, then en-ergy is efficiently transferred to chlorophyll (chl)to form singlet chl with a slightly higher energy.The physical structure of chloroplasts facilitatethe transference of energy absorbed by caro-tenoids to chl. In thylakoidal membranes, caro-tenoids are bound to chls and proteins to formspecific complexes called photosystem I (PSI)and photosystem II (PSII). It is known that PSIis a pigment protein complex functioning as a

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plastocyanin: ferredoxin oxidoreductase. Thisprocess is highly endergonic (∆G° = 77 kJ mole-

1) utilizing light energy to drive the reaction. PSIcomplex is composed of 11 to 17 differentpolypeptide subunits, about 90 chl “a” molecules,10 to 15 β-carotene molecules, two phylloquinonemolecules, and three [4Fe-4S] clusters.424 PSIIfunctions as a water-plastoquinone oxidoreduc-tase. It is a membranal complex that comprisesmore than 25 different proteins, and the heart ofthe complex is the reaction center (RC) consist-ing of the D1 and D2 proteins. In all the redoxprocess, quinones have an important participa-tion. It has been suggested that all pigmentsinvolved in photosynthesis are bound to proteinsof the individual thylakoid membranes, and cer-tainly in bacteria it has been established thatbacteriochlorophyll molecules are bound to Hisresidues of α and β apoproteins. Even less isknown about how carotenoids are bound to thephotosynthetic apparatus, but it has been pro-posed that carotenoids span the membrane, withthe groups interacting with polar amino acidsnear the membrane surface, and the middle partsmaking van der Walls contacts with hydropho-bic side groups of the protein.21 Main carotenoidin PSI is β-carotene and lutein in PSII. In PSII,all β-carotene is located in the complex nucleus,very near of the reaction center, while xantho-phylls are bound to the remaining chl “α” and“β” molecules in the energy-harvesting an-tenna.14,61,81,179,314,349 Wild-type Arabidopsisthaliana and ABA-3 mutant, which lacks ofepoxydase activity (contains only lutein and ze-axanthin), were used to study the function ofcarotenoids in the light harvesting complex II(LHCII). It was found the same chl a/b ratio inboth wild-type and ABA-3 mutant, but differ-ences were detected in the carotenoid levels.The wild-type ratio was 7/5/2/1 chl a/chl b/lutein/neoxanthin, whereas in ABA-3 mutant it was7/5/1.4/0.6 chl a/chl b/lutein/zeaxanthin. Thisstudy showed that the predominant sequentialenergy transfer is from carotenoids (predomi-nantly lutein) to chl “b” and then to chl “a” inwild-type Arabidopsis. In contrast, mutantsshowed substantial carotenoid to chl “a” energytransfer. It was suggested that zeaxanthin is thecomponent in ABA mutant that transfers energy

to chl a.84 The photosynthetic efficiency, O2 evo-lution, and nonphotochemical quenching wassimilar between mutants and wild type. How-ever, under strong light stress and long exposi-tion time, the ABA mutants showed an acceler-ated photoinhibition compared with wild-typeleaves.139

Carotenoid functions are greatly determinedby their associated proteins. These proteins aremainly membranal, usually hydrophobic, whichbound carotenoids by noncovalent bonds.183

Also, it has been established that protein cor-rect folding in the photosynthetic complexesrequires chl and carotenoids.186 A cDNA of pro-tein that bound fucoxanthin-chl was isolatedfrom Heterosigma carterae. This proteinshowed a high similarity with proteins that bindchl a/b. With this evidence, divergent evolutionwas suggested, while the energy-harvestingcomplexes of algae evolved their ability to bindaccessory pigments (chl’s and carotenoids) in-dependently to increase their absorption spec-tra and consequently to have a more efficientenergy utilization.131

On the other hand, Phaffia rhodozyma exposedto singlet oxygen increased the astaxanthin contentas a protection mechanism.428 Phytoene desaturasemutants of Chlamydomonas reinhardtii showed se-vere defects during PSII assembly, PSII repair dur-ing photoinhibition, degradation of D1 protein, andin the binding of D1 with PSII. Cells without β-carotene did not show PSII activity, and they did nothave D1 protein. It was concluded that, in additionto its role in photosynthesis and photoprotection β-carotene stabilizes PSII or permits the correct as-semble of D1 and PSII, protecting D1 of a fastdegradation. Also, it was proposed that phytoenedesaturase is plastoquinone dependent; plastoquinoneredox state could limit β-carotene biosynthesis, whichlimits the PSII assemble, and this regulates the plas-toquinone redox state. Consequently, PSII could beadjusted to illumination conditions by a fast turn-over of β-carotene, D1 protein, and its correspond-ing assemble into PSII.471 In addition, it has beenshown that synthesis of D1 protein increases duringthe transference of Chlamydomonas cells from lowlight to high light conditions, and it was suggestedthat it tends to favor the mitigation of photoinhibitionand to ensure the function of photosystem II.436

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c. Xanthophyll Cycle

When leaves are exposed to high illumina-tion, epoxy xanthophyll groups are removed ofviolaxanthin to initially form antheraxanthin andthen zeaxanthin. This is one of the plant protec-tion mechanisms against light damage. The num-ber of carotenoid molecules is higher in sun-ex-posed leaves than darkness maintained leaves.Also, xanthophyll cycle carotenoids (violaxanthin,antheraxanthin, and zeaxanthin) are increased insun-exposed leaves. This phenomenon is veryimportant, sun exposed leaves in a fast-growingstage use not more than 50% of absorbed energyduring the stage of maximum radiation (midday),and in some species only 10% is used. Thus, 50to 90% of absorbed light is in excess and must beeliminated in order to avoid cellular damage. Xan-thophyll cycle is a process that makes the energydissipation easy and protects the photosyntheticapparatus. It has been established that energy trans-ference from chl to zeaxanthin is theoreticallypossible, and this gives support to the observedzeaxanthin increments under high illumination.Moreover, xanthophyll cycle carotenoids are as-sociated with the energy harvesting complexesPSI and PSII.14,349

Xanthophyll cycle involving violaxanthin,antheraxanthin, and zeaxanthin is ubiquitous ofhigher plants and green and brown algae. InDunaliella salina Teod. and Dunaliella bardawilthe accumulation of β-carotene has been observedin response to a combination of high light, hyper-salinity, and nutrient stress. Also, substantialamounts of zeaxanthin and a continued operationof the xanthophyll cycle have been observed. In-terestingly, during diurnal illumination variations,stressed (nutrient and high salt concentration) cellsdid not show high xanthophyll levels at midday incontrast with nonstressed cells. It was explainedthat in stressed cells high levels of xanthophyllsare maintained, which work as an adaptative func-tion to protect the photosynthetic apparatus.369

d. Antioxidant

In vivo and in vitro studies have shown thatcarotenoid photoprotective role is related to its

antioxidant activity or with modulation of othercellular antioxidants. Also, it has been establishedthat carotenoid structure has a great influence inits antioxidant activity; for example, canthaxanthinand astaxanthin show better antioxidant activitythan β-carotene or zeaxanthin.281,300,355 Chloro-phyll-sensitized photooxidation (4000 lx) ofsoybean oil was studied in the presence ofcarotenoids (lutein, zeaxanthin, lycopene,isozeaxanthin, or astaxanthin). It was found thathigher carotenoid concentrations decreased theperoxide value of soybean oil by quenching sin-glet oxygen, and it was shown that longer chro-mophores favor this reaction. Thus, astaxanthinwith 13 conjugated double bonds was the best (inmethylene chloride).274 Packer352 evaluated theantioxidant activity of different carotenoids by invitro assays. It was reported that antioxidant ac-tivity depends on the used system.352 Other stud-ies indicated an isomer-specific activity of caro-tenoids: using in vitro assays, it was determinedthat cis-lycopene isomers showed better absorp-tion than β-carotene and were more efficient insinglet oxygen quenching.447

The antioxidant activity of lutein, lycopene,annato, β-carotene, and γ-tocopherol was evalu-ated on triglycerides by the effect of air and light.It was reported that lutein, lycopene, and β-caro-tene act as prooxidants, favoring the formation ofhydroperoxides; however, if a small quantity ofγ-tocopherol is added to these pigments, the phe-nomenon is reverted and they act as antioxidantswith a higher activity than γ-tocopherol. Annatoshowed antioxidant activity, however, this couldbe assigned to unknown fluorescent compounds.189

β-Carotene also showed prooxidant activity inoil-in-water emulsions evaluated by the forma-tion of lipid hydroperoxides, hexanal or 2-heptenal;the activity was reverted with α- and γ-tocopherol.With this evidence, it was suggested that toco-pherols effectively protect β-carotene against radi-cal autoxidation.204 Antioxidant activity ofcapsanthin and lutein was evaluated using chloro-phyll as photosensitizer. Capsanthin was a betterantioxidant, and it was concluded that the antioxi-dant activity depended on the number of doublebonds, keto groups, and that cyclopentane ringsin the carotenoid structure enhanced their activ-ity. It was suggested that carotenoids can be used

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in foods to prevent degradation of other compo-nents.100 Reactivity of singlet and triplet stateswas evaluated in carotenoid photodegradation; itwas found that canthaxanthin is more stable thanβ-carotene. β-Carotene could be degraded in oxy-gen absence (in toluene), but canthaxanthinshowed good stability. It was established thatcarbonyl groups facilitate energy transference bygiving greater stability because they have lessdiradical character, thus transformation betweenexcited states is easier. Also, it was suggested theuse of ketocarotenoids as food antioxidant. Inter-estingly, research has shown than astaxanthin is abetter agent to destroy free radicals than othercarotenoids.340 Miller et al.315 evaluated carotenoidantioxidant activity against radicals and estab-lished the following order of activities decreas-ing): lycopene > β-cryptoxanthin > lutein = zeax-anthin > α-carotene > echineone > canthaxanthin= astaxanthin. Lycopene showed three times moreactivity than γ-tocopherol, and it was concludedthat antioxidant activities are influenced by po-larities that are increased with the presence offunctional groups in terminal rings.315

e. Pharmacological Effects

Many diseases, such as cancer and strokes,involve oxidative processes mediated by free radi-cals. Carotenoids, by their antioxidant effect, canshow benefits in such diseases; however, this func-tion is not completely demonstrated in vivo. Caro-tenoids are an integral part of membranes.Carotenes are immersed in membranes, but xan-thophylls showed a variable membranal position,polar groups in xanthophylls affect their positionand mobility. Consequently, carotenes are able toreact efficiently only with radicals generated in-side the membrane. While zeaxanthin, with theirpolar groups aqueous exposed, is able to reactwith radicals produced in that zone. Additionally,it has been suggested that carotenoids influencethe strength and fluidity of membranes, thus af-fecting its permeability to oxygen and other mol-ecules. Also, it has been determined that caro-tenoids have a remarkable effect in the immuneresponse and in intercellular communica-tion.61,79,94,217 β-carotene, canthaxanthin, 4-hy-

droxy-β-carotene, and the synthetic retro-dehydro-β-carotene show an efficient induction of the gapjunctional communication (GJC) in murine fibro-blasts. The GJC stimulation was more than fivetimes, depending on the carotenoid. β-Caroteneand retrohydro-β-carotene were the most efficientinducers of GJC, indicating that the presence of asix-member ring is important in the GJC induc-tion; carotenoids with five-member rings showedlittle activity. Interestingly, the induction of GJCdid not show correlation with the quenching ofsinglet oxygen. Thus, it was suggested that GJCand antioxidant activities in cancer preventionoperate independently of each other.448 The im-portance of carotenoids with rings of six mem-bers was emphasized by the isolation of a caro-tenoid-binding protein (CCBP) of 67 kDa fromferret liver. Carotenoids with substituted β-iononerings, without β-rings, or with an intact β-ringwith a shorter lineal chain were not bonded byCCBP. Consequently, it was showed that CCBPbinds β-carotene mole per mole with high effi-ciency and specificity. Thus, CCBP may play amajor role in the storage, transport, and targetingof β-carotene in mammalian systems. Addition-ally, it was suggested that the high affinity be-tween CCBP and β-carotene may protect the caro-tenoid from degradation, and its antioxidantactivity must be better.388 There exists evidence ofthe effectiveness of β-carotene in the treatment ofcertain kinds of cancer, for example, smoking-related cervical intraepithelial neoplasia and cer-vical and stomach cancer.94 Siefer et al.438 showedthat β-carotene affects the immune response inrats, and by this means tumor growth is inhib-ited.438 In contrast, it was reported that retinoicacid and citral act as suppressors of this system.However, it was demonstrated that retinoic acidregulates the γ-interferon (IFN-γ) gene, which hasan important role in practically all the stages ofthe immune and inflammatory responses.78,217

More than 600 carotenoids are known, and 50of them are consumed in meals to be transformedinto the essential nutrient vitamin A. After theirabsorption, these carotenoids are metabolized byan oxidative rupture to retinal, retinoic acid, andsmall quantities of breakdown products. Caro-tenoids are transported by plasma lipoproteins.Carotenes are mainly associated with low-density

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lipoproteins, while xanthophylls show a uniformdistribution between the low- and high-densitylipoproteins.360 Vitamin A is required in the vi-sion process, epithelial maintenance, mucose se-cretion, and reproduction.348,385,460 In the processof senile macular degeneration, retinol is relatedto the induction of a gene cascade permitting thephagocytosis of damaged cell in retina; this pro-cess is critical for the photoreceptor survival.138

Also, it has been established that retinoids affectmany biological process, such as cellular prolif-eration, differentiation, and morphogenesis. More-over, retinoids have been used in treatments ofcertain kinds of cancers and some dermatologicalactivities. Additionally, it is mentioned that a dietwith deficiencies in vitamin A or supplementedwith an excess of retinoic acid could induce ter-atogenesis.306

Differentiation is a complex process, duringdevelopment of the monoblastic cell line U-937,the activation of both the retinoic acid receptor(RAR) and the 9-cis-RA receptor (RXR) are re-quired. Thus, it was suggested that different com-binations of retinoids produced different pharma-cological responses in the specificity and potencyin cancer therapy.53,133,187 Sun et al.453 found thatretinoic acid induces the response of proteins as-sociated with damage by ultraviolet light in F9and NIH3T3 cells. However, this induction doesnot show a correlation with the repairer of DNAdamage.453 Another research showed that retinoicacid conduces to differentiation of F9 cells by anincrement of cellular communication. It was sug-gested that this process is mediated by the proteinconnexin 43 that induces the expression of impor-tant molecules for cellular adhesion.79,94,133 Cur-rently, the effect of retinoic acid in arthritis treat-ment is controversial.267 In these studies, syntheticretinoic acid (Am-80) was used, and a suppres-sion in the incidence of arthritis, back-feet swell-ing, and bone destruction in collagen-treated ratswas observed. However, 13-cis-retinoic acid didnot show the above-mentioned effects, and thissuggest that Am-80 could pertain to a family ofantiinflammatory compounds but shows some sec-ondary toxic effects similar to those observedwith other retinoids.267 Ang et al.9 studied miceembryogenesis and found that retinoic acid has animportant role during the gastrulation process. In

that stage, alcohol dehydrogenase class IV wasidentified, and it was proposed that negative ef-fects of alcohol consumption during fetus devel-opment could be caused by the inhibition inretinoic acid synthesis, catalyzed by alcohol de-hydrogenase, which conduces to a failure in func-tion of retinoic acid receptor (necessary for nor-mal development).9

Retinoic acid (RA) has also been related tothe aging process. RA affects the gene expressionlevels through its interaction with triiodothyro-nine receptor (TR) and RAR. Interestingly, whenthe TR and RAR mRNA levels were analyzed inthe brain of young, adult, or aged rats, lowervalues were found. Additionally, it was showedthat RA supplementation increased the TR andRAR mRNA levels. Also, the transglutaminaseactivity was reduced in aged rats and recoveredafter RA treatment. Transglutaminase has involvedthe memory process.385

Prostaglandins are substances that have beeninvolved in several physiological process, and inparticular prostaglandin D2 has been involved inthe endogenous sleep promotion, modulation ofseveral central actions (regulation of body tem-perature, release of the luteinizing hormone), etc.Recently, it was shown that prostaglandin D (PGD)synthase binds retinoic acid and retinol. In addi-tion, it was demonstrated that all-trans-retinoicacid inhibits its enzymatic action but not retinol.With the generated information, it was suggestedthat retinoids may regulate the synthesis of PGD2,and that PGD synthase may be a transporter ofretinoids to the place where they are required.Thus, it was suggested that PGD synthase plays acritical role in regulating the development of neu-rons by the regulation of the transfer of all-trans-or 9-cis-retinoic acid to RAR or RXR in the im-mature nerve cell.457 Additionally, it has beenmentioned that the overexpression of thecyclooxygenase gene (a key enzyme in the forma-tion of prostaglandins) is an early and centralevent in colon carcinogenesis.217

In contrast with that discussed above, it wasshown in transgenic mice that inhibition of thetranscription activator protein-1 (AP1) has an im-portant antitumor promotion activity, but the ac-tivation of the retinoic acid-responsive elementhas not. AP1 regulates the expression of genes

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with the recognition sequence designed as 12-O-tertadecanoylphorbol-13-acetate (TPA)-respon-sive element (TRE) in their promoter region, andmany TPA-responsive genes include severalprotooncogenes (e.g., c-fos, c-jun). Thus, the de-sign of specific inhibitors of the transcription ofAP1may be an interesting strategy in the treat-ment of cancer.227

Carpenter et al.75 observed that mixtures ofcanthaxanthin with low-density lipoproteins (LDL)inhibited macrophage formation from humanmonocytes. However, if canthaxanthin and LDLwere added simultaneously (but without previousmixture) to cellular medium, it was not observedto any effect. The same was noted with β-caro-tene, but with zeaxanthin the opposite was found.It was explained that all evaluated carotenoidsshow good antioxidant activity, and observed dif-ferences are caused because they act at differentlevels: zeaxanthin can quench radicals in the aque-ous phase, while β-carotene inhibits lipidperoxidation; on the other hand canthaxanthinwas the most potent agent in the inhibition ofmethyl linoleate, and it was concluded that anti-oxidant activity depends on the particular system:radical, carotenoid, microenvironment, etc. Thus,diets with carotenoid mixtures are recommendedinstead of having just one particular carotenoid,because in vivo a great variability of radicals andmicroenvironments take place.

Carotenoids have been considered that pro-vide benefits in age-related diseases, against someforms of cancer (in especial lung cancer), strokes,macular degeneration, and cataracts. However,most studies relate dietary components with sick-ness incidence or symptoms; thus, these studiescannot establish a direct cause-effect relationship.On the other hand, it is clear that carotenoids inassociation with other components of fruits andvegetables seem to have a protective effect againstsome chronic diseases and precancerous condi-tions. Additionally, in some studies β-carotenewas supplied to smokers, and it was found thatcancer mortality indexes were higher in smokersthan in their respective controls.14,349,460,529 It hasbeen signaled that a combined supply of β-caro-tene, α-tocopherol, and selenium reduces stom-ach cancer mortality, and it was pointed out thatthe consumption of marine algae (especially

Phaeophyta) diminished the risks of being af-fected by certain types of cancer. Also, it wasestablished that the main antitumor agent is notβ-carotene, but other components (carotenoids)that are present in algae. In this sense, mixtures ofcarotenoids (α-carotene, fucoxanthin, andhalocintiaxanthin) have shown a higher inhibi-tory activity than β-carotene in proliferation ofhuman neuroblastoma cells. In addition, α-caro-tene showed higher antitumorigenic activity thanβ-carotene in rat cancer induced by glycerol, andit was mentioned that carotenoids with an ε-ring(absent in β-carotene) have higher inhibitory ac-tivity.94,337

The antimutagenicity of carotenoids in Mexi-can green peppers Capsicum annuum) were stud-ied. The experiment was carried out by determin-ing the number of revertants in the plateassay with Salmonella typhimurium. Theantimutagenicity inhibition by nitroarenes washigher than 90%. Pepper carotenoids were moreefficient antimutagens than pure β-carotene, sug-gesting that other carotenoids (e.g., lutein, zeax-anthin) in the pepper extracts showed a synergis-tic effect with β-carotene. Also, it was mentionedthat the antimutagen activity might be from block-ing the entrance of toxic compounds into the cellor by their antioxidant activity.384 Also, theantimutagenicity activity of carotenoids of Aztecmarigold (Tagetes erecta) was evaluated. It wasconcluded that lutein was the compound with ahigher activity on marigold extracts, but similarto the observation mentioned for the pepper ex-tracts, the mixture of carotenoids in the marigoldextract had higher antimutagenicity activity. Inaddition, it was suggested that lutein and1-nitropyrene (mutagen) formed an extracellularcomplex that limits the bioavailability of1-nitropyrene and consequently its mutagenicity.177

Böhm et al.44 indicated that carotenoids,α-tocopherol radicals, and ascorbic acid developtheir function by diminishing the content of harm-ful nitrogenous compounds. Also, a synergisticeffect between β-carotene and vitamins E and Cwas observed in cellular protection. It was ex-plained that β-carotene not only destroysoxyradicals but repairs tocopherol radicals pro-duced when α-tocopherol destroys oxyradicals.Additionally, it was suggested that low antioxi-

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dant levels (e.g., ascorbic acid) in smokers, incontrast with non-smokers, could be related withan apparent failure in the recycling of α-toco-pherol by β-carotene.44

Carotenoids protect lab animals of UV-in-duced inflammation and certain type of cancers.Historically, carotenoid supplementation has beenused in the treatment of diseases produced bylight sensitivity, which are usually hereditary: 84%of patients with erythropoietic protoporphyria,consuming diets supplemented with β-carotene,increased by a factor of 3 their ability to resistsunlight exposition without presenting symptoms.Also, carotenoids have been used in other photo-sensitivity diseases: congenital porphyria,sideroblastic anemia, and have shown only a lim-ited success in treatment of polymorphic lighteruption, solar urticaria Hydroa vacciforme, Por-phyria variegata, Porphyria cutanea tarda, oractinic reticuloid.300,529

Lutein and zeaxanthin have been consideredas protective agents against aging macular degen-eration and senile cataracts.460 Also, it has beensuggested that β-carotene suppress the incrementof hormones related to stress syndrome.197

Certainly, up to date studies on anti-carcino-genic activity have produced unexpected results.Massive studies the α-tocopherol, β-carotene[ATBC] cancer prevention study, the β-caroteneand retinol efficacy trial [CARET], and the phy-sicians health study) gave no results or do notinclusively give a higher incidence of cancer.Nowadays it is thus premature to enunciate finalconclusions regarding the potential role of caro-tenoids in the therapeutics of degenerative dis-eases. However, the consumption of fruits, veg-etables, and fortified foods with antioxidants isencouraged.94

6. Methodological Aspects

It is evident that new methodologies usuallygive better results and confidence when identify-ing and quantitating the carotenoid components,an important aspect if we consider that biologicalactivity of different carotenoids is different, forexample, not only β-carotene is source of vitaminA, and not all carotenoids show this activity.

Many methodologies used in the analyses oforganic compounds are inadequate for carotenoidanalyses. Their study is complicated by the greatvariability in nature, the existence of many isomers,great structural similarity, instability, presence ofsubstances that interfere the determination, and theabsence of an exact quantitation method.252,480

The stages to be considered for carotenoidstudies are extraction, saponification, separation,identification, and quantitation, and the develop-ment of a good work depends on our knowledgeof carotenoid properties:61,112

1. Carotenoids are lipids and thus soluble inother lipids or in nonpolar solvents.

2. Carotenoid color is imparted by conjugateddouble bonds that are mainly in trans con-figuration (in their natural form), in ex-tended form; hence, they are lineal andrigid molecules. The properties of cis-caro-tenoids show substantial differences withthe trans ones; they are more easily solubi-lized, absorbed, and transported. Factorsgenerating isomerization are free halogens,acids, excessive light, and high tempera-tures.

3. Conjugated double bonds in carotenoids arehighly delocalized, and consequently caro-tenoids have a low-energy excited state.Hence, the energy of transition is in thevisible region (400 to 500 nm); thus,they areintensely colored yellow, orange, or red).Also, because of this property carotenoidsare involved in photosynthesis andphotoprotection.

4. The polyene structure of carotenoids makesthemselves highly reactive molecules. Thestructure is rich in electrons and suscep-tible to be attacked by electrophilic reagentsresponsible for carotenoid instability againstoxidation and give them their radical char-acteristics. Carotenoids in the crystallinestate are susceptible to oxidation after theirisolation; their degradation is very quick ifstored in the presence of oxygen traces.Electrons are delocalized but not with auniform distribution, electronic density ishigher at molecule ends, and these are theirmore reactive part: deepoxydation reaction

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preferentially occurs in carbons 7 and 8,diminishing the reactivity by the presenceof keto groups (astaxanthin andcanthaxanthin).

a. Extraction

With solvents. Carotenoids are soluble inlipids or in nonpolar solvents, except when theyform complexes with proteins and sugars.Hence, they are extracted with nonpolar sol-vents. If the tissue is previously dried, thenwater-immiscible solvents are used such aspetroleum or ethyl ether; with the fresh materi-als acetone or ethanol are used, which have twofunctions, extracting and dehydrating solvents.Solvents used in extraction must be pure (with-out oxygen, acids, halogens) to avoid degrada-tion. Up to now, no solvent is optimal for theextraction of all carotenoids: carbon disulfideis the best solvent, but volatility, flammability,toxicity, and degradation limit its use. Chloridesolvents are good, but they show high toxicity;free peroxide ether, despite its efficiency, is notused because of its flammability and volatility;other solvents such as hexane, heptane, andisooctane are not so good for extraction, buttheir other characteristics are favorable. On theother hand, it must be considered which com-pounds will be extracted: polar solvents (suchas acetone, methanol, ethanol) are good withxanthophylls but not with carotenes. As a gen-eral rule, the extraction process consists of theremoval of hydrophobic carotenoids from anhydrophilic medium. The use of nonpolar sol-vents is not recommended because of penetra-tion through the hydrophilic mass that surroundspigments is limited, while slightly polar sol-vents dissolve poorly carotene in dried samplesand solubility diminish in fresh samples. Thus,it was postulated that complete extraction canbe reached by using samples with low mois-ture, and slightly polar plus nonpolar solvents.112

Literature describes different solvent systemsused in carotenoid extraction. In general, ex-traction must be carried out very quickly, avoid-ing contact with light, oxygen exposure, and

high temperatures in order to minimize degra-dation.60

Currently, industrial extraction consists ofpressing and solvent extraction of materials:material is milled and pelleted, mixed withhexane, and heated in a special recipient cov-ered with a vapor jacket; hexane is eliminatedin a film evaporator and afterward by vacuumdistillation, and the main problems to be solvedare to diminish pigment degradation, increaseextraction performance, and solve safety andenvironmental problems. More than 50% of thepigments is lost during this extraction process;oil extraction industries emitted into the envi-ronment 210 to 430 million liters of hexane thattogether with other organic compounds canproduce nitrogen oxides and other pollutants.These products in the last stage generate ozoneand other highly dangerous photochemical oxi-dants.403 With these perspectives, several re-searchers have proposed alternative extractionsolvents. Heptane has been used, and the fol-lowing characteristics have been mentioned:lower volatility than hexane, similar extractionefficiency, and good quality product (oil).83

When mixtures of heptane-isopropanol or onlyisopropanol are used, more antioxidants are ex-tracted and oils with enhanced stability are ob-tained, and considering that isopropanol haslower flammability than hexane, this solventcould be a good alternative in oil extraction.247,381

Enzymatic and/or aqueous extraction. Foodindustries have used enzymatic methods to obtaina diversity of products: maize starch, gluten andstarch of wheat, gelatin, deboned meat, amongothers. The main advantages of these proceduresare specificity, moderated temperature and pH,treatments are mild, secondary products are scarce,and the final product is almost not affected. Inenzymatic processing, enzymes with mixed ac-tivities are used because of cell wall complex-ity.106,119,132,298,353 Successful application of en-zymes have been reported in oil extractions, andextraction performance increments in the range50 to 90% have been reported, and byproductshave shown higher digestibility than control.124,403

Aqueous extraction has been proposed since1950 as an alternative to organic solvent. Thistechnology was implemented because of safety

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and the cheapness of the process, which is basedon oil-water insolubility and phases are separatedby differences in density. Up to now, many re-ports have been published on extraction by simul-taneous enzymatic and aqueous processes: in oilextraction of avocado, olive, coconut, rapeseed,maize, cocoa, among others.52,97,106 However, re-ports on the application of this technology inpigment extraction are scarce. Pommer377 proposedan extraction method of vegetable oleoresins, andbetween them pigments where plant material ismixed with water and an water-immiscible or-ganic acid. Organic acid was selected with a lin-ear chain of 6 to 12 carbons. Optionally, enzymesare used in the extraction process, and pigment-extraction efficiency was enhanced for pigmentsof marigold (Tagetes erecta) flowers. On the otherhand, Delgado-Vargas and Paredes-López120 re-ported a procedure in which enzymatic treatmentand aqueous extraction were carried out in orderto obtain marigold flower meals with a highercarotenoid content.

Supercritical fluid extraction (SFE). Theabove-described method appears to have the re-quirements to be considered a good extractionprocess: fast, simple, and cheap. However, it gen-erally implies that considerable time is needed forit to be carried out, and a concentration stage maybe necessary as well. Consequently, SFE has beenconsidered to perform a better extraction process.

Up to 1986, only two works using SFE hadbeen reported, but in the period 1986-mid-198926 works were published; this large incrementcould be explained because this technique showsthe following advantages: rapidity, solventstrength can be controlled, and the solvents usedare gases friendly to the environment and withlow toxicity. Interestingly, with this method theconcentration stage is avoided because solventsare eliminated immediately under environmen-tal conditions.104,203,387 With these characteris-tics, SFE has been used commercially to obtaincaffeine from coffee and hop oil.387 Lutein andcarotene were extracted from protein leaf con-centrate by using CO2 as a solvent in SFE, andit was shown that conditions can be manipulatedto make a selective extraction.143 β-Carotene SFEof sweet potato was three to five times moreefficient than traditional methods, but isomer-

ization was observed with this extraction pro-cess.445 SFE and traditional extraction processeswere compared in the extraction of carrot caro-tenoids. The highest efficiency was obtained byusing ethanol (10%) as a co-solvent, and it wasreported that the traditional method extracts moreα-carotene than SFE. Interestingly, vitamin Aequivalents obtained by SFE were higher thanby traditional methods and extraction time wasdiminished from 6 to 1 h.22 Despite the abovementioned, no commercial process of pigmentSFE is in use nowadays.

b. Saponification

As mentioned above, xanthophylls are usu-ally esterified,279 which produces additional analy-ses complications, for example, a pigment withtwo hydroxyl groups can be without one or twopositions esterified, which requires both separa-tion and identification. Thus, saponification ob-tains less complex mixtures when onlynonesterified pigments appear. Another advan-tage of saponification is chlorophyll destructionin the saponified samples.112

Most carotenoids are stable under alkalinetreatments; thus, the use of methanolic solutionsof potassium hydroxide is a common method ofsaponification, sometimes at environmental tem-perature or by heating.11,59,179,252 When carotenoidsare sensitive (e.g., astaxanthin and fucoxanthin),alternatively lipases have been used.59 Candidacylindracea lipase has been used in the saponifi-cation (darkness, under nitrogen atmosphere, and4 h) of red palm oil and neither loss nor isomer-ization of β-carotene was observed.282 In caro-tenoid extraction from paprika, it was reportedthat carotene is sensitive to alkaline saponifica-tion, thus mild conditions were evaluated andsamples were saponified with potassiummethoxide.237

c. Separation

Separation methods can be classified asnonchromatographic and chromatographic.Nonchromatographic method uses mainly phase

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partition, for example, by using petroleum etherand aqueous methanol (90%). Carotenoids aredissolved and nonpolar compounds recovered inepiphase, petroleum ether.59 Also, this methodhas been used in lutein purification by usinghexane:acetone:toluene:absolute ethanol (10:7:7:6v/v) and then hexane.478 Another example:xanthophyll esters are separated by Craigcountercurrent distribution using dimethyl-formamide:dichloromethane:hexane (8:2:10 v/v)as solvent.166

In chromatographic methods adsorbents havebeen used such as sucrose, cellulose, starch,CaCO3, Ca3(PO4)2, Ca(OH)2, CaO, MgCO3, MgO,ZnCO3, Al2O3, silicic acid, silica gel, kieselguhr,Microcel C, and mixtures. A general strategy forobtaining a pure carotenoid uses open columnchromatography in alumina followed by thin layerchromatography (TLC) in silica, MgO-kieselguhrG TLC, and silica TLC again, with different sol-vent systems. The criteria for choosing a solidsupport depends on the carotenoid to be purified;for example, alumina must not be used to separateastaxanthin because of oxidation problems; addi-tionally, alumina can produce isomerization ofother carotenoids. Magnesium oxide is not rec-ommendable when acetone is used as solvent; apossibility of solvent polymerization reactionexists. Alumina and silica separations are basedon polarity; on the other hand, magnesium oxideor calcium hydroxide permits separation by num-ber and type of double bonds in the structure;calcium hydroxide or zinc (II) carbonate sepa-rates cis-trans isomers. It is usually recommendedto use each one of these three supports (alumina,magnesium oxide, and calcium hydroxide) to reachthe separation, and, generally, preceded by silicaor alumina open-column chromatography.59,60

Nowadays, open column chromatography is usedas a prepurification stage to separate groups ofcarotenoids with similar characteristics in specialflash open column chromatography.264,308

In chromatography, TLC is still used becausenew advantages have been incorporated into thistechnique in addition to its main characteristics:low cost and simplicity. Also, it must be pointedout that this is the starting technique used inwhatever purification, and that is the wider usedtechnique.59,60,378 In addition, it is possible to use

reverse phase TLC chromatography to reach theseparation.235

High-performance liquid chromatography(HPLC) is the preferred column chromatographyto carry out the qualitative and quantitative analy-ses of carotenoids.60 This technique is ideal be-cause carotenoids can be monitored easily withthe UV-visible detector, and methodology hasconverted it into a powerful technique with theintroduction of the diode array detector (DAD),which permits detection at several wavelengthsand simultaneous tentative identification by UV-spectral analyses. In addition, information aboutpurity of compounds is obtained with a DADdetector.57,427,480 In general, HPLC shows the fol-lowing advantages over the traditional methods:greater sensitivity, resolution, reproducibility, andspeed of analysis; additionally, it is possible touse inert conditions.36,181,415,480

It has been reported that HPLC has reproduc-ibility advantages inclusive in relation tosupercritical fluid chromatography.36 Additionally,separation of carotenoid geometric isomers hasbeen tried with reverse phase C18 column but withlimited success.427 However, a successful separa-tion was reached by using a column with poly-meric surface C30. The resolution of isomers washigher for polar and nonpolar carotenoids thanC18 resolution.415 This column has been evaluatedin the separation of isomers of lutein, zeaxanthin,β-cryptoxanthin, α- and β-carotene and lycopene,which were produced by iodine isomerization.The main isomers identified were 9-, 9′-, 13-,13′-, 15-cis and all-trans carotenoids. It has beencommented that a good separation is a prerequi-site to make more exact determinations of provi-tamin A content, isomeric profiles in biologicaltissues, comparative physiological roles betweenthe cis and trans configurations, purity determi-nation, and purification of carotenoids.136 Thiscolumn has been used in the evaluation of bio-logical samples with excellent results.117,118 Whena C34 column was evaluated, better resolution wasobtained for only some isomers, and, in general,the results were comparable to those obtainedwith the C30 column.33 On the other hand, withCa(OH)2 columns, a good resolution has beenobtained in the separation of carotene geometricisomers with 5, 7, and 11 double bonds.425

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All the information indicates that HPLC analy-ses are required when analyzed carotenoids areminority compounds in a given sample.1 Also, itis necessary to establish the content of bioactivecompounds, such as provitamin A precursors. Theanalysis of leafy vegetables used in the Indone-sian food table (spinach, cassava, papaya, mango,sweet shoot, and jointfir spinach) showed thatwith HPLC were obtained lower values for provi-tamin A than previously reported values.228

d. Characterization

Spectroscopy. The most important techniquein carotenoid analyses is UV-visible spectroscopy,which gives information about the presence ofrings, carbonyl groups, and isomeric ef-fects.59,92,112,179,181 In this analysis, absorptionmaxima, form, and fine structure of spectra arecharacteristic of the molecule’s chromophore.Nowadays, the introduction of DAD permits tomake in situ identifications immediately afterHPLC separation.60

Infrared spectroscopy gives information onthe kind of bonds and atoms in the analyzed com-pound, while nuclear magnetic resonance (NMR)of proton and carbon permits the assigning ofthese atoms to a certain structure. However, thetechnique used most in carotenoid analysis is massspectroscopy (MS), mainly because of the samplequantity required for analysis is very small. Massspectroscopy provides information on carotenoidMW, and fragmentation pattern helps us to deter-mine the carotenoid structure.179 However, it isvery important to choose an adequate MS equip-ment because of carotenoid instability, and one ofthe initial and most successful MS ionization tech-niques is fast atom bombardment (FAB).200,426

The main advantage of this methodology is thatMW is determined unambiguously; the molecularion usually appears, and one of its disadvantagesis a poor fragmentation pattern, thus structuralelucidation limited and serial detectors must beused to differentiate between geometric isomers.Nowadays, new techniques have been introduced:HPLC coupled with an MS detector (LC/MS), avery convenient technique by considering intrin-sic carotenoid instability, and MS with an

electrospray detector that is 100 times more sen-sitive than conventional techniques (picomolar).480

Another ionization technique is Atmospheric Pres-sure Chemical Ionization (APCI), which producesa better fragmentation pattern than FAB orelectrospray and shows a good compatibility withHPLC equipment, and, recently, matrix-assistedlaser desorption ionization was introduced and itsdetection limit is in a femtomolar-attomolarrange.251,480 These detectors have been used inseveral studies to identify carotenoids from bio-logical samples, and it has been established thatcarotenoid identification must cover at a mini-mum the following criteria: co-chromatographywith authentic samples, UV visible, and massspectra.

RAMAN spectroscopy and circular dichro-ism have permitted the study of carotenoids inbiological systems.200,435 Kull and Pfander264 stud-ied canola carotenoids and used UV visible, MS,NMR, and circular dichroism to identify severalisomers of luteoxanthin and violaxanthin. Also, itwas mentioned that in nature both trans and cisisomers exist.

Photoacoustic spectroscopy (PA) was used toevaluate pigment composition of paprika. It waspossible to identify peaks or shoulders in the vis-ible spectrum region that corresponds to caro-tenoids or chlorophylls. With the generated infor-mation, it was possible to distinguish betweendifferent samples of paprika, and, when PA wasused in the near infrared region (800 to 1000 nm),semiquantitative information on total pigmentcomposition was obtained.489

Chemical tests. By considering carotenoidchemical structure, several simple tests are usedto corroborate the presence of chemical groups:5,6-epoxydes react with HCl to form 5,8-epoxydeisomers; this chemical modification is accompa-nied by a hypsochromic shift of 7 to 22 nm formonoepoxydes and 40 nm for diepoxydes. Allylalcohols treated with HCl are dehydrated, andanother double bond is formed with a consequentchange in their UV visible spectra. Another test toevaluate allyl alcohols is the reaction with HCl inmethanol to obtain methyl esters. To determinethe presence of aldehyde groups, aldol condensa-tion was carried out by treatment with acetone ina basic system producing an extension in the con-

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jugated system, then spectra showed a changetoward higher wavelengths. In carotenoids withcarbonyl groups, these can be identified by reac-tion with hydrides in ethanol or tetrahydrofuran;thus, a hypsochromic change of 20 to 30 nm andfinest spectra are observed. Another simple test isiodine isomerization, which produces a mixtureof equilibrium isomers (cis-trans); if the startingpigment is all-trans, a hypsochromic shift is ob-served (3 to 4 nm), while if it is cis, hyperchromic(1 to 3 nm).112,179

Silver nitrate is used to discriminate betweenβ and ε carotenoid rings. Carotenoids are sepa-rated by TLC (preferable in silica gel) and thendeveloped with a methanolic solution of silvernitrate. Carotenoids with β-rings produce abathochromic shift; the shift depends on the num-ber of β-rings, for example, after developmentzeaxanthin spot presents red tones, while luteinspot has yellow tones.236

Evaluation of pigmentation efficiency. Coloris a complex process and color evaluation also,because we need to evaluate eye perception andbrain interpretation, and pigment deposition andcolor perception do not show a direct relation-ship. All attempts are only approaches to reality.The most common used methods are (1) samplecarotenoid extraction followed by UV-visiblespectroscopy analysis,11,313 and (2) the use of pho-toelectric instruments that measure reflectance.Hunter-Lab is the most common equipment; it isbased on tristimulus effect.31,297 Several studieshave shown that reflectance colorimetry is a gooddescriptor of the pigment content from differentsamples (pepper, red beet, marigold). This re-search has given high correlation coefficients(> 0.9), and it has been concluded that the assess-ment of pigment content by color determinationis highly feasible at industrial level.121,230,394

7. Importance As Food Colors —Stability, Processing, and Production

Carotenoids have been used as food colorsfor centuries: saffron, pepper, leaves, and red palmoil have carotenoids as their main color compo-nents. Color of carotenoids, together with benefi-cial properties such as vitamin A precursors and

antioxidants, have led to their wide application inthe food industry; preparations to apply them inoily or aqueous media have been produced, in-cluding emulsions, colloidal suspensions, andcomplexes with proteins. These preparations havefound applications to pigment margarine, butter,fruit juices and beverages, canned soups, dairyand related products, deserts and mixes, preservesand syrups, sugar and flour confectionery, saladdressings, meat, pasta and egg products, amongothers,257 and, interestingly, other important areasof application of carotenoids have been emerged.It has been reported that corn carotenoids inhibitthe synthesis of aflatoxin by Aspergillus flavus(90%) and by most of the A. parasiticus (30%)strains. Interestingly, carotenoids with an α-iononering showed higher inhibition (25%) than theβ-ionone ring. Thus, lutein and α-carotene showedhigher activities than β-carotene or zeaxanthin,and it was determined that several corn inbredlines have high enough levels of carotenoids toaffect aflatoxin formation in the endosperm (0.09to 72 µg/g). It was shown that the antioxidantactivity of carotenoids are not responsible of inhi-bition of aflatoxin synthesis, and it was suggestedthat they may affect cell membranes enough toindirectly modify polyketide synthase activity(cytosolic enzyme) or by direct interaction withthe synthase or other enzymes of aflatoxin syn-thesis.344

a. Stability in Model Systems

In practice, it is very important to take intoaccount the carotenoid instability, and the estab-lishment of processing conditions must considerthis aspect.297 However, most studies have beencarried out with β-carotene. It has been reportedthat some pepper components facilitate β-caro-tene oxidation. β-Carotene oxidation was acceler-ated by increased levels of linolenic acid. Theascorbic acid effect depends on its concentrationand if copper ions are present. It was mentionedthat high aqueous activity favored prooxidantactivity. Additionally, high ascorbic concentra-tions (100 µmol/g of cellulose) in the presence ofcopper ions induce a greater antioxidant effect,and under these conditions prooxidant activity of

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peroxidase was inhibited. Moreover, it was con-cluded that ascorbic acid concentration neededfor food protection depends on the content andidentity of lipids present.250

At incubation conditions (37°C, 5% CO2, dark-ness), β-carotene was slightly stable during 48 hand showed fast degradation with the effect ofUV and visible light in the presence of oxygen;the degradation rate was increased with incre-ments in O2 turnover. β-Carotene was stabilizedwith antioxidants (e.g., BHT); it was thus con-cluded that degradation is by the effect of freeradicals.419

Another study evaluated the effect of tem-perature (100°C by different times) on aqueoussuspensions of β-carotene and lycopene. It wasconsidered that this treatment is representative ofthose commonly applied on vegetables. Volatilecompounds were trapped in column or in organicsolvents then analyzed by gas chromatography(flame ionization detector) and mass spectrom-etry. The main degradation products of β-caro-tene were identified as 2,6,6-trimethyl-cyclohex-anone; β-cyclo-citral; 5,6-epoxy-β-ionone anddihydroactinidiolide. The products of lycopenedegradation were 2-methyl-2-hepten-6-one;pseudo-ionone; 6-methyl-3,5-hepadien-2-one;geranial, and neral, and the following nonvolatilecompounds of β-carotene degradation were iden-tified: aurochrome; mutatochrome; 5,6,5′,6′-diepoxy-β-carotene and 5,6-epoxy-β-carotene.89

An oxidation study of β-carotene by peroxideradicals was carried out at 37°C in benzene, andalkylperoxyl radicals were generated by2,2′-azobis (2,4-dimethylvaleronitril). The analy-sis of oxidation products suggest that β-caroteneis destroyed by a free-radical-addition mecha-nism. Products were analyzed by HPLC, UV-visible spectroscopy, MS, and NMR (proton andcarbon-13).519

On the other hand, many studies have beencarried out to evaluate the kinetics of carotenoiddegradation and/or biosynthesis. Degradationmechanisms based on free radicals have beenproposed by using solid supports (carboxymeth-ylcellulose, Al2O3 or MgO).174 Also, it has beenestablished that low aw (around 0.3) diminish thecarotenoid degradation.174,192 Photodegradation2690 lx) of aqueous β-carotene has been studied.

The models were in matrixes of gelatin/sugar andacacia gum/sugar. In first-order kinetics in thedegradation and isomerization reaction was ob-served. Also, it was shown that in illuminatedaqueous β-carotene, the main isomers formed were9-cis-, 13-cis-, and all-trans-β-carotene. In dark-ness, 13-cis-isomer was favored, while 9-cis wasunder light conditions, showing a lower stabilityof 13-cis-β-carotene.

Another study showed that all-trans-β-caro-tene isomerization depends on which solvents weredissolved. Faster reactions were observed in non-polar solvents, identifying the higher rate of for-mation for the 13-cis-β-carotene.368 However,other studies showed contrasting results: β-caro-tene, diesterified capsanthin, and capsanthin dis-solved in anhydrous (cyclohexane) or aqueousethanol, water) solvents were illuminated(1000 l×). Degradation in anhydrous medium fol-lowed a zero-order kinetics and of first-order inaqueous medium. In addition, it was shown thatdeesterified pigments were more stable in aque-ous than in nonpolar medium, while at intermedi-ate polarity both forms esterified and deesterified)showed similar stability. β-Carotene was morelabile than capsanthin, and as in other studiesdegradation increased with temperature.319

Crystals of α- and β-carotene were isomer-ized by temperature, light, and iodine. The maincomponents in isomerized samples wereall-trans-, 15-cis-, 9-cis-, and 13-cis-β-caroteneand all-trans-, 15-cis-, 13-cis-, and 9-cis-α-caro-tene (in decreasing order). Minimal isomerizationwas observed in the range 50 to 100°C/30 min,but it was considerable at 150°C/30 min. Thephotoisomerization reactions were of first-order:reaction constant was higher for the conversiontoward all-trans-β-carotene, and with α-carotenethe 13-cis-isomer was favored. With iodine cata-lyzed reaction, β-carotene degradation followed afirst-order kinetics, while isomerization producedfirst- and second-order kinetics.98

Photostability (sunlight 8 h/day) of marigoldpigments was studied in aqueous emulsions withArabic gum and/or mesquite gum. Stability inemulsions with mesquite gum was better 3.83times) than with Arabic gum. Also the synergisticeffect between the two gums was observed. High-est stability was reached at pH 5.0. Kinetic deg-

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radation was of zero-order. It was concluded thatpolyelectrolytic nature and MW of gums are im-portant in color protection, and dark emulsion huecould act as a sun filter.486

b. Processing and Stability in Foods

Processing effects were analyzed in tomatoand green vegetables (broccoli, spinach, and greenbeans). Moderate processing did not produce caro-tenoid modifications, but a prolonged heating(1 h boiling) conduces to total destruction ofepoxycarotenoids; it was shown that under thiscondition only the most sensitive carotenoids arehighly destroyed. Boiled tomato showed an iden-tical carotenoid profile with fresh tomato, anddifferences in content were only detected.253 Asimilar result was obtained with vegetables usedin Spain, and it was mentioned that carotenoidsaponification results in losses that are more criti-cal in xanthophyll than in carotenes.184

The effect of freezing and canning of kiwiwas analyzed by TLC, HPLC, UV-visible spec-troscopy and chemical tests. The principal com-ponents of fresh and frozen kiwi were 9′-cis-neoxanthin, trans- and cis-violaxanthin,auroxanthin and lutein. In frozen kiwi stored for6 months (-18°C), the carotenoid profile was simi-lar to fresh fruit, but in addition antheraxanthinwas detected. On the hand, canned kiwi showed acomplicated carotenoid profile because of ther-mal treatment. Neoxanthin and violaxanthin weresome of the degraded xanthophylls.74

Processing of fresh pepper to obtain paprikawas studied. Carotenes and xanthophylls wereextracted with acetone and identified by UV-vis-ible, infrared spectroscopy, and chemical tests;quantitation was carried out by UV-visible spec-trometry. It was reported that fast drying inducesthe carotenoid destruction and increments wereobserved by slow drying of pepper. It was pro-posed that an adequate drying process could con-duce carotenoid synthesis: in bola pepper, incre-ments of red carotenoids were observed(capsanthin, capsorubin, and capsolutein); how-ever, this effect was not observed in agridulcepepper. Also, it was mentioned that during mill-ing process a high reduction in carotenoid content

was observed, and the most affected carotenoidwas β-carotene followed by β-cryptoxanthin andzeaxanthin, while the most stable were capsanthinand capsorubin. Also, it was observed that dryingprocess produces a net carotenoid biosynthesisthat is enhanced by illumination: drying underdarkness conditions increased the carotenoid con-tent in 15%, while under illumination it was 47%.Higher increments were observed in β-carotene,cryptoxanthin, zeaxanthin, and capsanthin, whileviolaxanthin and capsorubin were destroyed. Thebiosynthesis occurred in a first stage (35 to 40%of moisture), then degradation was observed.318,320

The effect of mango processing on carotenoidcontent was evaluated. Mango was sliced andstored at vacuum or frozen (-40°C) conditionsduring 6 months; other samples were commer-cially canned. Fresh and frozen fruits showedsimilar characteristics. Canned mango showedgreat changes in color and carotenoid profile. Themost stable carotenoid was β-carotene.73

All-trans-β-carotene isomerization sensitizedby chlorophyll was studied. Main formed isomerswere 9-, 13-, and 15-cis-β-carotene, with a higherproportion of the 9-cis-isomer. In control withoutchlorophyll, isomerization was imperceptible dur-ing the first 2, then a quick increment was ob-served during the first day and then remainedconstant. The main isomers under these condi-tions were 9- and 13-cis-β-carotene. The chloro-phyll sensitizer effect supports the idea that cis-isomers in photosynthetic tissues are notartifactual.350

Chen et al.99 studied the effect of juice carrotprocessing on its carotenoid content. Pasteuriza-tion 105°C/25 s) neither produced a considerablevariation in the isomeric profile nor in the caro-tenoid content. Treatment at 110°C/30 s producedthe degradation of 45% of β-carotene while theisomers formed were in decrement order): 13-cis-> 15-cis- > 9-cis- > 13,15-di-cis-β-carotene, α-Carotene pattern was similar, but the main isomerwas 15-cis-α-carotene. Lutein degradation wasaround 30%, and the main isomers produced were13-cis > 9-cis. At 120°C/30 s; 48% ofβ-carotene was lost. In the canning process (121°C/30 min), an increment in β-carotene content wasobserved, and the main isomers were 9-cis- and13,15-di-cis-β-carotene. α-Carotene was reduced

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in 60% and isomers were degraded after the can-ning. Destroyed lutein was around 50%. Colordiminution was higher in more drastic treatments.Also, vitamin A destruction showed a similarpattern: 55.7% of β-carotene was lost during can-ning.99 In contrast, it has been reported that can-ning of vegetables (e.g., broccoli, cantaloupe, car-rots, etc.) produces an isomerization all-trans- tocis-isomers of carotenoids. In sweet potatoes thehighest isomerization was observed and was thelowest in broccoli. In processed red, yellow, andorange vegetables, the 13- and/or 13′-cis-isomerare favored, while in green vegetables it is the 9-cis-isomer. The preponderance of the 9-cis-caro-tenoids in green vegetables was assigned to theeffect of chlorophyll as a sensitizer. Interestingly,the total carotenoid content was maintained aftercanning and the provitamin A concentration washigher. Later observation may be due to an in-creased extraction efficiency due to the disruptionof carotenoprotein complexes, inactivation ofcarotene-oxidizing enzymes, and/or loss of solublesolids into the liquid canning medium.277

Blanching (98°C /5 min), cooking (98°C for15, 30, and 60 min) and sun drying (25°C ± 6°C)are traditional processing practices. These meth-ods were used in the processing of Tanzanianvegetables used for human consumption (ama-ranth, cowpea, peanut, pumpkin, and sweet po-tato leaves). In general, blanching resulted in areduction of β-carotene and in an increment ofα-carotene concentration. Cooking enhanced thequantity of carotenoids extracted, and sun dryingreduced the concentration of carotenoids in all theevaluated vegetables. Also, it was shown thatthermal processing increased the vitamin A activ-ity of all vegetables, but in amaranth leaves areduction was observed. Thus, blanching andcooking could be considered as advantageousprocesses for increasing the amount of provita-min A available when vegetables are consumed.It was suggested that consumption of 100 g of dryweight of these vegetables cover the recommendeddaily intake for vitamin A for both children andadults.333 In the processing of bathua (Chenopo-dium album) and fenugreek (Trigonella foenumgraecum) leaves that are consumed in India,blanching, cooking (using hydrogenated fat), andsun drying resulted in the reduction of carotenoids.

It was shown that cooking in the presence of fat,carotenoid, and vitamin A reductions are lowerbecause fat may dissolve and protect carotenoids.It was recommended that blanching for a shorttime (5 min) and cooking in a pressure cooker arebetter processing conditions for the retention ofβ-carotene.512

During egg dehydration and storage, the ef-fect of α-tocopherol supplementation was stud-ied. It was shown that gas heater drying produceshigher loses than electric resistance drying. It isexplained that during gas drying higher quantitiesof nitrogen oxides and other free radicals areformed, pepper oleoresin acts as a prooxidant,and α-tocopherol stabilizes carotenoids and prob-ably permits the antioxidant action of caro-tenoids.269

In the processing of saffron, drying tempera-tures lower than 30°C or higher than 60°C pro-duced a greater carotenoid degradation. The opti-mum temperature of drying was 40 ± 5°C,independently of type of drying (sun or furnacedrying). Losses of 56.9 to 70.2% after 1 year ofstorage were observed (15 to 35°C, 10 to 12%moisture).386

To preserve color in carotenoid pigmented-foods, the antidiscoloring effect of green teapolyphenols [epigallocatechin 3-gallate (EGCg),gallocatechin (GC), gallocatechin 3-gallate (GCg),epigallocatechin (EGC), -)-epicatechin EC) or (+)-catechin (C)] in beverages and in margarine wasassayed. In general, it was observed that teapolyphenols and several tea catechins have stron-ger effects against the discoloration of β-carotenethan L-ascorbate (widely used to prevent discol-oration) in the aqueous (beverages) and oily (mar-garine) systems. The strength of antidiscoloringactivity was GCg, EGC, EGCg, GC >> gallic acid(GA) > L-ascorbate, EC, C. According to the ob-served order, it was established that activity de-pends on the presence of an hydroxyl group at the5′ position, and it was suggested that the hydro-gen radical from the phenolic hydroxyl group ofcatechin stabilizes the peroxide radical formedduring the oxidation process. Thus, tea polyphe-nols might be used as excellent antidiscoloringcompounds of β-carotene.479

Poultry feed pigmented with marigold oleo-resin (lutein is the main carotenoid) was exposed

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to sunlight. Poultry fed with sun-exposed feedshowed better pigmentation than control feed(maintained in darkness). It was proposed thatsunlight induces conversion of lutein to redderpigments (e.g., astaxanthin).149 However, in ourlab it was shown that sunlight illumination pro-duces a favorable equilibrium toward all-trans-lutein isomer (trans-isomer showed redder huesand high pigmenting efficiencies), but redder caro-tenoids were not observed. Additionally, weshowed a better egg yolk-pigmenting efficienciesof marigold meals exposed to sunlight than con-trols; and also we demonstrated that other compo-nents different to carotenoids are participating inpigmentation efficiency.268

Marigold pigments have also been used inthe pigmentation of shrimp (Penaeus vannamei).It was found that saponified extracts were betterpigmenting agents than esterified marigold ex-tracts; its study suggested that lutein and zeax-anthin (main carotenoids of marigold) can bemetabolized into astaxanthin and deposited inshrimp and better colorations could be obtainedthan with astaxanthin supplemented feed.487 Onthe other hand, in the pigmentation of white-fleshed fish, carotenoids must be eliminated fromthe corn gluten meal (CGM) used in feeds. In thecorn gluten bleaching, it was established thatcarotenoid extraction with ethanol is a good pro-cess, but economical benefits could be enhancedby using soybean flour (5% substitution level) asa bleaching agent by its content of lipoxygenaseand because the high content of protein in soy-bean.359

The use of high pressure (9000 times overatmospheric pressure) to kill contaminating or-ganisms in food has been proposed.210 It wasreported that sensorial characteristics, such asflavor and color, are unscathed. In Japan, thismethodology has been used in the preparationof fruit jams, yoghurts, and yomogimochi(steamed rice paste mixed with wild herbs).However, some vegetables showed undesirablecharacteristics after pressure treatment: grapeswere hardened, cabbage were softened, andmushroom and potato were oxidized. It wassuggested that the killing of organisms by pres-sure could be assigned to modifications of pro-tein structure by affecting ionic bonds and/or

ionic interactions, consequently, protein activ-ity is also modified. Another suggestion indi-cates that membranes are modified and the cellcontent is lost.

Kinetic studies of carotenoid degradation havebeen also carried out in food systems and in gen-eral, as in model systems, they are characterizedby first-order kinetics. When carrot juice wasused as model, photodegradation and photo-isomerization reaction were slower than in syn-thetic models, thus some protective factors mustbe present in juice. In carrot juice, the main iso-mer was 13-cis-β-carotene.367 In other studies,carrot juice was acidified, pasteurized, and thensubjected to light or dark storage at different tem-peratures (4, 25, and 35°C) for 3 months. Tem-perature had a negative effect on lutein content(higher degradation at 35°C), and it was observedthat the formation of 13-cis-lutein is favored.Under light conditions the behavior was similar,although lutein degradation was higher thanin darkness storage. α-Carotene showeda similar behavior than lutein; however,13-cis-α-carotene is formed preferentiallyin dark conditions (although the 9-cis-isomer increases also), and 9-cis-α-carotene is the preferred isomer in light conditions. Theconcentration change of β-carotene and its iso-mers was similar to the other carotenoids, but β-carotene was more susceptible to degradation.Vitamin A degradation showed a correlation withcarotenoid degradation.101 In saffron, pigment deg-radation showed a first-order reaction with theeffect of light, heat, and pH.475 During olive pro-cessing, lutein and β-carotene didnot show variations, while violaxanthin andneoxanthin content decreased in parallel withauroxanthin and neochrome increments. By con-sidering that total carotenoids remained constant,thus processing produced a conversion of 5,6-epoxyde carotenoids (violaxanthin, neoxanthin)to 5,8-epoxydes (auroxanthin, neochrome) withfirst-order kinetics.317

Koplas-Lane and Warthesen262 studied spin-ach and carrot photostability (2000 l × /8 days).Carotenoids were degraded with first-order kinet-ics. Lutein was the most and violaxanthin theleast stable carotenoids. Carrot carotenoids werenot affected by light and showed higher stability

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than spinach carotenoids. Interestingly, duringcarrot storage in darkness/6°C, chromogenesis wasobserved and samples showed 11% more totalcarotenoids after 60 days of storage.262

Stability of carotenoid extracts from saffron(Crocus sativus L.) was studied at different wateractivities (0.11, 0.23, 0.33, 0.43, 0.53, 0.64, and0.75) and temperatures (25, 40 and 60°C). Caro-tenoid degradation under all storage conditionsand with a first-order kinetics was observed. Also,a higher rate of degradation associated with incre-ments in water activities was shown. This resultcontrasts with those obtained with oil-soluble caro-tenoids. Crocin, the main carotenoid in saffron, iswater soluble, and it was suggested that moreavailable water could enhance the mobility ofsolution components (carotenoids and free radi-cals), inducing a higher degradation. Also, it wasmentioned that higher stability of carotenoidsmight be reached at temperatures below the glasstransition temperature, where solution componentsare at fixed positions and degradation reactionsare more difficult.474

c. Production of Carotenoids in Bioreactors

An important approach for carotenoid produc-tion consists of the culture of algae and bacterialstrains. The fungus Blakeslea trispora is a well-known β-carotene producer, and it has been shownthat cell growth and β-carotene production areenhanced by using surfactant agents such as Spanand Triton, except with Triton X-100.254 Anotherinteresting model for β-carotene production isPhycomyces blakesleeanus, and mutants carS havebeen reported that accumulate up to 100 times thewild-type level (2 to 5 mg β-carotene/g dry myce-lium). CarS mutants were exposed to N-methyl-N′-nitrosoguanidine and the mutant S276 was ob-tained. S276 contained 9.2 ± 1.6 mg of β-carotene/g dry mycelium, and it was suggested that mutatedgene product probably activates carotenogenesis.307

The alga Haematococcus lacustris contains largeamounts of astaxanthin esters and has been consid-ered as a potential source of astaxanthin. Recently,it was established that higher light intensities re-sulted in the accumulation of more astaxanthinesters in the cells: culture at 90 µmol m-2 s-1 of light

intensity and quickly rising to 190 µmol m-2 s-1

permitted obtaining the maximum carotenoid con-tent in H. lacustris. Also, echineone andcanthaxanthin were identified in the cultures, sup-porting the idea that astaxanthin is synthesizedfrom β-carotene via echineone and canthaxanthin.520

Other studies in vivo518 and in vitro110 have shownthat high astaxanthin production required high lev-els of oxygen (aerobic conditions). Additionally, ithas been shown that cell growth requires lowC/N ratios and astaxanthin production of high C/Nratios. The results showed that pigment productionby X. dendrorhous is affected by fermentation,respiration, and anabolism. It was suggested thathigh O2 levels are required for NADH oxidation,inhibiting the ethanol production (fermentation)and enhancing respiration. On the other hand,astaxanthin production required large amounts ofNADPH and high C/N ratios, and high oxygenlevels favor this condition. Thus, to optimize theastaxanthin production, it is necessary to optimizethe synthetic pathway of astaxanthin and optimizethe metabolic pathway anabolic and respiratory),and, consequently, it was proposed that optimalastaxanthin production is reached by a two-stagecultivation: the growth stage (first) in medium withlow C/N ratio and the astaxanthin production stage(second) in high C/N ratio.518 Also, it was sug-gested that the addition of ethanol during the sec-ond stage enhanced the production of astaxanthin2.2 times more than control without ethanol.517

Pine (Pinus pinaster) wood meals were used in theproduction of carotenoids. Pine meals were treatedwith acid and enzymatic hydrolysis. The enzymesused were cellulases from Trichoderma reesei(Celluclast) and cellobiase from Aspergillus niger(Novozym). This material was incubated withpreproliferated broths of Xanthophyllomycesdendrorhous (formerly Phaffia rhodozyma), and itwas determined that enzymatic hydrolysates be-have as suitable culture media for proliferation ofX. dendrorhous, reaching good growth rates (up to0.07 h-1) with high cell yield (0.47 g biomass/gconsumed glucose) and carotenoid concentrationsup to 1.8 mg carotenoids/l. Thus, it was suggestedthat wood hydrolyzates are potential substrates forcarotenoid production with X. dendrorhous.357

Astaxanthin production was also assayed in thinstillage as substrate and a nitrosoguanidine mutantof

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X. dendrorhous called JB2. The carotenoid pro-duction was increased 2.3 times in relation to thewild-type cells (1.54 µg carotenoid/mg dry weight).Thus, it was suggested that a strain like JB2 haspotential for commercial production of astaxanthinfrom corn byproducts.50 Also, Haematococcuspluvialis has been suggested for the production ofastaxanthin, and recently compactin-resistant mu-tants were isolated (compactin is an inhibitor of theHMGR that strongly blocks cholesterol formation).Selected mutants showed growth comparable withwild-type cells, while the astaxanthin content wasincreased in 1.4 to 2.0 times. The specific HMGRactivity was reduced in 14 to 27%, and it suggestedthe presence of specific HMGR for astaxanthinproduction.109

B. Anthocyanins

1. Definition

Anthocyanins are the most important groupof pigments, after chlorophyll, that are visible tothe human eye.195 Chemically, anthocyanins from

the Greek anthos, a flower, and kyanos, dark blue)are flavonoids (flavan like), and consequentlybased on a C15 skeleton with a chromane ringbearing a second aromatic ring B in position2 (C6-C3-C6) and with one or more sugar mol-ecules bonded at different hydroxylated positionsof the basic structure (Figure 15). Anthocyaninsare substituted glycosides of salts of phenyl-2-benzopyrilium (anthocyanidins).87,455

2. Classification

The basic C6-C3-C6 anthocyanin structure isthe source of an infinity of colors produced by itschemical combination with glycosides and/or acylgroups (Figure 15) and by its interaction withother molecules and/or media conditions.62

Harborne and Gryer195 mentioned the existence of17 anthocyanidins, with differences in the num-ber and position of hydroxyl groups and/or me-thyl ether groups, but six of them are the mostcommon anthocyanidin constituents of this kindof pigments (Table 4). From these 17 structurescombinations have arisen with at least one sugar

FIGURE 15. Basic structure of anthocyanidin pigments in which Rx could be H, OH, or OCH3 depending of theconsidered pigment. The most common accepted nomenclature for numbering carbons is indicated inside thestructure.

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TABLE 4Anthocyanidins Found in Nature

Substituted with a characteristic hydroxyl group

Namea Position of substitution Some of the produced colors

Apigeninidin 5,7,4’ OrangeAurantinidin 3,5,6,7,4’ OrangeCyanidin 3,5,7,3’,4’ Magenta and CrimsonDelphinidin 3,5,7,3’,4’,5’ Purple, mauve and blue6-Hydroxycyanidin 3,5,6,7,3’,4’ RedLuteolinidin 5,7,3’,4’ OrangePelargonidin 3,5,7,4’ Orange, salmonTriacetidin 5,7,3’,4’,5’ Red

Substituted with a characteristic methyl ether group

Capensinidin 5,3’,5’ Bluish redEuropenidin 5,3’ Bluish redHirsutidin 7,3’,5’ Bluish red

Malvidin 3,5’ Purple5-Methylcyanidin 5 Orange red

Peonidin 3’ MagentaPetunidin 3’ PurplePulchellidin 5 Bluish redRosinidin 7,3’ Red

a Underlined names represent the most common anthocyanidins. Adapted from Refs. 157, 193.

molecule to obtain anthocyanin compounds. Thus,anthocyanins have also been classified in agree-ment with the number of sugar molecules thatconstitute their molecules (i.e., monosides,biosides, triosides) and, interestingly, the numberof probable compounds is greatly increased bytaking into account the sugar diversity and all thepossible structural points of glycosylation, al-though the order of sugar occurrence in naturalanthocyanins is glucose, rhamnose, xylose, galac-tose, arabinose, and fructose. Additionally, manyanthocyanins have shown in their structures esterbonds between sugars and organic acids i.e.,acylated anthocyanins), and in nature the mostcommon acyl groups are coumaric, caffeic, feru-lic, p-hydroxy benzoic, synapic, malonic, acetic,succinic, oxalic, and malic.157 Moreover, substitu-tion of hydroxyl and methoxyl groups influencesthe color of anthocyanins. Increments in the num-

ber of hydroxyl groups tend to deepen the color toa more bluish shade. On the other hand, incre-ments in the number of methoxyl groups increaseredness (Table 4). With all of these facts in mind,it must be not difficult to understand the gamut ofcolors observed in nature that is produced from asingle structure.

3. Distribution

Anthocyanins are responsible for many ofthe attractive colors, from scarlet to blue, offlowers, fruits, leaves, and storage organs.193,195

They are almost universal in higher plants, butin general anthocyanins seem absent in the liv-erworts, algae, and other lower plants, althoughsome of them have been identified in mossesand ferns.455 The type of anthocyanins in plants

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is so variable that some ornamental plantspresent only one main type of anthocyanin(Dianthus, Petunia), whereas others have mix-tures (Rosa, Tulipa, and Verbena). On the otherhand, some fruits are a source of one anthocya-nin: cyanidin in apple, cherry, fig, and peach;delphinidin in eggplant and pomegranate; somefruits have two main anthocyanins such ascherry sweet and cranberry (cyanidin andpeonidin), while others have several anthocya-nins (grape). In general, the anthocyanin con-centration in most of the fruits and vegetablesgoes from 0.1 up to 1% d.w.455

Anthocyanins are vacuolar pigments andin this organelle the presence of membranebound bodies called anthocyanoplasts has beenproposed; these structures are formed whilepigment synthesis is in operation, and eventu-ally they are dispersed to produce a totallypigmented vacuole. In flowers, anthocyaninsare almost exclusively located in epidermalcells, and only occasionally in the mesophyll.On the other hand, in the leaves of rye (Secalecereale) they are restricted to the mesophyllcells.195 Interestingly, flavones co-occur withanthocyanins and participate in the color ofplants as copigments (substances thatcontribute to anthocyanin coloration by pro-tecting the anthocyanin molecules). Thecopigmentation mechanism is unique to theanthocyanin family. Anthocyanins also reactwith alkaloids, amino acids, benzoic acids,coumarin, cinnamic acids, and a wide varietyof other flavylium compounds. This weakassociation is termed intermolecular co-pigmentation. Intramolecular copigmentation isdue to the acylation in the molecule, and it ismore effective than intermolecular; in acylatedanthocyanins, it is suggested that acyl groupsinteract with the basic anthocyanin structure,avoiding the formation of the hydrated spe-cies. The basic role of copigments is to protectthe colored flavylium cation from the nucleo-philic attack of the water molecule.28,410 Otherimportant phenomena that show a great con-tribution to color is the self-association ofanthocyanins, and in some models the contri-bution of metal complexation have been sug-gested (Figure 16).63,146,157,193, 417

4. Biosynthesis: Biochemistry andMolecular Biology

a. Biochemistry

The phosphorylated compounds phospho-enolpyruvate (glycolytic pathway) and erythrose-4-phosphote (pentose phosphate respiratory path-way and Calvin cycle) are the precursors ofshikimic acid. Shikimic acid and acetate are theprecursors of the primary aromatic building stonesof many phenolic compounds, including antho-cyanins. Shikimate pathway gives several organicacids, such as cinnamic, p-coumaric, caffeic, feru-lic, chlorogenic, and phenylalanine. By usingchemogenetic studies and labeled precursors, itwas shown that the shikimic acid product, pheny-lalanine, is incorporated in the C6-C3 (B aromaticrings and carbons corresponding to the centralpyran ring) portion of the basic flavonoid struc-ture (Figure 3). On the other hand, ring A and theoxygen of the central pyran ring are provided byacetyl-CoA. In addition, it is remarkable that elu-cidation of anthocyanin biosynthesis and geneticshave been carried out mainly by using undifferen-tiated cells of parsley, maize, snapdragon, andpetunia.193,195,455

Anthocyanin biosynthesis pathway could bedivided into two main parts: (A) precursors of thegeneral phenylpropanoid metabolism, and (B) spe-cific steps toward flavonoid biosynthesis (Figure17). In the first part, phenylalanine is converted top-coumaryl-CoA by effect of three enzymes: phe-nylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumaryl-CoA ligase(4CL). p-Coumaryl-CoA is the main precursor offlavonoids, lignin, and other phenylpropanoids(Figure 17A). In the second part (Figure 17B),chalcone synthase (CHS), which is consideredthe key enzyme in flavonoid biosynthesis, cata-lyzes the condensation of three molecules ofmalonyl-CoA with 4-coumaryl-CoA to form theintermediate chalcone. Chalcone synthasehas been isolated and characterizedfrom several sources (Dianthus, Verbena,Anthirrhinum). In the next step, chalcone shows astereospecific isomerization to naringenin by theenzyme chalcone isomerase (CHI). Different CHIand tissue-specific isoenzymes have been charac-

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FIGURE 16. Suggested mechanism of formation for the complex anthocyanin-metal-ascorbic acid. (Adapted fromRef. 417.)

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terized. Narigenin is the common precursor toflavonoids and isoflavonoids; naringenin fla-vanone) is converted to dihydrokaempferol fla-vone), by a dioxygenase or a monooxygenase,depending on the tissue. In petunia, the enzymeflavanone-3-hydroxylase (F3H) (dioxygenase) isinvolved. In the next stage, dihydrokaempferolis converted to leucoanthocyanidin bydihydroflavonol-4-reductase (DFR), which isNADPH or NAD dependent according to the plantsource. This intermediate has been demonstratedby using acyanic flowers produced by differentgene blocks and by the characterization of theflavan-3,4-cis-diol-4-reductase (catalyzes the cat-echin synthesis). Further ahead in the pathway,leucoanthocyanidins are transformed to the col-ored anthocyanidins. This conversion is not wellcharacterized, but involves an oxidation and adehydration step; gene mutations correspondingto these activities have been identified in severalplant species (A2 in maize, Candi in snapdragon,ant17 in petunia), and this enzymatic activity hasbeen called as anthocyanidin synthase ANS). Theroute is followed by the anthocyanidin-anthocya-nin conversion that involves a glycosylation reac-tion. In this process the best described enzyme isthe UDP-glucose:flavonoid 3-O-glucosyl-trans-ferase (GT). Glycosyl transferases have shown apronounced specificity with regard to substrate,position, and sugar to be transferred. Recently,the first glycosyltransferase that catalyzesthe transfer of a sugar galactose) other than glu-cose to anthocyanidins was reported.The galactose:cyanidin galactosyltransferase(CGT) was characterized from a cell suspensionculture of Daucus carota L. In this system axylosyltransferase activity that is separatedby anion-exchange chromatography of thegalactosyltransferase activitywas also observed,suggesting the presence of two separate enzymes,each catalyzing a specific glycosyl transfer. Also,it was proposed that glycosyltransferases act insequential reactions, initiating with thegalactosyltransferase and followed by the reac-tion catalyzed by xylosyltransferase.401 Hydroxy-lation and methylation occur at different stages offlavonoid biosynthesis. In particular, anthocyaninmethylation is a late step, and the isolated fla-vonoid O-methyltransferases (MT) have shown a

great specificity, thus multiple methylation re-quires multiple enzymes. In Petunia hybrida flow-ers at least four methyltransferases have beeninvolved (Mt1, Mf1, Mt2, and Mf2).

On the other hand, it has been proposed thatacyl transferases show high specificity, andin some instances acylation must precedeglycosylationin the acylation of anthocyanins. Acyltransferases have been described in Silene,Matthiola, Callistephus, Dendranthema, and Zin-nia; they catalyze the transfer of 4-coumaryl orcaffeoyl groups to the corresponding acylated an-thocyanins, and succinyl transfer isa noncommon event. An acyltransferase from blueflowers of Centaurea cyanus was isolated. Thisenzyme catalyzes the transfer of the succinyl moi-ety from succinyl-CoA to 3-glucosides of cyanidinand pelargonidin, but not to3,5-diglucosides. Also, it was reported that thesame enzyme catalyzes the malonylation at similarrates of succinylation.513 Another important modi-fication is sulfation, and sulfate transferase cDNAwas isolated from Flaveria bidentis.6 The enzymesof anthocyanin synthesis are probably cytoplasmicand attached to the vacuolar membranes, and it islikely that after the glycosylation, anthocyanins aretransported into the vacuole.151,193,195,216

In anthocyanin-producing cells of sweet po-tato were identified vacuoles containing largeamounts of pigment and protein. This protein of24 kDa (VP24), which accounted for more than50% of the total protoplast protein showed a simi-lar pattern of accumulation to that of anthocyanin.It was observed that VP24 began to appear invacuoles at nearly the same time as the accumu-lation of anthocyanin became detectable, and itwas suggested that VP24 is involved in theintravacuolar accumulation of anthocyanin.345

b. Biosynthesis Regulation

Little is known about the regulation of thebiosynthetic enzymes of flavonoid synthesis, andmuch of the knowledge has emerged from mo-lecular biology studies.62,328 The pronounced sub-strate specificity of the 4-coumarate:CoA ligaseof different plants has been shown, indicating thatthis enzyme is an important regulation point to

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establish the distribution of CoA esters in plants.Additionally, external and developmental factorsshowed an interdependent response of flavonoidbiosynthetic enzymes. At least three photorecep-tors have been suggested that control flavonoidbiosynthesis (red/far red, blue UV-A, orcryptochrome and UV-B), and anthocyanin bio-synthesis showed different responses dependingon the light quality. In parsley plants, enzymeactivities of flavonoid synthesis are high only inyoung cotyledons and leaves, and a decrementwas observed in the later stages of develop-ment.195,216 In Arabidopsis, mutants in several ofthese photoreceptors have been identified andsome of them involve products of the Cop, Det,and Fusca genes.326 In Petunia, mutations at fourloci (An1, An2, An4, and An11) have regulatoryeffects on transcription of at least six structuralanthocyanin biosynthetic genes.216

c. Molecular Biology of AnthocyaninBiosynthesis

Flavonoid biosynthesis has been selected as afavorite pathway to carry out studies at the mo-lecular biology level; thus, this is the best knownpathway of secondary metabolism. Many cDNAand genomic clones involved in flavonoid synthe-sis have been characterized, and some of themhave been cloned in bacteria, providing an excel-lent model to produce the corresponding proteinand to induce the in vitro production of valuableflavonoid metabolites.151 In 1983, chalcone syn-thase gene was the first gene cloned of flavonoidbiosynthesis by using differential screening, whichwas also employed to isolate PAL, 4CL, and F3Hfrom parsley. Moreover, antibodies were used inan expression library to clone the genes CHI andF3H. Interestingly, flavonoid biosynthesis path-way was a model to design a new technique forcloning genes transposon tagging), and betweenothers several regulatory genes were isolated.125,481

In addition, new methodologies have been used toclone anthocyanin genes. Rapid amplification ofcDNA Ends (RACE) by PCR was used to isolatethe chalcone synthase gene 2 (chs2) from potato,and by Southern analysis it was shown that chsgenes of potato comprise a family of at least six

individual members.238 On the other hand, differ-ential display was used to identify four new chsgenes from morning glories.163 In addition, twoclones with high homology with flavonol syn-thase and anthocyanidin synthase were identifiedby using A. thaliana-expressed sequence tag(EST). This EST permitted the isolation of thefirst flavonol synthase gene.366 CHS is encodedby a multigene family in most of the speciesexamined to date. Only Arabidopsis and Antirrhi-num appear to contain single gene copies. It wassuggested that the evolution of CHS genes is ahighly fluid gene family where copy number var-ies greatly among plant lineages and where func-tional divergence may occur repeatedly.130 Inter-estingly, another chs-related cDNA was isolatedfrom Gerbera hybrida (Gchs2). Gchs2 did notshow correlation with the flavonol and anthocya-nin accumulation; its pattern of expression andaccepted substrates were different. Thus, it wassuggested that Gchs2 represents a new family ofgenes that have a common evolutionary originwith chs genes.205 In addition, it was proposed thatGchs2 emerged as the result of the duplication ofa single gene, followed by subsequent differentia-tion during the evolution of Gerbera.206

Other studies have evaluated the temporaland spatial control of flavonoid biosynthetic genes.Some genes showed organ-specific and others anstage-specific regulation, and several researchershave been characterizing the gene promoter re-gions.141,409 In maize it has been suggested thatchanges in kernel colors resulted from changes incis regulatory elements and not changes in eitherregulatory protein function or in the enzymaticloci, suggesting that differences in 5′ or 3′ regu-latory sequences or methylation patterns could beinvolved.191 The influence of DNA methylationwas also demonstrated in transformed petuniaplants. Petunia plants that show dihydrokaempferolaccumulation (defective in dfr gene) were trans-formed with the corresponding enzymes of maize(A1) and gerbera (gdfr). gdfr transformants showedhigher pigmentation than A1 transformants, and,particularly, it was observed that A1 transgeneinactivation was associated with a higher GCcontent and an increased methylation state.134 In-terestingly, studies on chalcone synthase (CHS)promoter of French bean showed that exists a

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differential utilization of cis regulatory elementsfor stress-induced (elicitors, wounding, and in-fection) and tissue-specific expression, and it wasindicated that this multiple combination of dis-tinct cis elements contributes to the complex ex-pression pattern of CHS genes.140 It has been es-timated that at least 30 genes are required forflavonoid biosynthesis, and within these differentmutations have defined regulatory proteins whoseabsence prevents the synthesis of multiple en-zymes (probable transcription factors). It has beenshown that chalcone synthase activity is under thecontrol of other genes (f in Matthiola, niv in An-tirrhinum, and c2 in Zea mays). In maize twodifferent families of regulatory genes have beenestablished, C1 and R. The expression of one ofthese genes induced a pleiotropic effect that insome instances is observed as an increment in theanthocyanin biosynthesis, and it has been ob-served that maize anthocyanin synthesis requiresthe expression of at least one gene of each fam-ily.125,328 Additionally, it has been observed thatregulatory systems of anthocyanin synthesis areconserved between plant families: transformationof petunia plants with genes of the C1 and Rfamilies induces the accumulation of anthocya-nins. In petunia three regulatory mutants(an1, an2, an11) showed accumulation ofdihydroflavonols, and it has been suggested thatAn2 gene is an R-homologue, while An1 is C1-homologue.125 Also, it was shown that the Anti-rrhinum del gene, tomato Lc gene, and the maizeR gene family are structurally and functionallyhomologous.175,178 When plants of two Solanaceaespecies (tomato and tobacco) were transformedwith the del gene, an increment in anthocyaninswas observed in the pigmented regions, butthis effect was not found in del-transformedArabidopsis, indicating that some genes may notbe able to function effectively in all plant hosts.329

To complicate more the understanding of theanthocyanin biosynthesis regulation, negativeregulators have been identified in Arabidopsisseeds (fus and banylus). The presence of fus andbanylus products prevents the accumulation ofpigments. Additionally, other negative regulatorshave been isolated (eluta from Antirrhinum flower,C1-1 from maize aleurone layer, fusca fromArabidopsis embryo). It has been proposed that

banylus can direct the biosynthetic pathway to-ward different metabolites.3 It is clear that regula-tion is a very complex process, and, as mentionedabove developmental (maturation, germination)and external signals (light, temperature) are in-volved: VP-1 is a pleiotropic protein that acti-vates C1 specifically, during seed maturation ofmaize, by interacting synergistically with one ormore ABA-regulated transcription factors. How-ever, there is no evidence that VP-1 can binddirectly to DNA, and it has been proposed thatprotein-protein interactions have a principal rolein the activation of promoters of downstreamgenes.304 In maize and petunia, it was shown thatduplicated sequences of regulatory genes exert acritical role in gene regulation. Therefore, thecopy number must be strictly controlled by as yetunidentified mechanisms, but indicating that DNAmethylation is involved.467 Moreover, it has beenestablished that maize-anthocyanin biosyntheticpathway can be considered as cold-regulationgenes that in addition are related with water stressbut not with heat stress. It was observed that amoderate cold (10ºC) enhances the transcriptionand/or stabilities for the anthocyanin biosynthesisgenes; however, at 5ºC the biosynthetic ability isdamaged. At 10ºC stress, it was observed a dra-matic increase in transcript abundance for antho-cyanin regulatory and structural genes, but only aslight increase in anthocyanin pigmentation thatis increased severalfold after plants are shiftedback to higher temperatures, showing that 10ºCstress impairs postranscriptional processes impor-tant for anthocyanin biosynthesis.108 By contrast,low temperature enhances the pigmentation andchalcone synthase gene expression in petunia flow-ers, similarly to the observed in apple skin and inflower buds.326,442

In eggplant hypocotyl-tissue was identified aP450 cDNA clone that is inducible by white light.At the same time, white light induces the accumu-lation of CHS and DFR mRNA in a coordinatedfashion, and it is proposed that this P450 enzymecatalyzes the hydroxylation of the B-ring in fla-vonoids, that is, the P450 isolated clone encodesfor a 3′,5′-hydroxylase that acts during anthocya-nin biosynthesis.468 Interestingly, it has been shownthat phytochrome stimulates anthocyanin biosyn-thesis through the induction of 3′,5′ cyclic mono-

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phosphate (cGMP) acting alone or together withcalcium.55 In Perilla frutescens, all the anthocya-nin biosynthetic genes were coordinately inducedby strong light (chs, f3h, dfr, ldox, 3gt, aat).176 InArabidopsis, it was shown that light-induced sig-nals converge on the fusca genes that relay theinformation to different plant responses (cell ex-pansion, chloroplast differentiation, tissue-specificchanges).322 In addition, it the hy4 gene that codesfor the CRY1 protein and have the characteristicsof a blue-light photoreceptor was identified. Thus,CRY1 must be responsible for anthocyanin bio-synthesis under UV-light irradiation.283 It wasfound that UV-A and UV-B induce different re-sponses. UV-A induction of CHS expression doesnot appear to involve calmodulin, whereas UV-Bresponse does. Consequently, the light signalingpathway showed differences between them andwith phytochrome-mediated response.27,107 On theother hand, the gibberellin GA3 stimulates theanthocyanin synthesis on petunia corollas, whileit is completely inhibited in cultured carrot cells.In petunia corollas, GA3 is essential for the in-duction of chs, chi, and dfr expression, probablyby effect of a regulatory protein receptor), be-cause after GA3 addition exists a lagging stageuntil the response can be observed. Interestingly,it was observed that the presence of sucrose isrequired to observe the effect of GA3.325,326,502

Similarly, it has been shown that the cytokininbenzyladenine (BA) treatment induces a red pig-mentation in Arabidopsis seedling, showing thatCHS and DFR appear to be transcriptionally ac-tivated, while PAL and CHI are controlledpostransciptionally.116

The last step in anthocyanin biosynthesis in-volves the transfer and deposition of the red andpurple pigments in vacuoles. In maize, this step iscarried out by the protein encoded by the bronze-2 gene (bz2), and it was shown that bz2 codes fora glutathione-S-transferase (GST) with activity inmaize, transformed A. thaliana plants, and E. coli.In bz2 mutants, cyanidin-3-glucoside is accumu-lated in cytoplasm, whereas in bz2 plants antho-cyanins are transferred to vacuole. It was sug-gested that anthocyanin import into vacuoles couldoccur as anthocyanin-glutathione conjugates, par-alleling the mechanism by which plants eliminateherbicides and xenobiotics.296

The world market of flowers is very impor-tant, and the knowledge of the biosynthetic routeof anthocyanins and molecular biology techniquesallows to modifying flower colors.215,220,243 Petu-nia DFR can not reduce dihydrokaempferol, thuswild type petunias do not produce pelargonidinpigments. Petunia plants were transformed withmaize DFR and mutant petunias that accumulatepelargonidin were obtained.312 On the other hand,petunia flowers with reduced pigmentation wereobtained by transforming with the chs gene ofpetunia in anti-sense.482 White-floweringtransgenic chrysanthemums have been producedby trans-inactivation of the main chs gene, and asimilar strategy was used to obtain a range ofcolor for carnation and rose flowers.88,188 Interest-ingly, the effect of anthocyanins on sexual pro-cess has been proposed to be manipulated bymolecular biology as an alternative to producemale sterile hybrid crops.326 In alfalfa f3h corre-lates with flavonoid accumulation, and particu-larly f3h expression in pollen grains and ovaryprobably could be associated with plant fertility.95

It was also demonstrated in tobacco that flavonoidsdo not only determine flower color, but also playan essential role in the development of male ga-metophyte. The stilbene synthase that competesfor primary precursors with chalcone synthasewas overexpressed, and, consequently, flavonoidproduction was impaired (reduced color) and male-sterile plants were obtained.147 On the other hand,the rapid quality improvement of some cultivarsrequires of selection at the seedling stage, andmolecular biology provides excellent tools todevelop this selection. Random-amplified poly-morphic DNA (RAPD) was used to identify red-der apple cultivars, and one marker was identifiedfor this characteristic. It was suggested that thismarker is associated with a regulatory gene of theanthocyanin biosynthesis.102

Another interesting function was suggestedby transforming a wine yeast strain with the an-thocyanin-β-glucosidase gene of Candidamolischiana. Transformed S. cerevisiae produceswines with quality characteristics comparable tothose obtained with the wild-type strain. This newstrain is of industrial relevance mainly in the pro-duction of white wines with lower red color, al-though they were obtained from red grape variet-

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ies. Additionally, transformed S. cerevisiae wasproposed for the production of wines with higherlevels of resveratol and terpenols from the hy-drolysis of their glycosidic precursors.414 As pre-viously mentioned, food industry is interested inpigments with higher stability and acylation ofanthocyanins produces more stable pigments.Anthocyanidin-3 glucoside rhamnosyltransferase(UFGT) of Antirrhinum majus was used to trans-form lisianthus (Eustoma grandiflorum Grise.).Transformed lisianthus plants were able to pro-duce a novel 3-O-glucosylated flavonols. Inter-estingly, C-3 glucosylated anthocyanins havenever been observed in this plant.433 On the otherhand, sometimes it is recommendable to diminishthe quantity of anthocyanins and to increase thecontent of other metabolites. The presence of con-densed tannins (CT) in alfalfa has supported ahigher sheep productivity higher weight and woolproduction) than CT-free alfalfa. Recently, it wasshown that cultivars with higher CT content couldbe obtained by molecular biology. It has beenestablished that dfr is involved in CT biosynthe-sis, and some Lotus corniculatus plants trans-formed with the Antirrhinum majus dfr cDNAshowed increased CT levels.

5. Functions

a. Color and Ecological Functions

As flavonoids, anthocyanins are benzopyranderivatives. Thus, anthocyanins also showed simi-lar functions in plants to those described in thecorresponding section for flavonoids: antioxidant,photoprotection, defense mechanism, as well asother ecological functions (symbiosis phenom-ena). In particular, anthocyanins are the mostimportant pigmenting compounds between theflavonoids, and consequently they show an inter-esting role in several reproductive mechanisms ofplants such as pollination, seed dispersal, andantifeedant. Interestingly, it has been observedthat cyanidin-3-glucoside is inhibitory to the lar-val growth in tobacco budworm Heliothisviriscens, and consequently anthocyanins couldbe considered agents of biological control. Addi-tionally, anthocyanins have been proposed as taxo-

nomic markers, although this goal has not beenachieved yet because of nowadays only a limitednumber of species levels within families havebeen investigated. However, interestingly, thepreferred presence of cyanidin has been suggestedas a marker of ancestral plants.193,195,455 In thisaspect, anthocyanins with substitution (sugar oracid) in the sugar 3-position have only been re-ported in the genus Allium;153 in the genus Bego-nia a pattern of six anthocyanins as marker (froma survey of 200 begonia cultivars)was suggested,105

and acylated anthocyanins with 5-O-(6-O-hydroxycinnamoylglucoside), wherehydroxycinnamic acids are coumaric or caffeicacids, have only been reported in the Gentianagenus.223

b. Marker for Good ManufacturingPractices in Food Processing

Anthocyanins have been used to evaluate theadulteration of some pigmented food products.56

Prune juice is a product in which brown color isdeveloped by the reaction of phenolic compoundsand/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 ofadulteration, it is believed that prune juice canhave only traces of anthocyanins, while the adul-terated juice will show increased levels.485 Also,anthocyanin profiles have been used to determinethe authenticity of fruit jams. With this kind ofanalyses, it was determined that labeled blackcherry jams in reality were prepared with com-mon red cherries (less expensive fruit). In addi-tion, it was suggested that adulteration of black-berry jams with strawberries can be detected withanalysis of the relation between pelargonidin andcyanidin 3-glucoside. Also, it was pointed outthat this methodology is very efficient becauseanthocyanins are pretty stable during jam manu-facture.165

c. Pharmacological Effects

Reports on biological activities of anthocya-nins are scarce, but considering the wide distribu-

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tion of anthocyanins, it is reasonable to assumethat humans are well conditioned to large con-sumptions of these compounds. In a survey withItalian subjects, anthocyanin daily intake was inthe range 25 to 215 mg/person, depending ongender and age, and this intake is largely enoughto induce pharmacological effects. The consump-tion of wine flavonoids has been correlated withlow incidences of coronary hearth diseases (Frenchparadox), and, similarly, chokeberry (Anoniamelanocarpa) extracts have shown very strongnutraceutical properties. Moreover, anthocyaninspossesses bactericidal, antiviral, and fungistaticactivities. They exhibit a strong antioxidant activ-ity that prevents the oxidation of ascorbic acid,provides protection against free radicals, showsinhibitory activity against oxidative enzymes, andhas 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′-OHin the B ring structure are determinants for theradical scavenging potential in flavonoids with asaturated 2,3-double bond, and that different pat-terns of hydroxylation and glycosylation maymodulate their antioxidant properties.499 It hasbeen mentioned that an increased number of hy-droxyl substituents on the B-ring produce higherantioxidant activities when are glucosides, but theactivity is weaker with aglycones.476 These twohydroxyl groups are important in protecting ascor-bic acid against oxidation by chelating metal ions.When the anthocyanin-metal chelate is formed, a20- to 25-nm shift in the visible spectra is ob-served, which in the presence of ascorbic acidinduces a further shift of 10 to 15 nm. It wasproposed the formation of the stable complexanthocyanin-metal-ascorbic acid, where ascorbicacid acts as a copigment. Thus, improved colorsare obtained with the intrinsic protection of ascor-bic acid from oxidation (Figure 16).417

The effect of pure anthocyanins against lipidperoxidation has been studied. Liposomes wereused to evaluate the inhibition in the productionof malondialdehyde. All evaluated anthocyaninswere better agents against lipid peroxidation thanα-tocopherol (up to seven times). Also, it wasdemonstrated that anthocyanins have scavengingproperties against ·OH and O2

-. In addition, it wasmentioned that ·OH scavenging is better with

aglycones of high number of OH groups in theB-ring, opposite to that observed with other fla-vonoids, while O2

- scavenging is independent ofthe glycosylation state but also increases with thenumber of hydroxyl groups, similar to the ob-served with other flavonoids.476 In the red colorantextracted from Anonia a mixture of anthocyanins(cyanidin, cyanidin-3-glucoside and cyanidin-3,5-diglucoside) and polyphenol substances calledbioflavonoids (leucoanthocyanidins, catechins,and flavonols) has been identified. In particular,bioflavonoids have shown activities to improvethe permeability and strength of capillaries, toaccelerate the ethanol metabolism, and to reduceinflammatory and edematic reactions.286 Similareffects have been observed with crude extracts ofRubus occidentalis, Sambucus nigra, andVaccinium myrtillus.157,195

6. Methodological Aspects

a. Extraction

Anthocyanin, like flavonoids in general, havearomatic rings containing polar substituent groups(hydroxyl, carboxyl, and methoxyl) and glycosylresidues that altogether produce a polar molecule.Consequently, they are more soluble in water thanin nonpolar solvents, but depending on the mediaconditions anthocyanins could be soluble in etherat a pH value where the molecule was unionized.These characteristics aid in the extraction andseparation of anthocyanin compounds.193 Conven-tional methods of pigment extraction usuallyemploy dilute hydrochloric acid in methanol.Methanol containing 0.001% HCl was the mosteffective, but HCl is corrosive, and methanol pro-duces toxic effects after human exposition; con-sequently, food scientists frequently prefer theuse of other extraction systems. Among othersolvents, one finds ethanol and water, 80 and 27%as effective as methanol, respectively. Addition-ally, it must be taken into account that aromaticacyl acid linkages are relatively stable in diluteHCl/MeOH mixtures, but aliphatic dicarboxyl acylgroups (malonic, malic, oxalic) are susceptible todiluted acids, and different methodologies mustbe considered. It is recommended to use weaker

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acids (acetic, formic, perchloric) during extrac-tions and to monitor the acidity during the pro-cess. With methanol, citric acid is the most effec-tive organic acid followed by tartaric, formic,acetic, and propionic; with water, the best acidsare acetic acid, citric, tartaric, and hydrochlo-ric.58,157,195,455 Recently, an aqueous extraction pro-cess for anthocyanins from sunflower hulls wasevaluated. It was shown that extraction with sulfu-rous water (1000 ppm SO2) was better than thetraditional extraction with ethanol:acetic-acid:water system. Also, it was mentioned that1 h of extraction was enough to reach a completeextraction of pigments. It was suggested that pos-sible reasons for the improved extraction withSO2 are the interaction of anthocyanins with HSO3

-

ions, which improves the anthocyanin solubilityand the diffusion through cell walls.164

b. Separation

The initial attempts for the separation of an-thocyanins were based on the adsorption in paperchromatography or in other suitable adsorbents.455

Nowadays, thin layer chromatography (TLC) iswidely used, because this technique has showncontinuous innovations and still keeps its advan-tages (practical and very cheap). For preparativework, droplet counter current chromatography hasbeen applied to separate the anthocyanins of blackcurrant. On the other hand, a general patent forthe purification of anthocyanins involves selec-tive absorption on a finely divided oxide such assilicic acid, titanium oxide, or alumina, which iscoated with a styrene polymer.157 However, un-doubtedly, the main developments of recent yearsin the research of anthocyanins is the introductionof HPLC for their separation and quantitation.193,195

Interestingly, it is possible to distinguish zwitteri-onic anthocyanins by their HPLC chromatographicseparation.195

c. Characterization

Spectroscopy. In general, color is evaluatedby spectrophotometry.157 Isolated pigments havebeen studied by UV-visible spectroscopy. All fla-

vonoids show high absorbance in the range of 250to 270 nm (UV region), and, particularly, antho-cyanins have an intense absorption in the range of520 to 560 nm (visible region). It has been sug-gested that UV absorption could be assignedmainly to ring A, while the visible to the pyranand ring B. With this methodology, researcherssuppose that identity between the isolated pig-ments and those found in living tissue is the same;however, the main problems are the generation ofartifacts during the extraction process. To solvethis problem, model systems such as anthocya-nins stored in synthetic vacuoles have been devel-oped. With UV-visible spectroscopy, it is alsopossible to detect glycosylation on B-ring, be-cause of the spectra show an hypsochromic shiftin relation to the unglycosylated B-ring. Antho-cyanin acylation is also observed by this method-ology: in the presence of AlCl3 a bathochromicshift is observed, only if the 3′- and 5′-OH groupsare free (nonacylated).462 Moreover, acylated an-thocyanins show a maximum absorbance in the310-nm region, and it has been found that theratio of absorbance at the acyl maximum wave-length to the absorbance at the visible maximumwavelength (Amaxacyl/ Amaxvisible ratio) is a mea-sure of the molar ratio of acyl group to anthocya-nin.173 Additionally, visible absorption is the besttool to observe the copigment effect: visible spec-tra of anthocyanins show a hyperchromic effect,increment of the intensity of this maximum re-sulting in a more colored species, and abathochromic shift by a solvation effect.62,63,195

Heating causes a hypsochromic and hypochromiceffect that is restored by copigment regenerationafter cooling in the malvin-rutin systems; how-ever, the reversibility was not observed in themalvin-quercetin system. However, in both sys-tems the copigment reaction was a spontaneousthermodynamic process (negative ∆G).20 On theother hand, visible absorption and RAMAN spec-troscopy have been used in the study of pigmentsin the living tissue. RAMAN spectroscopy hasbeen used to show the anthocyanin substitutionpattern. The presence of phenyl ring substitutionson benzopyrylium produces clear spectral modi-fications. On the other hand, the anthocyanin spec-tral features are modified by glycosylation, show-ing that perturbations are dependent of the sugar

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nature. Interestingly, 5-glycosides present highermodifications than 3-glycosides.63,309 The couplingof the diode array detector (DAD) into the HPLCmethodology has permitted the tentative identifi-cation of the separated anthocyanins, as was de-scribed with carotenoids. In addition, with theintroduction of innovated methodologies such asNMR and mass spectrophotometry, anthocyanincompounds have been identified conclusively.Proton NMR has been used to study the self-association of anthocyanin molecules, while car-bon-13 NMR spectroscopy has been used to de-fine the sequence, position, and configuration ofsugar residues in flavonoid glycosides. NMRmethodology has been used with heteronuclearshift correlation through multiple quantum coher-ence (HMQC) to show the configuration of an-thocyanin glycosides. β configuration wasobserved with glucosyl, galactosyl, andxylanopyranosyl groups and α configuration withrhamnosyl and arabinopyranosyl groups. More-over, 1H-1H COSY and DIFNOE have been usedin structure characterization to assign the NMRproton signals.365 In addition, mass spectrometryincreased its potential with the introduction of theFAB-mass detector, which permitted observing

an intense peak for the molecular ion, somethingdifficult with the previously designed detectors;anthocyanins are unstable and with low volatility.Nowadays, methodological advances have beenconducted on other mass detectors that inclusivelyare coupled with the HPLC equipment (see abovediscussion about carotenoids).19 Interestingly, newstructures have been elucidated, and anthocya-nins with caffeoylglucose and malonyl substitu-tions are perhaps the most notable.18,291 Anothermethodology used to study the copigmentationphenomena is circular dichroism.62,193,195

Chemical tests. A large number of chemicaltests have been developed to determine the antho-cyanin structure (some of them are shown in Table5), and a general procedure could be envisioned,but this must be modified depending on the ana-lyzed material.193 After separation, isolated antho-cyanins were hydrolyzed with mineral acidand glycoside bonds were disrupted, thenanthocyanidins were methylated, and after otheracidic hydrolysis the nature of the anthocyanidinand the positions of glycosylation were deter-mined.455 When anthocyanin molecules show ali-phatic acids in their structures, a zwitterion isobserved that can be detected by electrophore-

TABLE 5Some 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 greenand Mg (Wilstatter reaction) flavonoids)

Ethanolic solution with Flavonoids with o-hydroxyl Formation of a silver mirrorAgNO3 (12% in water) groups

Paper chromatography Flavonoids Dark or fluorescent colorsand treatment with ammoniacvapors

Sodium methoxide (2.5% in Flavonoids with hydroxyl Bathochromic change ofmethanol) groups at 3- and/or 4’- spectra and if intensity of

positions visible band do not decreasethus a 4’-OH is present

Sodium acetate (solid) Hydroxyl at 7-position Bathochromic change of theUV band

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

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sis.157 Additionally, to determine the occurrenceof phenolic acids as esters, the extract is concen-trated and resolubilized with 6N HCl and hydro-lyzed (100°C/60 min), the solution is extractedwith ethyl acetate and then evaporated undervacuum and the residue prepared to be analyzedby HPLC.19 The presence of B-ring-substitutedsugars is easily detected by an hypsochromic spec-tral shift.157

Evaluation of pigmenting efficiency. Inthe evaluation of visual color, reflectancecolorimetry has been the better approach be-cause it shows a good correlation with thechemical composition, and as above men-tioned, lightness has been used to determinethe pigment concentration; saturation (purity)reflects the spectral properties and the chro-maticity coordinates give an appreciation ofthe various mixed pigments.62,157 In this sense,a method has been developed to assess theanthocyanin concentration by reflectancemeasurements in red raspberry fruit, and it isreported that the best predictor is the hueparameter (r = 0.73).330

7. Importance as Food Colors —Stability, Processing, and Production

a. Stability in Model Systems

Anthocyanins are of great economic impor-tance as fruit pigments and thus also as pigmentsof fruit juices and wines.195 Interestingly, one ofthe main claims of the food industry is for naturalcolorants to replace synthetic red dyes, and antho-cyanins are the principal candidates, but, interest-ingly, enocyanin and lees (sediment of the grapejuice tanks) preparations are the only anthocyaninsources approved by FDA (in the U.S.) to be usedfor human food, while the main use is in theproduction of beverages and soft drinks.58,157 How-ever, anthocyanin instability limits their use, anddifferent preparations have been evaluated to avoidanthocyanin degradation.87 The color of antho-cyanins is provided by its resonating structure,but resonance phenomena also confers its intrin-sic instability. Moreover, it has been establishedthat instability has a direct relation with the num-ber of hydroxyl groups and indirect with the num-

ber of methoxyl groups. In addition, theglycosylation level showed a clear effect on sta-bility, diglucosides are more stable thanmonoglucosides, but browning is more favorablein diglucosides by the presence of the additionalsugar molecule. Also, it has been showed thathydroxylation at position 4 changes the colorstoward red tones.157

The equilibrium colored-uncolored anthocya-nin structures affects the stability of products onstorage, because the R+ form is the most stable.

R H O ROH + H

Colored catonic Uncolored

form pseudobase form

+2

++ →

The reactive species was found to be the an-thocyanin carbonium ion (R+), which reacts witha bisulfite ion to form a colorless 2-bisulfite ad-duct product.127

In solution, anthocyanin molecules arepresent in an equilibrium between the coloredcationic form and the colorless pseudobase.This equilibrium is directly influenced by pH.Acidic pH is favorable for the colored formthat diminished with pH increments. Someanthocyanins are red in acid solutions, violetor purple in neutral solutions, and blue in al-kaline pH. This is the reason that most colorantscontaining anthocyanins can only be used atpH values below four.58 Acylation hindershydrolysis of the red flavylium cationic form,allowing preferential formation of the bluequinonoidal bases. Interestingly, acylated pig-ments retain more color at the higher pH val-ues than unmodified anthocyanins. In addi-tion, acylated anthocyanins show an improvedresistance to other factors such as heat, light,and SO2. In particular, in Vitis vinifera wineswere identified the anthocyanins vitisins inwhich C-4 positions of their structures aresubstituted, and consequently these pigmentsgive greater colors, higher resistance to colorloss with sulfur dioxide, and higher pH val-ues.18 Thus, this group of anthocyanins hasbeen proposed as potential food colorants.Moreover, it is also very important that acyla-tion produces a major absorption band at pHvalues above four, and, consequently, acylatedanthocyanins will be highly colored at pH > 4,

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contrasting with the conventional compoundsthat are uncolored at these condi-tions.58,127,157,161,193 As a matter of fact, a greatnumber of studies have been carried out tofind new acylated pigments, and several oneswith novel characteristics not previously re-ported) have been characterized (Table 6). In-terestingly, malonylated anthocyanins are pro-duced in purple sunflower seeds, which is oneof the most important oilseed crops, and, con-sequently, the purple-hulled residue obtainedafter oil extraction from seeds provides anexcellent source of red colorant (cyanidin de-rivatives) that has been suggested as a colorantof food, pharmaceutical, and cosmetic prod-ucts.303 In addition, several of the acylatedanthocyanins have shown highly desirable sta-bility, and the structures of some of them arepresented in Figure 18. In particular, the im-proved stability of the Ajuga reptans antho-cyanins was assigned to the presence of twocinnamic acid groups in the anthocyanin struc-ture,461 and it has been suggested that long

acylation groups permitted anthocyanins toadopt a folded conformation, which protectsthe basic anthocyanin structure of degradativefactors.146,458 To corroborate the effect of acy-lation on anthocyanin stability, anthocyaninextracts of Sambucus nigra and S. canadiensiswere compared. Anthocyanins of S. nigra havea free hydroxyl group on C-5, while in S.canadiensis a glucosyl residue acylated withp-coumaroyl is present. It was observed thatS. canadiensis extracts were more stable thanthose of S. nigra. It was shown that C-5glucosylation does not represent a crucial fac-tor on heat stability, but acylation does. How-ever, it was also shown that C-5 glycosidation,as well as acylation, has an importantcontribution to color stability for light. In ad-dit ion, the following decreasing orderof stability tolight was reported: acylateddiglucosides > nonacylated diglucosides > monoglucosides.232 A comparable high stabil-ity of C-5 acylated anthocyanins was also ob-served with the pigments from red radish.173

TABLE 6Reports 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 222Hyacinthus 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 8victorialis 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 amorliacea) bathochromic shift (5 nm) on the maxima of the

visible spectra

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FIGURE 18. Acylated anthocyanins with improved stability under different process conditions. These pigments wereobtained from (A) Ajuga reptans, (B) Bletilla striata, (C) Eichhornia crassiped, (D) Ipomoea purpurea, and (E)Tradescantia pallida.

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On the other hand, anthocyanin production inleaves of Ajuga pyramidalis was comparedwith that produced in cell cultures. The mainanthocyanin was 3-O-(6-O-(E)-ferulyl)-2-O-[(6-O(E)-ferulyl)-β-D-glucopyranosyl-β-D-glucopyranosyl]-5-O-(6-O-malonyl)-β-D-glucopyranosylcyanidin; and it was determinedthat anthocyanins from cell cultures showedhigher stability than those obtained from invivo extracts. It was explained that in cell cul-tures copigmenting agents such as flavonols,phenolic acids, and tannins could accumulate,which may be absent in plant.292

Condensation reactions have been reportedby the reaction of acetaldehyde, anthocyanins,and flavan-3-ols, producing the increment of color(up to seven times). It is believed that acetalde-hyde forms a bridge between the two flavonoids,and consequently condensation reactions couldproceed and contribute to the polymericcolor.157,193,242 Moreover, the formation of newanthocyanins by the reaction of malvidin 3-monoglucoside and procyanidin B2 in the pres-

ence of acetaldehyde 15ºC/4 months) was ob-served. Three new pigments were observed andtheir visible spectra showed a bathochromic dis-placement, in relation to the anthocyanin by thecondensation of these compounds. Interestingly,these compounds showed an improved stability.155

Compounds with similar characteristics to thesynthesized ones have been detected in red wines.

b. Processing and Stability in Foods

Anthocyanin pigments can be destroyed easilyduring the processing of fruits and vegetables, andconsidering food color as an appealing characteris-tic, many studies have been carried out to under-stand the anthocyanin properties and to obtain bet-ter products with minimal degradation (Table 7).High temperature, increased sugar level, pH, andascorbic acid can affect the rate of destruction.157,510

Temperature has been reported to induce a loga-rithmic destruction of pigment with time of heatingat a constant temperature. Bleaching by effect of

TABLE 7Processing and Stability Studies on Anthocyanin Pigments in Different Food Systems

Model Conditions Effects Ref.

Blackberry Different Disappearance rate followed a first-order 114juice 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 effectwas assigned to inhibition of degradativeenzymes and to steric interference withcondensation reactions

Grape musts Addition of Quinones are involved in anthocyanin 103glutathione degradation because of this is inhibited by

glutathione

Barley Heating (40–100ºC) Hordeumin anthocyanin showed an 347improved stability because of it isconstituted by a molecular complexbetween anthocyanins and polyphenols

Marashino cherries Brined cherries The coloration imparted by RAE and 173pigmented with red FD&C Red No. 40 was similar; theradish anthocyanin kinetics of degradation followed first-extracts (RAE) order kinetics

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heat occurs because of the above-described equi-librium is changed toward the uncolored forms. Ithas been suggested that flavonoid structure isopened to form chalcone, which is degraded fur-ther to brown products.157 However, interestingly,it has been observed that optimal conditions permitthe regaining of color on cooling if there is suffi-cient time (several hours) for the reconversion.

In slightly alkaline solutions (pH 8 to 10)highly colored ionized anhydro bases are formed.At pH 12, these hydrolyze rapidly to fully ionizedchalcones.58

Knowledge of anthocyanin characteristics haveallowed the development of a large number of prod-ucts and processing conditions that obtain high-quality colored products. Pomace extracts were freezedried on DE 20 maltodextrin, and this preparationshowed good shelf life (2 months at 50°C/0.5 awand more than 5 years with aw< 0.3). Also, improvedstability was obtained in concentrated extracts offermented elderberries. The described process wasas follows: berries were homogenized, fermentedwith C. cereviceae var. malaga (room temperature,pH = 4.5), and a concentrated juice is obtained (4%d.w. of anthocyanins). Additionally, the use of thisproduct to enhance the color of spiced wines ismentioned. Four patents have been developed toextract the colorant from blue corn by using a simpleacidified aqueous extraction. A patent to obtain thesorghum colorant involves alcoholic extraction andpurification on a silica gel column. While otherpatents have been developed for anthocyanin ex-traction from black mulberries, elderberries, Vibur-num berries, rowanberries, red sweet potatoes (Ipo-moea batatas), and for a variety of plant sources. Inaddition, a successful extraction procedure to isolatepigments using 350 ppm SO2 in water and ion ex-change chromatography was reported. Also, a puri-fication process was implemented that involves se-lective absorption on inorganic oxides such as silicagel, titanium oxide, or alumina, all of them coatedwith a styrene polymer.157

During processing and commercialization offresh products, color is the main factor of choice,and several attempts have been carried out topreserve the “fresh” appearance of fruits whenstored. In fresh strawberry fruits, CO2 treatmentshave been assayed to preserve their attractiveappearance. It was found that skin color was sig-

nificantly affected by different CO2 treatments,evaluated by reflectance colorimetry; however,internal color decreased markedly. Color disap-pearance was correlated with low anthocyaninlevels. Additionally, it was suggested that thisopposite behavior (skin and internal) could beproduced by different anthocyanin profiles in theanalyzed structures; higher concentrations of cya-nidin 3-glucoside are observed in external tissue(higher stability), while pelargonidin glycosidesare in the internal tissue. Moreover, the accumu-lation of phenolic compounds is greater in exter-nal tissue, in agreement with the protective roleassigned previously.170 In addition, other method-ologies have been proposed to change metabolicpathways to reach a higher color preservation. Infrozen strawberries, it was shown that sugar addi-tion has an stabilizing effect on the total mono-meric anthocyanins, suggesting that sugar can beemployed to enhance the shelf life of coloredproducts (Table 7).511 Litchi is a tropical fruit andwithin 2 to 3 days after harvest its pericarp be-comes desiccated and turns brown. To diminishthe losses, litchis were coated with chitosan(1.0 to 2.0%) and stored (4ºC/90% relative hu-midity). The use of chitosan delayed changes ofcontents of anthocyanin, flavonoid, and total phe-nolics. Interestingly, the activities of polyphenoloxidase and peroxidase, which have been involvedin anthocyanin degradation, are inhibited. It issuggested that a plastic coating forms a protectivebarrier on the surface of the fruit and reduces thesupply of oxygen for enzymatic oxidation of phe-nolics.523 Also, anthocyanin profiles have beenproposed as an indication of inadequate process-ing conditions. In red raspberry juices, it wasfound that bad processed samples showed higherlevels of polymeric color instead of the mono-meric anthocyanin pigments, and also this wasobserved as a product of poor quality.56 Anotherimportant factor to preserve color during fresh-fruit storage is the elimination of mold contami-nation, which has a deleterious effect onanthocyanin polymerization.405 In general, it is considered that light has deleterious effectson anthocyanin stability and light exposure ofnatural colored beverages must be avoided. It hasbeen reported that other flavonoids flavone,isoflavone, and aurone sulfonates) increase the

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photostability.157 On the other hand, light inten-sity has a profound effect on an apple’s color,because the light-exposed peel contains twice asmuch anthocyanin as a shaded peel. Interestingly,a correlation was shown between UFGT(UDPGal:flavonoid-3-O-glycosyltransferase) ac-tivity and anthocyanin accumulation with anUFGT activation by ethephon; it suggests a regu-latory importance of UFGT in anthocyanin syn-thesis in apple.245 Also, in apple it has been ob-served that methyl jasmonate is associated withthe ethylene production, and thus with anthocya-nin accumulation; this is the explanation for theapplication of methyl jasmonate during the initialstages of apple development to produce bettercolored fruits.142 To modify the apple fruit appear-ance, the use of magnesium and urea as a sourceof nitrogen was attempted, and a better final col-oration was observed, but this was mainly af-fected by the increase in chlorophyll and caro-tenoid content, while the anthocyanin levelremained the same.393 Oxygen has a negative ef-fect on anthocyanin stability, while ascorbic acidcould have a negative or positive effect, depend-ing on the media conditions. Additionally, it hasbeen suggested that flavonols act as free radicalscavengers to protect anthocyanin molecules.Anthocyanins are very reactive toward metals,and they form stable complexes with tin, copper,and iron; it has been proposed that metal com-plexes could be used as colorants.417

The overall color changes may be due to anumber of factors such as pigment degradation,pigment polymerization, reactions with other com-ponents of the formulation, nonenzymatic brown-ing, oxidation of tannins, and other reactions com-pletely unrelated to the added colorant.157

Anthocyanins can be degraded by a number ofenzymes found in plant tissue: glycosidases,polyphenoloxidases, and peroxidases. Glycosi-dases produce anthocyanidins and sugars, andanthocyanidins are very unstable and rapidly de-graded. Polyphenoloxidase catalyzes the oxida-tion of o-dihydrophenols to o-quinones that fur-ther react to brown polymers; however, thisreaction is more favorable for other phenols. Thus,pigment loss can be reduced by blanching to in-activate these enzymes. Addition of 30 ppm SO2

will inhibit phenolase activity in sour cherry juice.

Gallotannin will inactivate phenolase through adirect condensation system. The destruction ofanthocyanins by enzymatic activity could be im-portant in the design of an extraction procedureand perhaps in the final formulation in a food.157

c. Production of Anthocyanins by PlantTissue Culture

Plant tissue culture is a potential source ofproduction of anthocyanins, in view of the quotedprice ($1250 to $2000/kg) and the expanding mar-ket of natural anthocyanins. Moreover, plant cellculture could ensure a continuous supply of uni-form-quality anthocyanin pigments that cannotbe produced by other biotechnological approaches;however, to date no food colorant obtained by thissystem has been commercialized, and the mainbottleneck is the low yield of secondary metabo-lites.58,157,202

It has been considered that bioreactor produc-tion is prohibitive because present technological limi-tations make the process very expensive; a commer-cial pigment production by plant cell tissue culturecould be achieved only when a fully automatedpredictable process with a more advanced technol-ogy could be established.442 One of the main prob-lems with plant cell suspensions is the heterogeneityof media due to the existence of cells as large aggre-gates that cause problems of oxygen diffusion, masstransfer, cell ejection of the liquid media, and re-duced growth rates. Additionally, the conditionsfavoring high growth do not favor production, andfrequently high production conditions are detrimen-tal for growth. In particular, maximal growth ofstrawberry cells in suspension cultures was found at30ºC, but lower temperature induced an increasedlevel of anthocyanin pigment (the maximum at20ºC).524 Thus, processes by stages have been devel-oped (a first stage for cell growth and a second onefor anthocyanin production), and models have beendesigned for batch and semicontinuous anthocyaninproduction to introduce the concept of primary andsecondary metabolism because of the continuouscompetence for precursors.441

Anthocyanin production by cell cultures hasbeen evaluated from grapes (2 to 3 mg/g freshweight),514 Euphorbia milli,516 and carrot,492 among

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others. In general, stable production by plant cellcultures has not been reached. However, somecultures of Ajuga reptans have produced antho-cyanins for more than 10 years, but the analysesof five lines showed quantitative differences be-tween the lines, and it was observed that 5′-sub-stituted anthocyanins seem to decrease more eas-ily during repeated subcultures.67 In wild carrot(Daucus carota), clonal variability has been ob-served to obtain low- and high-pigmented cells.Interestingly, it was shown that high-pigmentedcells have an intermediate limitation reflected bya considerable increment in pigment productionafter supplementation with dihydroquercetin,naringenin, and 4-coumarate.492

Remarkably, anthocyanin production is lightdependent. Darkness cell cultures showed an in-tense pigmentation after illumination,418 althoughdarkness cultured cells, Aralia cordata amongothers, have shown anthocyanin accumulation.Cultured cells of Perilla frutescens need lightirradiation for 7 days from the initial cultivationperiod for the formation of anthocyanin pig-ments.526 Interestingly, it was observed that lightcultured cells are positively influenced by thetreatment with auxins and cytokinins, while cul-tures in darkness are not affected.411 In callus cul-tures of Oxalis linearis, anthocyanin productionwas promoted by cytokinins but repressed byauxins such as NAA and 2,4-D.311 Another factoranalyzed in plant cell pigment production is thepH, and considering the stability characteristicsof anthocyanins the optimum pH is mainly acidic(pH < 7), but it was found that suspension cul-tures of strawberry cells showed the highest an-thocyanin production at pH 8.7. It was observedthat the time profile ratio of pigmented cells didnot change with the variation in initial mediumpH, suggesting that initial pH affected anthocya-nin synthesis by changing the anthocyanin con-tent inside the pigmented cells only.525

Several substrates have been assayed to in-duce increments in anthocyanin production. Incell suspension cultures of Vitis, phenylalanineaddition induces the cessation of cell division andthe induction of anthocyanin biosynthesis. Addi-tionally, it was shown that Vitis cell division islimited by nutrients in medium, such as sucrose,phosphate, and nitrate or ammonium, and that

nutrient limitation induces the activities of PALand CHS enzymes, suggesting an association ofphenylalanine addition with enzyme induction,and a similar behavior was observed with straw-berry cells.115,246,524 With Fragaria anansa cellcultures, riboflavin addition had a 3.2 times greaterproduction than in control medium, but only un-der light conditions.331 In carrot cell, suspensioncultures were found that favor fructose growth,while anthocyanin production is better with glu-cose, suggesting quantitative differences in themetabolism of these two sugars.530 In culturedcells (e.g., Vitis vinifera, Aralia cordata, Fragariaanansa), high sucrose and low nitrate aredesireable for anthocyanin production. It has beenconsidered that although sucrose is an essentialnutrient, it also acts as an osmotic agent whenused at high concentrations. Interestingly, it wasobserved that high ammonium concentration(24 mM) promotes the anthocyanin acylation,which is important for the food industry based onthe improved stability of acylated pig-ments.213,331,332,411,418 It has been proposed that re-duced nitrate favors anthocyanin synthesis byfavoring the uptake into vacuoles by a tonoplast-associated transport mechanism.213

Recently, the use of extracts of cultures orconditioned media to improve the anthocyanin pro-duction by tissue cultures has proposed. Culturefiltrates and cell extracts of B. cereus,S. aureus and E. coli (among others) were used aselicitors of anthocyanin production in carrot cellcultures, obtaining increments up to 77%.454 It hasbeen found that conditioned media (CM) fromstrawberry cell cultures promote anthocyanin ac-cumulation in cell suspensions of strawberry. Theanthocyanin accumulation was sixfold higher inCM than in control media. On the other hand, rosecells were transferred to CM from strawberry cells,and it was observed that anthocyanin accumulationwas induced. Thus, it the presence of some un-known material(s) released (heterologous condi-tioning factor) during culture that contributed toanthocyanin accumulation was proposed.332,412

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C. Betalains

1. Definition

The term “betalains” was introduced by Mabryand Dreiding;288 this was supported by structuraland biogenetic considerations. In a strict sense,betalains do not belong to alkaloids because theyare acidic in nature due to the presence of severalcarboxyl groups. Originally, betalains were called“caryophyllinenroth” and successively renamed“rübenroth” and “chromoalkaloids”.371,395

Chemically, betalain definition embraces allcompounds with structures based on the generalformula shown in Figure 19; therefore, they areimmonium derivatives of betalamic acid.375,451 Thechromophore of betalains can be describedas a protonated 1,2,4,7,7-pentasubstitued1,7-diazaheptamethin system.371

Betacyanin structures (Figure 20A) show somevariations in the acyl groups and sugar moieties,while betaxanthin (Figure 20B) exhibits the samedihydropyridine moiety but show conjugation withseveral amines and amino acids.

Betanidin is the basic structural unit of mostof the betacyanins, followed by its C15 epimer, theisobetanidin.374 A considerable number of differ-ent betacyanins can be obtained with glycosidationof one of the hydroxyl groups located at positions5 and 6 (Figure 20A).

Betaxanthins are constituted of differentproteinogenic and nonproteinogenic amino acids, aswell as biogenic amine-conjugated moieties ofbetalamic acids. More than 200 amino acids foundin plants may potentially give rise to betaxanthinstructures.451 The archetypal compound represent-ing betaxanthins is the indicaxanthin, isolated fromprickly pear (cactus fruits of Opuntia ficus-indica).371

FIGURE 19. Betalain general formula. (A) Betalamic acid moiety is present in all betalain molecules. (B) Thestructure will represent a betacyanin or a betaxanthin, depending on the identity of the R1 and R2 residues. (Adpatedfrom Ref. 46.)

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FIGURE 20. Betanidin is an example of betacyanins (A), while miraxanthin II is of betaxanthins (B). (Adapted fromRefs. 375, 451.)

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2. Classification

They can be divided into two structural groups,the yellow betaxanthins (from Latin beta, red beetand Greek xanthos, yellow) and red-purplebetacyanins (kyanos, blue color), depending onR1-N-R2 moieties. More than 50 betalains are wellknown, and all of them have the same basic struc-ture, in which R1 and R2 may be hydrogen or anaromatic substituent. Their color is attributable tothe resonating double bonds.46 Conjugation of asubstituted aromatic nucleus to the1,7-diazaheptamethinium chromophore shifts theabsorption maximum from 480 nm in yellowbetaxanthins to 540 nm in red-purplebetacyanins.451

In some earlier papers, the terms “betalaines”,“betacyanines”, and “betaxanthines” were used;the terminal letter “e” was added by Fisher andDreiding148 so the names would conform to theI.U.P.A.C. nomenclature; at present, these termscan be used without the terminal “e”.

Betacyanins and betaxanthins can be classi-fied using their chemical structures. Betacyaninstructures show variations in their sugar (e.g.,5-O–D-Glucose) and acyl groups (e.g., feruloyl),whereas betaxanthins show conjugation with awide range of amines (e.g., glutamine) and aminoacids (e.g., tyrosine) in their structures. Table 8shows some well-studied betalains.

3. Distribution

Among higher plants the occurrence ofbetalains is restricted to the Caryophyllales289 andthose found in certain higher fungi such asAmanita, Hygrocybe, and Hygrosporus.451

Betalains of higher plants are in different organs.402

They produce red, yellow, pink, and orange col-ors in Aizoaceae and Portulacaceae flowers,473

and purple pigmentation in Cactaceae fruits andin red-beet root (Chenopodiaceae).370 Betalainsare in bracts, for example, Bougainvillea(Nyctagynaceae) possesses a wide range of col-ors;372 they are also in seeds of Amaranthus,37 inleaves of Teloxis and in stems.12

Common names and classification of differ-ent betacyanins and betaxanthins are standard-

ized, and they are usually assigned in agreementwith their botanical genus.371 In the betacyaningroup, amaranthin-I was obtained fromAmaranthus tricolor, betanin from Beta vulgaris,and gomphrenin-I from Gomphrena globosa.While in the betaxanthin group, miraxanthin oc-curs in flowers of Mirabilis jalapa, vulgaxanthin-I and II have been found in root of Beta vulgaris,and portulaxanthin has been isolated from thepetals of Portulaca grandiflora.375

Up to date more than 50 structures of natu-rally occurring betalains have been identified. Aconsiderable number of different betacyanins maybe derived from two basic compounds, betanidin(2S, 15S) and isobetanidin (2S, 15R) byglycosidation of one of the hydroxyl groups lo-cated at position 5,374 for example, betanin, whichoccurs as the 5-O-glucoside, and the less-occur-ring position 6, for example, gomphrenin-II,which is a 6-O-glucoside. No betacyanin isknown to have both positions substituted withsugar residues.375 A few biosides areknown: amaranthin the betanidin 5-O-[2–O-(β-D-glucopyranosyluronic acid) β-D-gluco-pyranoside], and its epimer isoamaranthin is inthe leaves of A. caudatus.38 Moreover, twoepimeric betacyanins, bougainvillein-r-I andisobougainvillein-r-I, have been isolated from B.glabra.372

The isomeric 5-O-β-cellobiosides have beenproduced by deacylation of the pigments aleracin-I and II from P. oleracea.45 The only known6-(2G-glucosylrutinosides) of betanidin andisobetanidin have been obtained by deacylation ofbougainvillein-V. Betalain glycosides can be es-terified with hydroxycinnamic acids (ferulic and p-coumaric acids), for example, celosianin-I4-coumaroylamaranthin) in Chenopodium rubrumand Lampranthus sociorum;48 in addition, sulfuryland malonyl betacyanins are known, for example,rivianin (betanin-3′-sulfate) from Riviana humilisand phyllocactin (malonic acid 6′-half-ester ofbetanin) from Phyllocactus hybridus.370 Thedecarboxybetanidin, isolated from Carpobrotusacinaciformis, is unique among betacyanins incontaining a modified aglycone moiety.371

There are about 15 naturally occurringbetaxanthins; the indicaxanthin from Opuntia fi-cus-indica was the first crystallized.375 In total,

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TABLE 8Some Fully Identified Naturally Occurring Betalains

Betalaina Residueb Ref.

Aglycones

Betanidin — 375

Betanin group

Betanin 5-O-Glc 46Phyllocactin 5-O-Glc 373Lampranthin-I 5-O-Glc 48

Amaranthin group

Amaranthin 5-O-Glc-2-O-GlcU 451Celosianin II 5-O-Glc-2-O-GlcU 449

Bougainvillein

Bougainvillein 5-O-Glc-2-O-Glc 372

Gomphrenin group

Gomphrenin-I 6-O-Glc 316

Betaxanthins

DOPAxanthin DOPA 371Indicaxanthin Proline 371Portulaxanthin-II Glycine 473Vulgaxanthin-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-tain non-protein amino acids.472

4. Biosynthesis: Biochemistry andMolecular Biology

a. Biochemistry

Initial stages (Figure 21A). The determina-tion of the chemical structure of betalains and oftheir biosynthetic intermediates contributed to theestablishment the corresponding biosynthetic path-way; in addition, feeding experiments with isoto-

pically labeled precursors and in vitro cell cul-tures were important tools in such discovering.46,375

However, very few enzymes involved in betalainsynthesis have been purified and character-ized.211,273,434,451 Figure 21 summarizes the pro-pounded biosynthetic pathway. In detail, betalainsare considered secondary metabolites; they de-rive from shikimic acid and from the tyrosineaminoacid.371 In their basic structure, the phenylgroup is bonded to a lateral n-propyl chain givingplace to a C6-C3 unit.35 Biogenesis of betalainsfrom tyrosine has not been thoroughly under-stood, and only a few enzymes involved in thebiosynthetic route have been identified.47,273

Two molecules of tyrosine are required in thebiosynthesis of one molecule of betacyanin or

n-

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FIGURE 21. Pathway proposed for betalain biosynthesis. (A) Intial stage, (B) betacyanin biosynthesis. Betacyaninsmay 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 betalainbiosynthesis are (1) DOPA 4,5-dioxygenase, (2) glycosyltransferase, and (3) acyltransferase. (Adapted from Refs.46, 451.)

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betaxanthin. Initially, two molecules of 3-hydroxy-L-phenylalanine (L-DOPA) are formed.335,375 Thehydroxylation of tyrosine to L-DOPA is recog-nized as the first step in the biogenesis of betalainsbecause experiments with radioactive precursorshave shown good incorporation of labeled [C14]-tyrosine into amaranthin and betanin, the majorbetacyanins in Amaranthus tricolor seedlings andred-beet (Beta vulgaris), respectively.135 The as-sumption has been that the first enzyme is a phe-nol-oxidase complex catalyzing both the conver-sion of tyrosine to L-DOPA by a monophenoloxidase and the dehydrogenation of the latter toO-quinone by a diphenol oxidase.

Recent work on the enzymology of betalainbiosynthesis has focused on the toadstool “flyagaric” (Amanita muscaria) mushroom of theAgaricales. In this basidomycete, the accumula-tion of betalain pigments is restricted to the cu-ticle of the cap and its subject to developmentalregulation.451 There is a rapid increase in betalainlevels during the process of development fromyoung fungi, which are still entirely covered bythe veil, to mature specimens. Recently, Müelleret al.335 characterized a tyrosinase from pileus ofAmanita muscaria. This enzyme was located onlyin the colored parts of the fungi, and it was dem-onstrated that tyrosinase catalyzes the reaction oftyrosine hydroxylation to L-DOPA, confirmingits involvement in betalain biosynthesis. Tyrosi-nase also shows diphenolase activity, and it seemsto be an heterodimer of two subunits with mo-lecular weights of 27 and 30 kDa, somethingunusual for tyrosinases.

On the other hand, one L-DOPA molecule istransformed into DOPA-quinone, which is spon-taneously converted to cyclo-DOPA, whilebetalamic acid is formed from the second mol-ecule of L-DOPA by means of a reaction cata-lyzed by DOPA-dioxygenase.336 In the proposedmetabolic pathway betalamic acid arises from L-DOPA. L-DOPA is cleaved at the C4-C5 bond ofthe aromatic ring to form an intermediate(4,5-seco-DOPA) that is cyclized, forming an het-erocyclic system. It has been suggested that4,5-seco-DOPA is produced by an “extradiol-cleavage” of L-DOPA.463

The oxidative disruption of the L-DOPA ringappears to be analogous to the aromatic rings

fission of catechols, and the last reactions arecatalyzed by specific dioxygenases. These en-zymes contain Fe ion (both ferric and ferrousstates) as an essential cofactor. Girod and Zrÿd171

isolated the DOPA 4,5-dioxygenase fromA. muscaria. This enzyme as other extradiol-cleaving dioxygenases is an oligomer that cata-lyzes the 4,5-extradiol disruption of L-DOPAleading, via 4,5-secoDOPA to betalamic acid.463

It was shown by affinity chromatography thatDOPA 4,5-dioxygenase is composed of a vary-ing number of identical 22-kDa subunits.Another DOPA dioxygenase (DOPA 3,4-dioxygenase) was also extracted from A.muscaria; this enzyme catalyzes the 2,3-ring-opening reaction that yields the muscaflavin pig-ment, a compound that has never been found inplants. The betalamic acid may condense withthe imino-group of cyclo-DOPA to produce thered-purple betacyanins (Figure 21B) or with theimino or amino group of amino acids to give theyellow betaxanthins (Figure 21C).451

Betacyanin biosynthesis (Figure 21B).Betacyanins are formed through the reaction ofcyclo-DOPA with betalamic acid followed byglycosylation, or by condensation of the cyclo-DOPA glycosides with betalamic acid.209 In gen-eral, it could be mentioned that condensation ofbetalamic acid with cyclo-DOPA to betanin for-mation and the subsequent glucosylation reactionto betanidin 5-O-β-glucoside, main compoundof red-beet, remain unknown. Completeglycosylation takes place with cyclo-DOPA fol-lowed by condensation with betalamic acid. Onthe other hand, free betanidin can be stored as themain component in betalain-producing cells, andit has been shown that betanidin can be the mainreceptor of UDP-glucose from glucosyl-transferaseduring betanin biosynthesis.208,209 It may be pos-sible that the sequence of reactions depends onthe plant genus.

Betanin glucosylation catalyzed by uridine5 ′-diphospho-glucose: betanidin 5-O-β-glucosyltransferase (5-GT) was demonstrated byHeuer and Strack,208 who described the occur-rence of one of the two proposed pathways ofbetacyanin formation, the transfer of glucose to5-hydroxy group of betanidin in the formation ofbetanin.

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On the other hand, Heuer et. al.209 describeda new glucosyltransferase, the 6-O-glucosytransferase (6-GT) that catalyzes theregiospecific transfer of glucose to the 6-hydroxygroup of betanidin in the formation of gomphreninI, analogs to 5-GT. Both GT were extracted fromcell cultures of Dorontheanthus bellidiformis. The5-GT showed three isoforms and the 6-GT onlyone; both enzymes are monomers with a molecu-lar weight near 55 kDa. Nowadays, it has beenshown that both enzymes catalyze the indiscrimi-nate glucose transfer from UDP-glucose to hy-droxyl groups of betanidin, flavonols, anthocya-nins, and flavones. Interestingly, it was observedthat GT catalyzes the formation of 7-O-gluco-sides, but in a minor extent.493 Based on theseresults, it could possibly be that 5-GT and 6-GThave a phylogenetic relation with flavonoidglucosyltransferases. Subsequently, acylation ofglycosylated betanidins is through acyl groupdonation from 1-O-acylglucosides. It is importantto point out that this reaction catalyzed by anacyltransferase seems to be an exclusive biosyn-thetic mechanism of betalain-producing plants,contrasted with the analogous reaction in the bio-synthesis of flavonoids where acylated flavonoidsare produced by the hydroxycinnamoyl-coenzyme-A pathway.47

Another enzyme involved in betacyanin for-mation is an acyltransferase (HCA), which cata-lyzes the transfer of hydroxycinnamic acids from1-O-hydroxycinnamoyl-β-glucose to the C2 hy-droxy group of glucuronic acid of betanidin 5-O-glucuronosylglucose (amaranthin) in Chenopo-dium rubrum;48,49 the products formed arecelosianin I (4-coumaroylamaranthin) andcelosianin II (feruloylamaranthin), as shown inFigure 22. This enzyme exhibits a molecularweight near 70 kDa.

HCA could also catalyze the formation of4-coumaroyl and feruloyl-derivatives in Beta vul-garis (lampranthin II), Gomphrena globosa(gomphrenin III), Lampranthus sociorum(celosianin I and II), and Iresine lindenii(lampranthin II).47

Betaxanthin biosynthesis (Figure 21C).Little is known about the betaxanthin biosynthe-sis.472 However, it has been suggested the inter-change of basic compounds as one of the main

routes.451 A spontaneous condensation betweenbetalamic acid and an amine group inside thevacuole has been based on genetic and biochemi-cal studies on clones of P. grandiflora. In a recentwork, Hempel and Böhm207 found two newbetaxanthins in hairy root cultures of Beta vul-garis var. Lutea, vulgaxanthin III, and IV, whenthe culture media was supplemented with thecorresponding L-amino acids. This feeding ex-periment provides arguments for a spontaneouscondensation of betalamic acid with amino acidsor amines in the course of betaxanthin biosynthe-sis.

In other experiments, the administration ofL-DOPA to violet petals of flowers in Portulacagrandiflora led to the biosynthesis of betaxanthinsnot present in natural plants. These results showthat L-DOPA administration elicits the formationof betaxanthins, which are absent in untreatedflowers.397 In addition, nine L- and D-isomers ofamino acids were supplied to seedlings and indi-vidual hairy roots strains of Beta vulgaris, var.Lutea; the increment of betaxanthin concentra-tion and also the appearance of new betaxanthinswas observed.207 Therefore, the theory of a spon-taneous condensation of betalamic acid with aminoacids or amines in the course of betaxanthin bio-synthesis is supported.

On the other hand, the activity of a betacyaninand betaxanthin-decoloring enzymes has been pos-tulated in Beta vulgaris,271,437 Amaranthus tri-color,135 and Phytolacca americana;265 the resultssuggest there is an enzyme complex with twoacidic and two basic enzymes that contain a metalion in the active site. Earlier works described thiscomplex like a “peroxidase” enzyme.231 Parra andMuñoz361 confirmed that horseradish peroxidasecatalyzes the oxidation of betanin. HPLC analysisshowed a red intermediate and several yellowproducts (presumably of a polymeric nature) be-ing betalamic acid, one of the final products.Zakharova et al.521 described the occurrence of abetalain oxidase in three species of Amaranthus.This enzyme was found mainly in cell walls ofA. caudatus, and it was suggested that pigmentyield can be increased by regulating the activityof this enzyme during plant tissue extraction.

Watson and Goldman500 reported dominantalleles at two tightly linked loci (R and Y) of red

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FIG

UR

E 2

2 A

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the

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yme

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rase

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beet. These alleles are involved in the productionof betalain pigment. In addition, it has been shownthat several alleles in these loci influence theproduction and distribution of betalains. The au-thors also suggested the existence of genes thatplay an important role in betalain synthesis.

b. Biosynthesis Regulation

In plants, betalain biosynthesis is subject tocomplex regulation. Pigments are accumulatedonly in certain tissues and at specific stages ofdevelopment. Their synthesis has been shown tobe regulated by light and cytokinins.135 It has beenshown that effects of DOPA, light, and kinetinexposure are different between models producingbetacyanins or betaxanthins. The betanin synthe-sis was only induced under the influence of DOPAor with the combination of kinetin and light. Onthe other hand, betaxanthins were synthesized onlywhen the seedlings were fed with DOPA, and asignificant increment of this production was ob-tained by light and kinetin supplementation.37

The presence of free betalamic acid in plants,which produce betaxanthins or betacyanins andbetaxanthins, and its absence in plants, whichproduce only betacyanins, suggest a regulatorymechanism during its biosynthesis.451 A coordi-nated condensation mechanism of betalamic acidwith cyclo-DOPA or glycolized-cyclo-DOPA hasbeen suggested in plants that produce betacyanins;then the accumulation of betalamic acid is ar-rested. On the other hand, control mechanisms inbetaxanthin-producing plants must be different toallow the accumulation of betalamic acid (Muelleret al., 1997b).46 Regulatory mechanisms on betalainformation are largely unknown, but the involve-ment of photoregulation and hormone control hasbeen suggested.395

Cell lines produced from red beet showed arange of cell colors via specific induction meth-ods. Phenotypic color ranged from white/greenthrough yellow, orange and red to deep violet,representing all types of pigments found in red-beet plant. Differences in callus phenotypes wereassociated with cells of markedly different mor-phologies; cells were classified into two groups:white, orange, and violet phenotypes, and green

yellow and red phenotypes. The selective expres-sion of betaxanthin and betacyanin appeared tooccur through a limited number of discrete, stable,and differentiated states, because only four col-ored phenotypes were isolated.171

In absence of specific information, it is onlypossible to speculate about the regulatory mecha-nism affecting the gene expression during pheno-typic transitions of plant cell cultures. It has beenproposed that DNA transposition or specific rear-rangements such as translocations, inversions,breakage, and fusion are involved. It has beenalso suggested that such rearrangements couldresult in the relocation of particular genes, eitherwithin the same chromosome or to another chro-mosome. Moreover, it is possible that regulationof pigment synthesis is so tightly coupled to cel-lular morphology that an enhancement of geneexpression never occurs in the yellow and redcells.171

Some inhibitors of cell division were used inPhytolacca americana suspension cultures, and areduced betacyanin accumulation was observed,even though their modes of action are known tobe different. It was suggested that inhibitory ef-fects on betacyanin accumulation are due to theinhibition of cell division as a result of lack ofDNA synthesis and that betacyanin biosynthesisand accumulation are correlated not only withDNA synthesis, but also with the progression ofthe cell cycle.212

c. Molecular Biology of BetalainBiosynthesis

Up to now, very few of the enzymes involvedin betalain synthesis have been purified and char-acterized, despite the importance of betalains asnatural food colorants.211 The only enzyme activi-ties that have been described from higher plantsare enzyme preparations that catalyze theglycosylation of betanidin and enzymes involvedin betalain degradation, but work regarding genesis scarce.

Two clones encoding polyphenol oxidase wereisolated from a cDNA library constructed from alog-phase suspension culture of Phytolaccaamericana, producing betalains. Spatial and tem-

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poral expressions were investigated by Northernblot analysis of total RNA from various organs ofPhytolacca plants. Transcripts of the two cloneswere found to be 2.1 and 2.3 kb. Both transcriptswere present only at substantial levels in the rip-ening of betalain-containing fruits.244

Hinz et al.211 described the cloning and regula-tion of the gene dodA from basidiomyceteA. muscaria. This constitutes the first effort toclone genes of the betalain biosynthetic pathway.dodA codes for a DOPA dioxygenase. The cDNAlibrary was constructed from the cap tissue of youngspecimens where betalain synthesis had not beenyet begun. Southern blot analysis showed thatDOPA dioxygenase is encoded by a single-copygene in A. muscaria.211 Müeller et al.334,336 trans-formed white petals of Portulaca grandiflora withthe dodA gene of A. muscaria, and the formation ofyellow and violet spots that contained betalain andmuscaflavin pigments was observed, indicating thatthe fungal enzyme DOPA dioxygenase was ex-pressed in an active form in plants.

Notwithstanding these promissory results, itis obvious that much more research work is neededin order to unravel the processes involved in bio-synthesis and regulation of betalains.

5. Functions

a. Taxonomic Markers

Even before the structure of betalains wasevident, the importance of betalain pigmentsin plant taxonomy and systematic distributionwas clear. Betalains are in eightfamilies: Amaranthaceae, Aizoaceae, Basellaceae,Chenopodiaceae, Cactaceae, Nyctaginaceae,Phytolaccaceae, and Portulacaceae. Nowadays,it is known that 9 of 11 families of theCaryophyllales order contain betalains.402 Therecent addition to the list of betalain families isDidieraceae, a small family from Madagascar.451

Only two families of Caryophyllales, viz.,Caryophyllaceae and Molluginaceae, lackbetalains and possess anthocyanins. This suggestsan early differentiation of the Caryophyllales intogroups with different kinds of pigments. Rosendal-Jensen et al.402 constructed a phenogram that shows

the relationship between betalains, glucosinolates,and polyacetylenes in flowering plants.

This remarkable correlation between chemi-cal and morphological characters has led to pro-pose that the order Centrospermae, includingCactaceae, must be reserved for betalain-contain-ing families, while the anthocyanin-containingones (Caryophyllaceae and Molluginaceae) mustbe separated into the related but distinct orderCaryophyllales.375

The totally different chemical structure ofbetalains and anthocyanins,158 the fact that theyare mutually exclusive, and the restricted distri-bution of betalains are good arguments in favor ofthe paramount taxonomic significance of thesepigments.

Such arguments are not lessened by the find-ing of their occurrence in the Basidiomycetes ofhigher fungi, which do not have any phylogeneticrelation to flowering plants. This coincidentaloccurrence could be a case of chemical conver-gence under evolutionary phase.371 Consideringthe crucial dependence of a living system on theeconomy of energy and our growing knowledgeabout the chemoecology of secondary products innature, this assumption can be feasible.451

b. Ecological and Physiological Aspects

As in the case of other secondary metabolites,it is impossible to assign a definite function tobetalains in the economy of the organisms thatproduce them.375 When pigments are in flowers orfruits they may have a role as attractants for vec-tors (insects or birds) in the pollination processand in seed dispersal by animals, such as antho-cyanins.371,503

The occurrence in other plant parts (e.g.,leaves, stem, root) may be devoid of immediatefunction. However, it has been suggested thatbetalain accumulation in red beet root is related tothe storage of carbohydrates as a physiologicalresponse under stress conditions.260 In addition,the transient coloration of many seedlings and thereddening of senescent leaves of several plants ofCaryophyllales order (e.g., Kochia scoparia) haveno obvious physiological or ecological reasons.Whatever its significance, the process resembles

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the analogous phenomenon observed in antho-cyanin-producing species. Betalains are also pro-duced in injured tissues, normally not pigmented,possibly as a defense mechanism against infec-tion. This physiological response was only ob-served in plants possessing specific factors thathave been associated with two novel antifugalproteins.263

Interestingly, it must be mentioned thatbetalains from Beta vulgaris (betanin andvulgaxanthin) are effective inhibitors of indoleace-tic acid (IAA) oxidase and that betanin counter-acts the inhibitory effect of IAA on wheat rootelongation.375 It means that betalains could beconsidered as potential modifiers of auxin me-tabolism; notwithstanding, there is no evidencethat supports the betalains role as in vivo regula-tors of IAA activity.

c. Pharmacological Effects

Although structurally related to alkaloids,betalains have no toxic effects in the human body,as can be deduced from the fact than they arepresent in considerably high amounts in certainfoodstuffs, such as red-beet, prickly pear fruits,and Amaranthus seeds.46 Therefore, betalains rep-resent a safe natural alternative to some syntheticcolor additives that are currently in use. Interest-ingly, there is no upper limit to the recommendeddaily intake.158

On the other hand, in betanin tested for mu-tagenic and carcinogenic activity an absence ofmutagenicity in five Salmonella typhimuriumstrains was observed, and it did not initiate orpromote hepatocarcinogenesis in levels of 50mg/kg of weight of pure betanin or diets contain-ing 2000 mg/kg of betacyanin.429,431 Notwithstand-ing, after ingestion of these products (particularlyred beet), betanin occasionally appears in the urine,an effect known as beeturia or betaninuria. Theetiology and mechanism of this disorder are stillcontroversial.371 There are a very few pharmaco-logical applications of betalains. Recently, theyhave received attention because betanin has shownantiviral and antimicrobial activities (e.g., Pythiumdebaryum, a pathogenic fungi in red-beet). Insome places in Mexico, an infusion of Bougain-

villea bracts mixed with honey is used widely fora cough. However, in both cases the action mecha-nisms are still unknown.

Finally, in a recent work, the importance ofsome natural pigments as nutraceutical ingredi-ents was reviewed.382 It was suggested thatbetalains like anthocyanins, β-carotene, and vari-ous vegetable and fruit extracts must be used fortheir potential health benefits. For example, yel-low betaxanthins, in addition to their potentialrole as natural food colorant, may be used as ameans of introducing essential dietary amino ac-ids into foodstuffs, giving rise to an “essentialdietary colorant”.273 Another new interesting areaincludes foods that can generate their own light(“biolume products”) in which betalains couldplay an important role, suggesting how excitingthe area of natural colorants has become. As othertopics, it is necessary to emphasize the need ofmore research in these new areas.

6. Methodological Aspects

a. Extraction

Betalains-containing material (raw plant orcell culture) are generally macerated or ground.Pigments can be extracted with pure water, cold,or at room temperature, although in most casesthe use of methanol or ethanol solutions (20 to50% v/v) is necessary to achieve complete extrac-tion.375 Sometimes, the necessity of anaerobic juice fermentation (e.g., Saccharomycescerevisiae, Aspergillus niger) in order to reducefree sugars and then to increase the betacyanincontent has been reported.379 In both procedures,the inactivation of degradative enzymes by a shortheat treatment of the extract (70ºC, 2 min) couldbe desirable, although this may destroy some ofthe pigments. Betacyanins can be precipitated bya slight acidification with hydrochloric acid orwith acidified ethanol (0.4 to 1% HCl); subse-quently, by the addition of 95% aqueous ethanolyields betaxanthins.39,373

Degradation of betanin may occur very fastand destruction of complex pigments should alsobe avoided, because acid-acylated betacyanins arerapidly deacylated and such pigments can be over-

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looked.451 In such a situation, extraction should becarried out with cold water for long-term anddarkness conditions.

b. Separation

Ion-exchange and column chromatography.Ionic-exchangers are the most widely usedadsorbents in fractionation, as much as in separa-tion; then gel filtration is used.374 In a simple andrapid procedure, plant extract must be stirred withthe ion-exchanger resin (e.g., Dowex 50W-X2,Merck I, DEAE-Sephadex A25, etc.), whichadsorbs the betalains (nonionic interaction). Sub-sequently, resin is washed with aqueous HCl (0.1%v/v) and pigments are eluted with water followedby final separation on a chromatographic column(e.g. Polyamide, Polyclarc-AT, or polyvinylpyr-rolidone, Sephadex G-15 and G-25).

The chromatographic and electrophoreticproperties from unknown plant materials can becompared with those reported in the literature forknown pigments (e.g., Piatelli and Minale,374 forbetacyanins; Minale et al.316 for acylated betalains;Piatelli and Imperato;370 von Elbe et al.;496

Piatelli;371 and Steglich and Strack449 for betalainsin general).

Electrophoresis and thin layer chromatog-raphy (TLC). Paper electrophoresis using pyri-dine and formic or acetic acid as solvents or incellulose are common and reliable methods forbetacyanin detection,380 because they migrate firstas immobile zwitterions (pH 2), followed asmonoanions (pH 2 to 3.5), and finally as bis-anions (pH 3.5, 7.0). In the case of betaxanthins,the mobility may be related to indicaxanthin, andbetacyanins are related with the mobility ofbetanin.374 Electrophoresis can be carried out us-ing pyridine-citric acid solvent, voltage gradientof 5.6 volts/cm, and a temperature of 4ºC.496 Re-cently, capillary zone electrophoresis (CZE) hasbeen used for the analysis of betalains, particu-larly from Beta vulgaris.452 This technique wascarried out with a fused-silica capillary at 15ºCand at a constant voltage of -22 kV, and it haspermitted the separation of betanin, isobetanin,and their corresponding aglycones. CZE has beenused successfully for the separation and charac-

terization of betalains; however, time analysis islonger, but it could be shorten if separation isfocused on the two major red pigments, leavingthe aglycones unseparated. Betaxanthins can alsobe quantitated by CZE with an acceptable resolu-tion.

By contrast, thin layer chromatography (TLC)is not widely used for betalains because its low Rfvalues; however, Bilyk41 developed a preparativeTLC system in a 0.5-mm cellulose-coated plateusing two different mobile phases: isopropanol-ethanol-water-acetic acid in a ratio of 6:7:6:1(v/v) in the first solvent mixture and a 11:4:4:1 (v/v) ratio in the second one. When acid is incorpo-rated in the developing solvent, betalain mobilityon the TLC plate is facilitated due to protonationof the betacyanin carboxyl group. The acid anionprovides an electrically neutral system by its in-teraction with the quaternary nitrogen. Thesame effect occurs with betaxanthins.41 Goodbetaxanthin separations was also obtained ondiethylaminoethyl cellulose plates using isopro-panol-water-acetic acid (13:4:1 v/v).451 No indi-cator is needed for visualization of the separatedpigments because they are clearly observed on theTLC plate with their natural colors.

High-performance liquid chromatography(HPLC). The HPLC technique has become themethod of choice for chromatographic separa-tion, rapid quantification, and tentative identifica-tion of betalains. The first application was doneby Vicent and Scholz488 using a C18 column witha gradient run using tetrabutylammonium in pairedion system as the mobile phase. The most usefulcolumn supports are C8 and C18 reversed phase(e.g., Nucleosil, LiChrosorb, µBondpack, etc.),with particle sizes between 3 to 10 µm, while themost used solvents are water-methanol or water-acetonitrile mixtures, acidified with acetic, for-mic, or phosphoric acid.451 HPLC elution order ofpure crystalline pigments was as follows: betanin,betanidin, isobetanin and isobetanidin.432 Thisevidence was based on an acid hydrolysis of theglycosides to yield aglycones and isomerizationof betanin to isobetanin occurring.488

More recently, Pourrat et al.379 analyzed afermented red-beet root extract using a reversed-phase C18 column and ion-pairing and methanol-water as mobile phase, and the elution order was

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betanin, isobetanin, betanidin, isobetanidin, andprebetanin for the betacyanins and vulgaxanthin Ifollowed by vulgaxanthin II for the betaxanthins.Another good example of betaxanthin character-ization was carried out by Trezzini and Zrÿd.472

Betalamic acid was conjugated with both proteinand non-protein amino acids to yield a series ofbetaxanthins. They described retention character-istics for 15 naturally occurring pigments such asportulaxanthin-I, miraxanthin-II, vulgaxanthin-I,among others. Such products could be used asHPLC standards for unknown pigments.

c. Characterization

General procedures. In most cases it is im-possible to distinguish between anthocyaninsand betalains visually. However, the extract sourceis an indication for the presence of betalains oranthocyanins because in plants the presence ofone is mutually exclusive to the other. It is impor-tant to remember that betalains are characteristicpigments in plants members of Caryophyllales.Preliminary tests have been developed to easilydistinguish between betacyanins and anthocya-nins (Table 9) using the color exhibited at differ-ent pHs and their temperature.451

Spectroscopy. Betalain analysis as that ofother colored compounds has been based basi-cally on UV-visible spectroscopy. As a matter offact, red violet betacyanins absorbs around λmax= 540 nm, while yellow betaxanthins at λmax =480 nm, and the starting studies of betalain iden-tification were supported in this methodology. Inaddition, structural modifications of betalains havebeen followed by UV-visible spectroscopy.287,288,375

However, in the 1980s spectroscopy showed enor-mous progress and nowadays chemical character-ization must be carried out considering at leastHPLC separation and UV-visible, MS, and NMRspectroscopies: rigorous characterization ofbetanin, lampranthins, cellosianins, neobetanin,among others, was established thanks to thesemethodologies.449,451,452

Chemical tests. Chemical methods for thesynthesis and degradation of pigments are veryimportant in betalain research. Some of these es-tablished methods are well described in more detailby Strack et al.,451 and they are briefly summa-rized in this section. A number of color reactionsbased on changes in pH have been proposed todistinguish between betalains and anthocyanins.

Acid hydrolysis (dilute aqueous HCl) ofbetanin gave a mixture of both aglycones,betanidin, and its 15R epimer isobetanidin; this

TABLE 9Differentiation 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.

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methylneobetaninidin dimethyl ester in good yield(Figure 23), exhibiting an absorption at 403 nm,which can be shifted, with the addition of acid, to513 nm.375 Also, neo-derivatives can be obtainedfrom betaxanthins on treatment withdiazomethane, whereas esterification withCH3OH-HCl or CH3OH-BF3 affords the normalesters.

Quantification and pigmenting efficiency.Nilsson342 developed a method to measure bothred and yellow betalains of Beta vulgaris withoutprior separation of betacyanins and betaxanthins.Their quantitative determination mainly involvedspectrophotometry, where the absorbance at themaximum wavelength (λ) is translated into con-centration by means of the appropriate absorptivi-ties. Another method is based on electrophoreticseparation of individual pigments followed by themeasurement of the color intensity of the sepa-rated bands in a densitometer. The result wasexpressed as peak area in cm2, which was deter-mined with the aid of an integrator after correct-ing the baseline; thus, the results are translated toconcentration comparing them with a betanin stan-dard curve.496

A computer-aided determination, based onpreviously reported absorptivity values, has beenperformed by Saguy et al.408 This method uses anonlinear curve fitting of the spectrum with apredicted function of the individual pigments (e.g.,betanin, betalamic acid, vulgaxanthin-I). The pro-posed procedure is rapid and accurate, avoidingthe laborious and time-consuming separation steps.Schwartz and von Elbe432 developed a method toquantify individual betalains by HPLC using themolar absorptivity of each pigment instead ofabsorptivity values. This method provides a moreaccurate determination of the total betalain con-tent.

It must be pointed out that discrepanciesbetween spectrophotometric and HPLC meth-ods have been observed. Differences up to 15%have been reported after an extended heat treat-ment of betalains. Such differences have beenattributed to degradation products or interfer-ing substances formed during processing.430 Pre-vious fermentation of red beet juice is recom-mended for betalain quantification because freesugars are degraded and betalain content of the

mixture is easily separated by chromatographicmethods. On the other hand, enzyme-catalyzedhydrolysis produces only betanidin.374 Moreover,heating by prolonged time produces the cleavageof betanin into betalamic acid and cycloDOPA5-O-glycoside.371 After alkali fusion, betanidin wassplit into 4-methylpyridine-2,6-dicarboxylic acid,5,6-dihydroxy-2,3-dihydroxyindol, and formicacid; together, these fragments revealed the car-bon structure of betanidin. Epimerization at C15 isalso observed by betanidin treatment with dilutedalkali or citric acid solution (5% aqueous); in bothreactions a betanin-isobetanin ratio of 3:2 wasobserved, while the isobetanin produced a 2:3ratio under the same treatment conditions. In ad-dition, alkaline treatment, for example, alkalinehydrolysis during deacylation of gomphrenin-IIto gomphrenin-I, a 1:1 betacyanin-isobetacyaninratio can be obtained.

On the other hand, betaxanthin analyses in-volve methodologies for amino acid analysis. Theyare hydrolyzed with 1 N aqueous HCl or 0.6 Nammonia to obtain betalamic acid and free aminoacids.375 The reaction of betanin in ammonia alka-line solution with an excess of amino acids areused in betaxanthin synthesis. This reaction isfollowed by monitoring the increments of thebetaxanthin maximum (absorption at 475 nm) ordecrements of betanin maximum at 540 nm. Thus,vulgaxanthin II can be obtained mixing betanin in0.6 ammonia solution with an excess (10 M) ofglutamic acid; this base exchange method hasalso been applied for the synthesis ofindicaxanthin, miraxanthin, and otherbetaxanthins. However, nonnaturally occurringbetaxanthins can be obtained with serine, pheny-lalanine, threonine, and lysine.472

Indicaxanthin oxidation with peroxyacetic acidyields L-aspartic acid, a reaction used to demon-strate the 11S configuration of this betaxanthin.Betanin has the 15S configuration at thedihydropyridine ring, as indicaxanthin was ob-tained via amino acid exchange from betanin.This is in accord with the isolation of S-betalamicacid after the degradation of many betaxanthinswith alkali treatment.451 The formation of neo-derivatives demonstrated the tendency of thedihydropyridine ring to amortize. Thus, treatmentof betanidin with diazomethane gives di-O-

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extract is enriched; thus, HPLC is more easilyperformed.379

Interestingly, it has been shown that betalainquantitation by capillary zone electrophoresis isin close agreement with the HPLC determina-tion.452

On the other hand, pigment efficiency is usu-ally measured in terms of CIELAB parameters. Itmeans that triestimulus colorimetry is the bestmethodology to carry out such measurements.158,230

Values provided by universal colorimeters aredescribed in previous sections (“L”, “a”, and “b”).

Sapers and Hornstein416 reported the Huntercolor values for standardized dilutions of juicefrom 48 red beet cultivars. “a” values varied overa relatively narrow range being slightly lower insamples with higher “b” values. The analyses ofthe 20 most highly pigmented cultivars producedvalues in the range 8.5 to 17.2. Interestingly, itwas reported that “b” values could be used in theestimation of the betaxanthin-betacyanin.

7. Importance as Food Colors —Stability, Processing, and Production

a. Stability in Model Systems

When betalains are used as food colorants,color stability is a major concern. There are sev-eral factors that have been recognized to affectthe stability of these pigments:pH. The hue of betalains is unaffected at the pHbetween 3.5 to 7; the values of most foods are inthis range. Betalain solutions in this pH rangeshowed a similar visible for betacyanins andbetaxanthins. Betacyanin maximum is in the wave-length (λ) range 537 to 538 nm, while betaxanthinmaximum is between 475 to 477.497 Below pH3.5, λ shifts toward a lower wavelength, andabove 7 the change is toward a longer wave-length; out of the pH range 3.5 to 7 the intensityof the visible spectra decreases.225

Stability of betanin solutions is pH depen-dent. Huang and von Elbe225,226 have shown thatoptimal pH for maximum betanin stability in thepresence of oxygen is between 5.5 to 5.8. Redbeet solutions showed their maximum stability atpH 5.5, the normal pH for beets. In addition,

vulgaxanthin I was most stable between pH 5.0 to6.0, and it was more stable in juice than in puri-fied extracts, while optimal pigment stability inreconstituted powders was noted at pH 5.7.440

Temperature. Temperature also shows a cleareffect on betalain stability.129 Thermal kinetic deg-radation of betanin has been evaluated by severalauthors.4,129,225,226,408,495 It has been reported thatthermostability of betanin solutions is pH depen-dent and partially reversible. Heating of betaninsolutions produces a gradual reduction of red color,and eventually the appearance of a light browncolor. von Elbe et al.495 observed a first-orderreaction kinetics for betanin degradation by heat-ing.

Saguy et al.408 developed a thermal kineticdegradation model using a nonlinear least-squaretechnique; this model enables one to predict theretention of betalains under variable conditions oftemperature and time. Thermal degradation ofbetalains produced activation energies EA) in therange 17 to 21 Kcal·mol-1 for the forward reac-tion, whereas the reverse reaction showed valuesbetween 0.6 to 3.5.225,408 It was also reported thatEA values showed a pH dependence. Betalamicacid and cycloDOPA-5-O-glycoside have beenreported as the probable intermediates for betanindegradation.226,408 Interestingly, the regeneration(reverse reaction) involves a Schiff’s base con-densation of the amine group of cycloDOPA-5-O-glycoside with the aldehyde group of betalamicacid; betanin is rapidly formed when both com-pounds are mixed in solution.226 Altamirano etal.4 reported the lowest stability of betanin and thelowest EA in a water-ethanol model system, sup-porting the idea that the first step of the thermalbetanin degradation is the nucleophilic attack onthe >N+ = CH- structure of betanin. Ethanol hasa high electron density on the oxygen atom; there-fore, it is a strong nucleophilic agent that dimin-ishes the betanin stability.Light. Von Elbe et al.495 found that rate of betanindegradation increased 15.6% after pigment day-light exposure at 15ºC. Degradation of light-ex-posed betalains followed a first-order kinetic. Inaddition, it was observed that degradation washigher at pH 3.0 (k = 0.35 days-1) than at pH 5.0(k = 0.11 days-1), when betacyanins were exposedto fluorescent light. On the other hand, at dark-

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ness conditions betacyanins were most stable (k= 0.07 days-1).416 Attoe and von Elbe16 showedan inverse relationship between betalain stabilityand light intensity in the range 2200 to 4400 lux).It is explained that visible light absorption ex-cites π electrons of the pigment chromophore toa more energetic state (π*). This would cause ahigher reactivity or a lowered activation energyfor the molecule (EA = 25 Kcal·mol-1 in darknessand 19.2 in illumination). The effect of UV andgamma irradiation on betanin stability was re-ported by Aurstad and Dahle;17 total pigmentdestruction was reported by the treatments with120 h of UV radiation or with 100 krad of gammaradiation. Nevertheless, these results, thephotodegradation mechanisms for betalains re-main to be determined.Water activity. Recognizing the importance ofwater in many degradation reactions, it is notsurprising that water activity (aw) is includedamong the primary factors affecting the betalainstability and/or color of a food product contain-ing these pigments.494 Because the degradationreaction does involve water, the greatest stabilityof betalains has been reported in foods or modelsystems of low moisture and aw.82 Pigment deg-radation follows first-order kinetics, and stabil-ity increases with decreasing aw.407 It has beenestablished that aw has a pronounced exponentialeffect on pigment stability. Pigment stability de-creases in one order of magnitude when aw wasincreased from 0.32 to 0.75.82

On the other hand, Simon et al.439 studied theinfluence of aw on the stability of betanin in vari-ous water-alcohol model systems. In all cases, itwas observed a rate-constant dependence withaw.439,494 The increase in stability of betanin withdecreasing aw may be attributable to reducedmobility of reactants or limited oxygen solubility.

Consequently, high moisture content pro-duces a high degradation rate. Furthermore, speci-fication of aw alone without the moisture contentis not enough to predict pigment stability.Oxygen. Oxygen causes a product darkeningand loss of color. Von Elbe et al.495 stored buff-ered betanin solutions at pH 7 under atmosphereof air and nitrogen for 6 days at 15ºC; it wasobserved that color degradation increases up to15% due to air conditions. Betanin reacts with

molecular oxygen, producing pigment degrada-tion in air-saturated solutions.15 Degradation ki-netic under air atmosphere follows a first-ordermodel, but deviates from first-order in the ab-sence of oxygen. As mentioned above betanindegradation is a partially reversible reaction,15

and it has been reported that in order to increasethe recovering of pigment it is necessary to havethe samples under low levels of oxygen. Thus,heated betanin solutions (pH 4.75, 130 min, 15ºC)under low oxygen levels showed an increasedbetanin retention from 54 to 92%.225 Reactionreversibility was responsible for the deviation fromthe first-order degradation kinetics of betanin inthe absence of oxygen.

Several methods have been reported to preventthe destruction or to improve the stability of pig-ments, including degassing, addition of antioxidantsand stabilizers, control of pH, minimal heath treat-ment, among others,5,15,40,190,363 and these efforts havebeen directed to their application in food products.

b. Processing and Stability in Foods

The sensitivity of betalains to different fac-tors suggests that their application as food colorantsis limited. Based on these properties, betalainscan be used in foods with a short shelf-life, pro-duced by a minimum heat treatment, and pack-aged and marketed in a dry state under reducedlevels of light, oxygen, and humidity.390,497

Betalains have several applications in foods,such as gelatins desserts, confectioneries, drymixes, poultry, dairy, and meat products.87,497 Table10 summarizes some applications of betalain pig-ments in food products. The amount of pure pig-ment required in these foods groups to obtain thedesired hue is relatively small and for most appli-cations does not exceed 50 ppm of betalains, cal-culated as betanin. Problems associated withbetalain degradation and pigment recovery dur-ing the processing operations are of economicimportance and must be solved to betalains dis-place the application of synthetic dyes in somefood products. The effectiveness of commercialbetalains depends largely on a continuous avail-ability of highly pigmented sources, the use ofcold and modified storage atmospheres prior to

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processing, efficient enzymatic control, handlingpractices, extraction procedures, purification, con-centration, and finishing operations e.g., freeze,spray, and vacuum drying).

Nowadays, beet roots represent the main com-mercial source of betalains (concentrates or pow-ders).382 Many factors during the pre- and post-harvest period and during processing influencethe recovery of these natural beet colorants. Inaddition, recent efforts are centered around thebetalain content in red beets through selectivebreeding. Initially, high pigment content is veryimportant. The average pigment content of beetsis approximately 130 mg/100 g freshweight,416,496,497 but new red beet varieties producearound 450 to 500 mg/100 g fresh weight. Fur-

thermore, this value is increasing as advancedselection is developed.382

Commercial preparations of beet pigmentfor use as food colorants are available as eitherjuice concentrates (produced by concentratingjuice under vacuum to 60 to 65% total solids) orpowders (produced by freeze or spray drying).These preparations contain from 0.3 to 1% ofpigment.42,76,77,497 They show a variety of colors,depending on their content of yellow pigments,and may have a beet-like odor and flavor. Theremainder of the solids is mainly sugars (75 to80%), ash (8 to 10%), and protein (10%). On alaboratory scale, betalains can be obtained byemploying reverse osmosis,276 ultrafiltra-tion,30,391,392 solid -liquid extraction,275,505 and dif-

TABLE 10Applications 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.

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fusion.504 These processes have been shown tobe efficient on the recovery of betalains fromraw beet tissue when compared with conven-tional hydraulic techniques.391 As approximately80% of beet juice solids consist of fermentablecarbohydrates and nitrogenous compounds, a fer-mentation process to remove these materials hasbeen widely employed.128 The yeast Candidautilis and Saccharomyces cerevisiae have beenused in the fermentative process, whereas a strainof Aspergillus niger not only destroyed free sug-ars but also enriched the colorant in betanin.379

The powder obtained from fermented juice con-tained five to seven times as much as thebetacyanin obtained in the powder from raw juice(on a dry weight basis).

c. Production of Betalains by Plant TissueCulture

Cell tissue culture has been a very useful toolin the study of various aspects of biochemistry,enzymology, genetics, and biosynthesis ofbetalains,273 and, interestingly, betalain produc-tion by plant cell culture will represent an excel-lent option in the future; it has a number of advan-tages over conventional procedures. Mainly withthis methodology, it is possible to control qualityand availability of pigments independently ofenvironmental changes.126 Nevertheless, the pro-ductivity of the bioreactor systems must be in-creased over 0.168 mg/g dry weight/day240,446 andthe cost reduced below $0.15 U.S. dollars/l inorder to be considered economically feasible.241

Thus, a successful betalain production will de-pend on process optimization to maximize yields,and, consequently, suitable downstream recoverytechniques must be available.382

Betalain production has been detected in cellcultures of plant species belonging to five fami-lies of Caryophyllales.46 Betalain accumulationof betalains in beet callus culture was reportedby Constabel and Nassif-Makki85 and also hasbeen demonstrated in cell cultures of P. grandi-flora,137 A. tricolor,37 O. microdasys,239 Ch.rubrum,35 and P. americana.212 Plant cell cul-tures are generally deep-red or purple colored; itmeans that betacyanins are dominant over

betaxanthins. Some models do not producebetaxanthins. It has been also established thatmain betacyanin in most of the studied models isbetanin. Schwitzguébel et al.434 observed thatindividual Beta vulgaris callus cultures containedcells exhibiting a variety of colors either non-pigmented, yellow, orange, red, or purple. Therange of observed pigmentation was due to thepresence of the deep red-purple betacyanins andto the yellow betaxanthins contained within thecell vacuole.171

Recently, plant hairy roots have become ofinterest as an alternative for cell culture becausetheir infinite and active proliferation in a phyto-hormone-free medium and their ability to synthe-size and accumulate valuable betalains at compa-rable levels to those found in plants.459 Extracellularproduction of betalains accompanied by pigmentrelease has been obtained in hairy root culturesunder oxygen starvation.255 However, both plantcell and hairy root cultures are influenced by avariety of physical and chemical factors impli-cated in the production of betalain pigments, andsome of them, such as growth regulators, light,nitrogen, carbon, and microelements, are widelydiscussed by Böhm and Rink46 and Leathers, etal.273

In addition, it is important to consider thatbetalain production by cell or hairy root culturesare mainly empirical, and it is not supported by astrong knowledge of the underlying mechanismsof biosynthesis and regulation. Nevertheless, insome instances, such as in the production ofB. vulgaris betalains, it is possible to obtain cul-tures producing specific pigments in comparableor even larger quantities than in the tissues of theoriginal plant.446

Recently, Hempel and Böhm207 adminis-trated nine L-amino acids to hairy root culturesof Beta vulgaris var. Lutea. Two betaxanthins,portulaxanthin II and vulgaxanthin I, were pro-duced predominantly, while minor quantitiesof muscaauri-VII, dopaxanthin, andindicaxanthin were synthesized de novo. Theseresults are very important because the possibil-ity of betaxanthin production at commerciallevel is opened, and, interestingly, red beet isone of the best known betalain production mod-els, being easiest the methodology standardiza-

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tion. Nowadays, the selection of a bioreactorand cultivation techniques for optimal culturegrowth and betalain production is one of themost important issues to be solved.241 Muchdata are now available about the growth andproduction kinetics in suspension cultures;273

however, most of them are not suitable for thedesign of large-scale processes, and the growthand production kinetics must be studied underwell-defined conditions and at different steadystates using the type of bioreactor selected forthe large-scale process.241

V. FUTURE TRENDS

Since the 1960s people have shown a clearpreference for natural products, including pig-ments, because more nutritious and healthy char-acteristics have been associated with them. Re-markably, food scientists consider that thisconsumer trend will be maintained in the future.In this sense, food technologists will continueaffronting the problems of availability and stabil-ity of natural pigments in order to replace thesynthetic ones. Moreover, one of the main prob-lems to be solved, that natural colors approved byFDA and European Union do not cover all rangesof colors (e.g., blue). Thus, many research groupsare looking for new sources of natural pigments;however, these efforts have vanished, becauseunder the current legislation the FDA or the Eu-ropean Union approval of new natural sources ofpigments is very difficult, contrasting with poli-tics followed in Japan. Consequently, it is ex-pected that the world global market must contrib-ute to the implementation of more realistic laws.On the other hand, it has been clearly establishedthat technology development has introduced newmethodologies or processes to avoid the intrinsicinstability and solubility problems of natural pig-ments, and today most of these problems can besolved through technological processes. In addi-tion, the pigment research area is very wide, andnatural pigments with improved characteristics(new colors and improved stability) have beendiscovered, being that their application differedby the above-mentioned reasons. Thus, a moreadequate and updated legislation will provide a

broad range of natural colors, and importantlywith better stability characteristics.

It is convenient now to point out that plantpigment production is limited, being that pig-ments are secondary metabolites. Furthermore,agriculture must continue focusing on food pro-duction for a growing population, and, undoubt-edly, food color does not matter for millions ofpeople all over the world as much as basic foodproduction. However, the demand for better natu-ral-colored foods by an important sector of thesociety will be increased because it is highly likelythat future studies will increase people’s con-science about the positive health benefits of natu-ral pigment consumption. Consequently, betterproduction systems for natural pigments will berequired higher productivities in smaller areas).Then, research interests will evolve, and two ar-eas will be the future of natural pigment produc-tion: the generation of crops with improved char-acteristics, and pigment production at theindustrial level and under controlled conditions.An interesting goal of food research will continuebeing the production of highly consumed crops(e.g., rice, corn, wheat, oat, bean) with improvedchemical composition, plus better functional per-formance, and at the same time favoring theirnutraceutical properties, and genetic-engineeredplants with higher productivity and with modifiedbiosynthetic pathways as well. In order to pro-duce such crops, the understanding of the in-volved metabolic pathways must be wider anddeeper. In this sense, more studies must be carriedout to have a complete vision of the biosynthesisand regulation of carotenoid and betalain plantproduction, while nowadays those performed onthe anthocyanin biosynthetic pathway will con-tinue focusing on regulatory aspects. Remark-ably, the most impressive advances in these as-pects have been reached using molecular biologytechniques, and this will be so in the near future.

Interestingly, model systems for pigment pro-duction under controlled conditions are now avail-able, but production at the industrial level has notbeen feasible yet. Thus, carotenoid production byyeasts, bacteria and fungi, and anthocyanin andbetalain production by plant tissue cultures re-quire the development of better biotechnologicalapproaches.

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ACKNOWLEDGMENTS

FDV, ARJ, and OPL acknowledge the finan-cial support received from the Consejo Nacionalde Ciencia y Tecnología, México (CONACYT);ARJ also acknowledges the support from theInstituto Politécnico Nacional (IPN) on naturalpigment studies.

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

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