Biosynthesis of Food Constituents: Natural Pigments. Part 2 – a Review

Czech J. Food Sci. Vol. 26, No. 2: 73–98

Jan Velíšek, Jiří Davídek and Karel Cejpek

Department of Food Chemistry and Analysis, Faculty of Food and Biochemical Technology, Institute of Chemical Technology in Prague, Prague, Czech Republic

Abstract

Velíšek J., Davídek J., Cejpek K. (2008): Biosynthesis of food constituents: Natural pigments. Part 2 – a review. Czech J. Food Sci., 26: 73–98.

This review article is a part of the survey of the generally accepted biosynthetic pathways that lead to the most important natural pigments in organisms closely related to foods and feeds. The biosynthetic pathways leading to xanthones, flavonoids, carotenoids, and some minor pigments are described including the enzymes involved and reaction schemes with detailed mechanisms.

Keywords: biosynthesis; xanthones; flavonoids; isoflavonoids; neoflavonoids; flavonols; (epi)catechins; flavandiols; leucoanthocyanidins; flavanones; dihydroflavones; flavanonoles; dihydroflavonols; flavones; flavonols; anthocyanidins; anthocyanins; chalcones; dihydrochalcones; quinochalcones; aurones; isochromenes; curcuminoids; carotenoids; carotenes; xanthophylls; apocarotenoids; iridoids

The biosynthetic pathways leading to the tetrapyr- restricted in occurrence to only a few families of role pigments (hemes and chlorophylls), melanins higher plants and some fungi and lichens. The (eumelanins, pheomelanins, and allomelanins), majority of xanthones has been found in basi- betalains (betacyanins and betaxanthins), and cally four families of higher plants, the Clusiaceae quinones (benzoquinones, naphthoquinones, and (syn. Guttiferae), Gentianaceae, Moraceae, and anthraquinones) were described in the first part of Polygalaceae (Peres et al. 2000). Xanthones can this review (Velíšek & Cejpek 2007b). This part be classified based on their oxygenation, prenyla- deals with the biosynthesis of other prominent tion and glucosylation pattern. The majority of natural food colorants, xanthones, flavonoids, xanthones isolated so far are of the tetraoxygen- curcuminoids, carotenoids, isochromenes, and ated-type. Xanthones posses a number of remark- iridoids. able pharmacological activities (Peres et al. 2000) and have been used as, e.g. cardiovascular pro- 5 Xanthones tective agents (Jiang et al. 2004) and antitumor promotors (Ito et al. 1998). The root of Yellow

Xanthones, represented as the C6-C1-C6 system Gentian (Gentiana lutea, Gentianaceae) contains (Figure 30), are a group of about 70 yellow pigments several yellow xanthones, exemplified by gentisin

Partly supported by the Ministry of Education, Youth and Sports of the Czech Republic (Project No. MSM 6046137305).

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10 HO O OH 4 5 HO HO O OH O HO O 3 6 ABC O OCH3 2 9 7 OH OH OH OH O 1 8 OH O O OH O HO OH

xanthone gentisin mangiferin mangostin

Figure 30

(1,3,7-trihydroxyxanthone), that are also found in benzophenone 3'-hydroxylase and converted to the Common Centaury (Centaurium erythraea, the 2,3',4,6-tetrahydroxyphenone intermediate, Gentianaceae) (Figure 31). The Gentiana root is common to both pathways. The crucial step in this bitter and becomes the principal vegetable bitter biosynthesis is the cyclisation of the benzophenone employed in medicine. Before the introduction to a xanthone, catalysed by xanthone synthases. of hops, Gentian, with many other bitter herbs, While in C. erythraea the xanthone synthase con- was used occasionally in brewing. It is a princi- verts this tetrahydroxybenzophenone intermediate pal ingredient in angostura bitters. 1,3,6,7-Tet- regioselective to 1,3,5-trihydroxyxanthone, the rahydroxyxanthone, the aglycone of C-glucoside xanthone synthase of H. androsaemum converts mangiferin occurs in mango leaves (Mangifera it to the 1,3,7-trihydroxyxanthone. The last step indica, Anacardiaceae) and has been used as a in the biosynthesis of the tetrahydroxyxanthones yellow textile dye. It is also known to exhibit anti- is the introduction of a hydroxyl group to the inflammatory, antioxidant, and antiviral effects. C-ring of the xanthone skeleton. This reaction Mangostin is the major xanthone constituent of is carried out by the plant-specific cytochrome the mangosteen tree (Garcinia mangostana, Clu- P450-dependend monooxygenase xanthone 6-hy- siaceae) (Bennet et al. 1990). droxylase to form 1,3,5,6-tetrahydroxyxanthone in The biosynthesis of xanthones (Figure 31) origi- C. erythraea and 1,3,6,7-tetrahydroxyxanthone in nates mainly from a mixed shikimate-acetate path- H. androsaemum. Another representative of the way; however xanthones entirely derived from Gentianaceae, chirayta (Swertia chirata), hydroxy- acetate have also been reported in lower plants lates 1,3,5-trihydroxyxanthone in the C-8 position (Peres et al. 2000). The main steps in the xanthone of the C-ring resulting in the major xanthone of biosynthesis involve the condensation of shikimate this species 1,3,5,8-tetrahydroxyxanthone (Wang and acetate moieties, which constitute a benzo- et al. 2003a; BIOCYC 2007). phenone intermediate followed by a regioselective, oxidative mediated intramolecular coupling to 6 Flavonoids form the xanthone ring (Peters et al. 1998). The shikimate derivatives used as entry compounds Flavonoids comprise a widespread group of more for the tetrahydroxyxanthone biosynthesis differ than 4000 higher plant secondary metabolites. in the individual plants. Many flavonoids are easily recognised as water- In the Common Centaury, the biosynthesis of soluble flower pigments in most angiosperm fami- tetrahydroxyxanthone is obtained via 3-hydroxy- lies (flowering plants), which themselves provide benzoic acid (that appears to originate directly the major source of food plants (fruits, vegetables, from the shikimate pathway) by the stepwise con- and grains). Molecules of flavonoids posses fif- densation of 3-hydroxybenzoyl-CoA (formed un- teen carbon atoms and contain two benzene rings der catalysis of 3-hydroxybenzoate-CoA ligase, (rings A and C, respectively) connected with a EC 6.2.1.-) with three malonyl-CoA to form an chain built up of three carbon atoms that becomes intermediate 2,3',4,6-tetrahydroxybenzophenone a part of O-heterocyclic (pyrane) cycle (ring B). (benzophenone synthase, EC 2.3.1.151). In Hyperi- The skeleton of flavonoids can be thus represented cum androsaemum (Clusiaceae), the biosynthesis as the C6-C3-C6 system (1,3-diarylpropanoids). of this tetrahydroxyxanthone proceeds via ben- Many flavonoids are hypothetically derived from zoic acid and benzoyl-CoA (benzoate-CoA ligase, flavan (IUPAC 2006) (Figure 32). EC 6.2.1.25). The new intermediate, 2,4,6-tri- Various subgroups of flavonoids are classified hydroxybenzophenone is further hydroxylated by according to their substitution pattern of the ring B.

74 Czech J. Food Sci. Vol. 26, No. 2: 73–98

HOOC OH HOOC

3-hydroxybenzoic acid benzoic acid ATP + HS-CoA

EC 6.2.1.25 ATP + HS-CoA AMP + PP EC 6.2.1.- OO 3 AMP + PP CoA OH

malonyl-CoA 4 HS-CoA + 3 CO 2 HO OH

S S CoA OH CoA EC 2.3.1.151 O O OH O 3-hydroxybenzoyl-CoA benzoyl-CoA 2,4,6-trihydroxybenzophenone OO

NADPH + O2 3 CoA OH malonyl-CoA

4 HS-CoA + 3 NADP + H O EC 2.3.1.151 CO 2 2

HO OH

OH OH O NADPH + O2 NADPH + O2 2,3´,4,6-tetrahydroxybenzophenone

NADP + 2 NADP + 2 H2O H2O

OH HO O HO O

OH OH O OH O

1,3,5-trihydroxyxanthone NADPH + O2 1,3,7-trihydroxyxanthone

NADPH + O2 NADPH + O2 NADP + H2O

NADP + H2O NADP + H2O

OH OH HO O HO O OH HO O OH

OH OH O OH OH O OH O

1,3,5,8-tetrahydroxyxanthone 1,3,5,6-tetrahydroxyxanthone 1,3,6,7-tetrahydroxyxanthone

Figure 31

Both the oxidation state of the heterocyclic ring the three rings and oxidation of the C-4 hydroxyl and the position of the ring B substituents are group. Examples of the seven major subgroups of important in the classification. Derivations include the most important structural types are given in oxidation of the 2(3)-carbon-carbon double bond Figure 33. The compounds are listed according in flavan, hydroxylation at various positions of to their oxidation degree that concerns the three

75 Vol. 26, No. 2: 73–98 Czech J. Food Sci.

3´ nylbenzopyrylium or 2-phenylchromenylium), 2´ 4´ 1 anthocyanidins became the most prominent group 8 C O 5´ of water-soluble plant pigments as they are re- 7 1´ A B 2 6´ sponsible for blue, purple, violet, magenta, pink, 6 3 and red coloration.17 5 4 In a few cases, the six-membered heterocyclic flavan ring B occurs in an isomeric open form (chalcones and dihydrochalcones) or is replaced by a five- Figure 32 membered ring (aurones). Chalcones and aurones belong to the prominent flower pigments that are central carbon atoms (ring B). In the rows, the responsible for their golden and yellow colora- oxidation degree increases from the left to the tion. Isoflavonoids or 3-phenylchromen-4-ones right while in the columns the oxidation degree (1,2-diarylpropanes, colourless to light yellow is equal. In the same direction their coloration compounds derived from isoflavan) and neoflavo- increases, too. Catechins and leucoanthocyanidins noids or 4-phenylcoumarins (1,1-diarylpropanes are uncoloured compounds, flavanones are from derived from neoflavan) are less common flavo- uncoloured to light yellow like flavanonoles, fla- noids having the ring C attached to the ring B at vones and flavonols are yellow. Due to the system C-3 and C-4, respectively (Figure 34). Isoflavonoids of conjugated doubleO bonds (flavilium or 2-pheO - and neoflavonoidsO can be regarded as abnormalO

OH OH OH OH OH O O flavanols (catechins) flavandiols (leucoanthocyanidins) flavanonoles (dihydroflavonols) flavonols O O O O O O O O

OH OHO OHO OH OH OH OH OH OH O O OH O O flavanols (catechins) flavandiols (leucoanthocyanidins) flavanonoles (dihydroflavonols) flavonols flavanols (catechins) flavandiols (leucoanthocyanidins) flavanonolesO (dihydroflavonols) flavonolsO flavanones (dihydroflavones) flavones O O O O O

O O O O flavanones (dihydroflavones) flavones OH flavanones (dihydroflavones) flavones anthocyanidins

O O Figure 33 OH OH 3 4´ anthocyanidins 1 8 2 4 anthocyanidins O O O 3´ 5´ 7 2 3´ B B 2´ A B 2´ OH 5 OH 6´ 3 4´ 2´ 6 1 3´ 1 1´ 4 A 6 7 5 1´ 5´ C D O D 6 O 6´ 4´ 1´ 2 6´ A 5´ O O 5 3 O 4 chalcones dihydrochalcones aurones isoflavonoids neoflavonoids

Figure 34

17According to their colour, flavonoid pigments have been classified into two large groups, the red to blue anthocyanins and the yellow anthoxanthins. Chalcones and aurones are categorised into a class of flavonoids called anthochlors or anthochlor pigments.

76 Czech J. Food Sci. Vol. 26, No. 2: 73–98 flavonoids18 and are of marginal importance as 6.1 Chalcones and flavanones plant pigments. Most of the naturally occurring flavonoids con- Flavonoids (and stilbenes that are represented tain multiple hydroxyl or methoxyl substituents at as the C6-C2-C6 system) are products from the various positions of the rings A, B, and C. Their shikimate pathway (Dewick 2002; IUBMB 2007) basic structures may be further modified by conju- (Figure 35). The cinnamoyl-CoA starter is produced gation reactions like glycosylation, acylation, and from l-phenylalanine (phenylalanine deaminase, polymerisation. The major proportion of flavonoids EC 4.3.1.5) and hydroxylated (cinnamate 4-hy- occur naturally as glycosides (Harborne 1980; droxylase, EC 1.14.13.11) to yield 4-hydroxycin- Hendry & Houghton 1992; Velíšek 2002). namic (4-coumaric, p-coumaric) acid, which is

NH3 NADPH + H + O2 NADP + H2O HS CoA + AT P + PP AMP O COOH COOH COOH CoA S

NH2 EC 4.3.1.5 EC 1.14.13.11 HO EC 6.2.1.12 HO L-phenylalanine (E)-cinnamic acid 4-coumaric acid 4-coumaroyl-CoA OO 3 CoA EC 2.3.1.74 HO S Claisen condensation malonyl-CoA

3 HS CoA + 3 CO2 OH OH HS CoA H2O O S HO O HO OH CoA OH O EC 2.3.1.74 EC 5.5.1.6 OH O OH O OO naringenin naringenin chalcone (isosalipurpol)

NADPH + H + O2 EC 1.14.13.21 EC 1.14.13.88

NADP + H2O OH OH OH NADPH + H + O2 NADP + H2O HO O HO O OH OH

EC 1.14.13.88 OH O OH O

eriodyctiol dihydrotricetin

Figure 35

18The colourless catechins undergo enzymatic browning reaction becoming the substrates of various oxidoreductases. Fermentation of tea leaves (Camellia sinensis), for example, leads to black tea pigments called oxytheotannins. They are classified as theaflavins (orange to red dimeric structures in which oxidation reactions lead to the formation of seven-membered tropolone rings) and thearubigins (heterogenous yellow, red, and brown high molecular weight pigments). Furthermore, catechins form oligomers (the condensed tannins) that contribute astringency to many fruits, foods, and drinks of plant origin. Some flavanone-7-glycosides, e.g. naringin (naringenin-7-neohesperidoside) occurring in grapefruit (Citrus paradisi, Rutaceae) and neohesperidin (hesperetin-7-neohesperidoside) from bitter orange (C. aurantium), are intensely bitter compounds. The conversion of these flavanones into dihydrochalcones produces intensely sweet compounds used as sweetening agents. Isoflavonoids are regarded as phytoestrogens as they show estrogenic activity. Some flavonoids (flavanones and flavonols) have been also commonly referred to as bioflavonoids and also referred to as vitamin P. These compounds have been appreciated as they influence the blood capillaries permeability, prevent their fragility and show activity as antioxidants and scavengers of free radicals.

77 Vol. 26, No. 2: 73–98 Czech J. Food Sci. subsequently transformed to 4-coumaroyl-CoA 6.1.1 Quinochalcones (4-coumaroyl-CoA synthetase, EC 6.2.1.12). The chain extension is provided using 3 molecules of The petals of safflower (Carthamus tinctorius) malonyl-CoA (chalcone synthetase, EC 2.3.1.74). contain yellow quinochalcone pigments such as This initially gives a polyketide, which, depend- safflor yellow A, safflor yellow B, precarthamin, ing on the enzyme available, can be folded in two and red carthamin. Traditionally, safflower has different ways. These allow either aldol conden- been used for its flowers in cooking as a cheaper sation yielding finally stilbenes or Claisen-like substitute for saffron (sometimes referred to as condensation generating chalcones that act as bastard saffron), textile dye, and herbal medicine precursors for a vast range of flavonoids. in oriental countries. Nowadays, the plant is culti- Flavanones are formed from the primary reaction vated mainly for the vegetable oil used quite com- products chalcones (chalcone-flavanone isomerase, monly as an alternative to sunflower seed oil. Its is EC 5.5.1.6) by Michael-type nucleophilic attack of supposed that these pigments are biosynthesised, a phenol group on the unsaturated ketone.19 The analogously to the chalcone naringenin, from the key flavanone naringenin (5,7,4'-trihydroxyfla- cinnamoyl-CoA starter and three malonyl-CoA vanone) may be oxidised (hydroxylated) at C-3' of extenders yielding first safflor yellow A, which is the ring C by flavonoid 3'-hydroxylase (flavonoid transformed to safflor yellow B, the precursor of 3'-monooxygenase, EC 1.14.13.21) or flavonoid precarthamin (Figure 37). Precarthamin forms from 3’,5’-hydroxylase (EC 1.14.13.88) to eriodictyol safflor yellow B by oxidation of the open-chain (5,7,3’,4'-tetrahydroxyflavanone). The enzyme d-glucose residue. Oxidative decarboxylation of flavonoid 3’,5’-hydroxylase (EC 1.14.13.88) may precarthamin catalysed by precarthamin decar- further oxidise eriodictyol to dihydrotricetin boxylase yields carthamin (Cho & Hahn 2000). (5,7,3’,4’,5'-pentahydroxyflavanone). Many flavonoid structures loose one of the hy- 6.2 Flavones, flavanonoles, flavonols, droxyl groups, so that the acetate-derived aromatic leucoanthocyanidins, (epi)catechins, ring (ring A) has a resorcinol oxygenation pattern and anthocyanidins rather than the phloroglucinol system (Dewick 2002). This modification has been tracked down Flavanones can give rise to many variants of the to the action of a reductase enzyme 6'-deoxychal- basic skeleton, e.g. flavones (flavone synthase, cone synthase (EC 2.3.1.70), and thus a chalcone EC 1.14.11.22), flavanonoles (flavanones 3-hydroxy- isoliquiritigenin (2',4,4'-trihydroxychalcone) is pro- lase, EC 1.14.11.9), flavonols (flavonol synthase, duced rather than naringenin chalcone (2',4,4',6'-te- EC 1.14.11.23), leucoanthocyanidins (dihydrofla- trahydroxychalcone) (IUBMB 2007) and yields vanol 4-reductase, EC 1.1.1.219), anthocyanidins liquiritigenin (4’,7-dihydroxyflavanone), the pre- (anthocyanidin synthase, EC 1.14.11.19), cate- cursor of isoflavones (Figure 36). chins (leucoanthocyanidin reductase, EC 1.17.1.3),

⊕ ⊕ 3 malonyl-CoA + NADPH + H 4 HS-CoA + 3 CO2 + H2O + NADP 3 malonyl-Coa + NADPH + H 4HS-Coa + 3 CO2 + H2O + NADP OH OH

O HO OH HO O CoA S EC 2.3.1.70 EC 5.5.1.6 HO O O 4-coumaroyl-CoA isoliquiritigenin liquiritigenin

Figure 36

19This isomerisation can occur non-enzymatically under acid conditions that favour the flavanone whereas basic conditions favour the chalcone. The enzyme-catalysed reaction is stereospecific and results in the formation of a single flavanone enantiomer, (2S)-flavanone.

78 Czech J. Food Sci. Vol. 26, No. 2: 73–98 and epicatechins20 (anthocyanidin reductase, thocyanidin 5,3-O-glycosyl transferase (EC 2.4.1.-) EC 1.3.1.77) (Figure 38). (IUBMB 2007; BIOCYC 2007). Modifications to the hydroxylation patterns in the two aromatic rings may occur, generally at 6.3 Aurones the flavanone, flavanonole, or flavone stage, and methylation, O- glycosylation, C-glycosylation, and Aurones belong to a small class of plant flavonoids dimethylallylation are also common, increasing that, in the glycosylated form, provides the bright the range of compounds enormously. Figure 39 yellow colour of some important ornamental flowers, shows, as an example, the pathways leading to such as snapdragon (Antirrhinum majus, Scrophula- the most common methylated and glycosylated riaceae) and plants belonging to the Asteraceae family apigenin derivatives. The enzymes involved are (e.g. Coreopsis sp., Comos sp., and Dahlia sp.). apigenin 4'-O-methyltransferase (EC 2.1.1.75), The pivotal precursors of aurones are chalcones. flavone 7-O-β-glucosyltransferase (EC 2.4.1.81), The minor 4,4’,6-trihydroxyaurone can form from flavone 7-O-β-glucoside 6''-O-malonyltransferase naringenin chalcone by a not yet characterised ox- (EC 2.3.1.115), and flavanone-7-O-glucoside ygenase enzyme. The enzyme aureusidin synthase 2''-O-β-l-rhamnosyltransferase. (a copper-containing glycoprotein and a homo- The pathways leading to the most common cya- logue of plant polyphenol oxidase, EC 1.21.3.6) nidin glycosides are given in Figure 40. The un- catalyses the 3-hydroxylation of the ring B and stable anthocyanidins are thus coupled to sugar the oxidative cyclisation (2',α-dehydrogenation) molecules to yield the final relatively stable gly- of chalcones carrying hydroxyl functions at their cosides anthocyanins. The enzymes involved in C-2' and C-4 positions. Aurone formation from these pathways comprise anthocyanidin 3-O-glu- chalcones having a 4-hydroxy B-ring must be cosyltransferase (EC 2.4.1.115), anthocyanidin accompanied by the oxygenation of the ring B, 3-aromatic acyl transferase (EC 2.3.1.-), and an- whereas aurones formation from chalcones with

OH CH2OH OH HO HOH HOH HOH O OH 2 D-Glc D-Glc H O HOH 2 O O O OH OH O O HO OH HO OH O OH O HO OH HO OH OH HO OH OH HO OH O O OH HO HO HO O OH OH OH 2,4,6,4’-tetrahydroxychalcone2,4,6,4´-tetrahydroxychalcone safflorsafflor yellow yellow A A safflorsafflor yellow yellow B B

OH OH OH OH oxidation

HO O O O CO2 O O

O O HO O O HO OH OH O O OH O OH HO OH decarboxylation OH OH OH HO OH HO OH HO HO HO O OH oxidation HO O OH OH OH carthamincart ham in precarthamin

Figure 37

20The products are (2S)-flavan-4-ols, (2R,3R)-dihydroflavonols, leucoanthocyanidins (cis-flavan-3,4-diols), catechins (flavan-3-ols), (+)-catechins, and (–)-epicatechins. Formally, 4'-hydroxyderivatives are derived from 4-hydroxybenzoic (p-benzoic) acid, 3',4'-dihydroxyderivatives from protocatechuic acid, and 3',3',5'-trihydroxyderivatives from gallic acid.

79 Vol. 26, No. 2: 73–98 Czech J. Food Sci.

1 1 1 R R R 2-oxoglutaric acid + O2 2-oxoglutaric acid + O2 OH OH OH succinic acid + CO2 succinic acid + CO2

HO O 2 HO O 2 HO O 2 R R R

EC 1.14.11.9 OH EC 1.14.11.23 OH OH O OH O OH O

flavanones (dihydroflavones) flavanonoles (dihydroflavonols) flavonols

R1 = R2 = H, naringenin R1 = R2 = H, aromadendrin (dihydrokaempherol) R1 = R2 = H, kaempherol R1 = H, R2 = OH, eriodictyol R1 = H, R2 = OH, taxifolin (dihydroquercetin) R1 = H, R2 = OH, quercetin R1 = R2 = OH, dihydrotricetin R1 = R2 = OH, ampelopsin (dihydromyricetin) R1 = R2 = OH, myricetin

NAD(P) 2-oxoglutaric acid + O2

EC 1.14.11.22 EC 1.1.1.219

succinic acid + CO2 + H2O NAD(P)H + H

1 1 1 R R R OH OH OH NADP NADP H + H

HO O 2 HO O 2 HO O 2 R R R

OH EC 1.17.1.3 OH OH O OHOH OH

flavones leucoanthocyanidins (flavandiols) catechins (flavanols) 1 2 1 2 R = R = H, apigenin R1 = R2 = H, leucopelargonidin R = R = H, afzelechin 1 = 2 1 2 R H, R = OH, luteolin R1 = H, R2 = OH, leucocyanidin R = H, R = OH, catechin 1 2 1 2 R = R = OH, tricetin R1 = R2 = OH, leucodelphinidin R = R = OH, gallocatechin 2-oxoglutaric acid + O2

EC 1.14.11.19 succinic acid + CO2 + H2O 1 1 1 R R R OH H2O OH OH OH HO O 2 HO O 2 HO O 2 R R R

OH OH O OHOH OHOH OHOH

1 1 1 R R R OH 2 NAD(P ) OH OH 2 NAD(P)H + H H2O

HO O 2 HO O 2 HO O 2 R R R

OH EC 1.3.1.77 OH OH OH OH OHOH

epicatechins (flavanols) anthocyanidins

R1 = R2 = H, epiafzelechin R1 = R2 = H, pelargonidin R1 = H, R2 = OH, epicatechin R1 = H, R2 = OH, cyanidin R1 = R2 = OH, epigallocatechin R1 = R2 = OH, delphinidin

Figure 38 a 3,4-dihydroxy B-ring is not necessarily accom- droxyl group. The subsequent two reactions leading panied by B-ring oxygenation (Sato et al. 2001; to aureusidin may then proceed spontaneously. Nahayama et al. 2001; Nahayama 2002). Thus, When 2',3,4,4’,6'-pentahydroxychalcone becomes aureusidin may be formed from naringenin chal- the substrate of aureusidin synthase, analogous re- cone or 2',3,4,4',6'-pentahydroxychalcone. It is actions yield a mixture of aureusidin and bracteatin supposed that 3-hydroxylation proceeds through (Figure 41). Aurones with no or only one hydroxyl an intermediate with an o-quinone structure, group in the ring B are produced by alternative which induces the Michael-type addition of 2'-hy- mechanisms.

80 Czech J. Food Sci. Vol. 26, No. 2: 73–98

OCH3

HO O

OH O acacetin SAH OH EC 2.1.1.75 HO SAM OH OH O OH OH UDP UDP-D-Glc HO HO HO O HO O O O O OH EC 2.4.1.81 enzyme not yet HO characterised OH O OH OH O OH O

apigenin 7-O -E-D-glucopyranoside (cosmosiin) apigenin vitexin (apigenin 8-C -E-D-glucopyranoside)

malonyl-CoA enzyme not yet characterised EC 2.3.1.115

HS-CoA O COOH OH OH O OH HO O O HO O O O O O OH OH HO UDP- L-Rha UDP HO HO O HO O O OH O OH O OH OH O OH CH3 EC 2.4.1.236 HO OH OH OH

apigenin 7-O -(6´´-malonyl-E -D-glucopyranoside) apigenin 7-O -neohesperidoside isovitexin (apigenin 6-C -E-D-glucopyranoside)

Figure 39

6.4 Miscellaneous flavonoids an intermediate able to cyclise intramolecularly, which is followed by aromatisation to yield santalin Red sandalwood, called also red sanderswood, A. Its methylation at C-13' hydroxyl gives santalin sanders or saunders, and rubywood, is the dark B. Hydroxylation of the isoflavilium salt followed red heartwood of the leguminous tree Pterocarpus by oxidation yields santalin AC (Figure 42). santalinus (Fabaceae) of India. Sandalwood red is a pigment obtained by extracting red sandalwood 7 Isochromenes with water. It was used as a wool colouring and also a food colouring in medieval and renaissance food Red yeast rice, red koji rice, or ang-khak is a (jellies, custards) and has been used in Ayurvedic bright purplish-red fermented rice, which acquires medicine as an anti-septic wound healing agent and its colour and a subtle but pleasant taste by culti- in anti-acne treatment. Its major red components vation with the mold Monascus purpureus (syn. are lipophilic substances santalin A and santalin B. M. albidus, M. anka, M. araneosus, M. major, Some other structurally similar compounds (e.g. M. rubiginosus, and M. vini). It was commonly used santalin AC and other similar compounds) were for red food coloring in East Asian and Chinese identified (Kinjo et al. 1995). cuisine prior to the discovery of chemical food It is supposed that the first phase of santalins A and colours and is still used to colour a wide variety B biosynthetic mechanism starts with isoflavilium of food products including pickled tofu, red rice salts as potential electrophiles and an open chain C15 vinegar, Peking duck or chinese pastries that re- unit as a nucleophile (Kinjo et al. 1995). After the quire red food coloring. It is used traditionally in initial coupling of the isoflavilium ion and this open the production of several types of Chinese wine, chain unit, a carbonium ion at C-4' could generate Japanese sake (akaisake), and Korean rice wine

81 Vol. 26, No. 2: 73–98 Czech J. Food Sci.

HO O OH OH O OH OH O O O OH

OH cyanidin 3-O -(6´´-O-4-p -coumaroyl-E -D-glucopyranoside) HS-CoA

EC 2.3.1.- p-coumaroyl-CoA

OH OH OH UDP- D-Glc UDP UDP- D-Glc UDP HO O HO O HO O OH OH OH OH OH HO OH EC 2.4.1.115 O EC 2.4.1.- O OH OH OH OH O cyanidin HO O HO O OH O OH UDP OH UDP- D-Glc SAM cyanidin 3-O - E-D-glucopyranoside HO enzyme not yet OH characterised D UDP UDP- -Glc cyanidin 3,5-O - E-D-diglucopyranoside SAH EC 2.4.1.- OH EC 2.4.1.-

HO O OH OH

HO O HO OH OCH3 OH O O O OH OH OH HO HO O OH OH

peonidin 3-O - E-D-glucopyranoside cyanidin 5-O - E-D-glucopyranoside

Figure 40

(hongju) giving them a reddish colour (Ma et al. ketide chromophore is possibly synthesised via monas- 2000; Wild et al. 2002). It has also been used in cusone A. The side chain of these pigments forming Chinese herbal medicine.21 a fused γ-lactone system is derived by condensation The major Monascus pigments (called monas- of 2-oxohexanoic acid and 2-oxooctanoic acids syn- cus red or just monascus) are a mixture of li- thesised via fatty acid synthetic pathway (Figure 44). pophilic azaphilones. They comprise yellow Nitrogen analogues of azaphilones (Figure 45) form pigments monascin and ankaflavin, their oxi- by their reactions with amino acids (Ogihara et al. dised forms (orange pigments) monascorubin 2000; Jongrungruangchok et al. 2004). and rubropunctatin, and their nitrogen analogues (red pigments) rubropunctamine and 8 Curcuminoids monascorubramine) which are derived from 1H-isochromene (Figure 43). Curcuminoids comprise a group of polyphenols 22 The main polyketide chain of the azaphilone related to lignanes. Molecules of curcuminoids pigments is assembled from acetic acid (the starter contain two benzene rings connected with a chain unit) and five malonic acids molecules (the chain built up of seven carbon atoms (diarylheptanoids), extender unit) in a conventional way. The hexa- which can be thus represented as the C6-C7-C6 21Recent discoveries of cholesterol-lowering statins (a class of hypolipidemic agents used to lower cholesterol levels in people with or at risk for cardiovascular disease) produced by the mold and other fungi has prompted research into the red yeast rice possible medical uses (Furberg 1999). 22In filamentous fungi, the polyketide pathway is the major route for the biosynthesis of secondary metabolites, including various mycotoxins.

82 Czech J. Food Sci. Vol. 26, No. 2: 73–98

O OH HO HO O OH O O 2 H2O HO OH HO OH HO O HO O H EC 1.21.3.6 H O O OH O OH O OH OH naringenin chalcone aureusidin

1/2 O2 H2O OH OH EC 1.21.3.6 HO OH 1/2 O2 H2O OH O2 H2O HO O HO OH HO O OH

O EC 1.21.3.6 O OH OH O OH

4,4´,6-trihydroxyaurone 2´,3,4,4´,6´-pentahydroxychalcone bracteatin

Figure 41 system. Curcuminoids are responsible for the transferase) to yield demethoxycurcumin, which yellow colour of turmeric (Curcuma longa), a on further hydroxylation and methylation yields member of the ginger family (Zingiberaceae). Its curcumin (the enzymes were not yet characterised). dried roots are ground into a deep yellow spice23 Curcumin can exist in at least two tautomeric that is a significant ingredient in most commercial forms, keto and enol. The enol form is more stable curry powders and has found application in canned in solutions. beverages, dairy products, ice creams, yoghurts, It is speculated that demethoxycurcumin can biscuits, sweets, cereals etc. Curcumin, i.e. (1E,6E)- alternatively form from 4-coumaroyl-CoA and 1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-di- caffeoyl-CoA instead of two molecules of 4-cou- ene-3,5-dione, is the principal lipophilic pigment maroyl-CoA by the direct action of polyketide of turmeric (less then 2% dry matter). Curcumin synthase. Analogously, curcumin might be formed is accompanied by two other minor pigments from two molecules of feruoyl-CoA (Ramirez- (about 0.6% dry matter), demethoxycurcumin and Ahumada et al. 2006). The enzymes involved in bisdemethoxycurcumin. the biosynthesis of these phenolic acid derivatives Similarly to flavonoids, curcuminoids are derived from 4-coumaroyl-CoA are shikimate O-hydroxy- from the intermediates in the phenylpropanoid cinnamoyltransferase (EC 2.3.1.133), p-coumaroyl- pathway such as 4-coumaryl-CoA that condenses 5-O-shikimate 3'-hydroxylase (EC 1.14.13.36) and with malonyl-CoA (perhaps involving a diketide caffeoyl-CoA O-methyltransferase (EC 2.1.1.104) intermediate) yielding bisdemethoxycurcumin (IUBMB 2007). The biosynthesis of shikimic acid (Ramirez-Ahumada et al. 2006). Enzymes similar envolved in these reactions was already reviewed to polyketide synthase (called curcuminoid syn- (Velíšek & Cejpek 2006). thase) are most likely responsible for the formation of the basic backbone structure of curcuminoids 9 Carotenoids (Figure 46). Bisdemethoxycurcumin is then hydroxylated (cytochrome P450 hydroxylase) and Carotenoids are a group of more than 700 very methylated (using SAM-dependent O-methyl- common yellow, orange and red (exceptionally

23Turmeric is also thought to have many medicinal activities such as antiseptic and antibacterial. It is currently being investigated for possible benefits in Alzheimer’s disease, cancer, and liver disorders. Colourless tetrahydrocurcumi- noids from turmeric may have antioxidant, skin lightening properties, and may be used to treat skin inflammations, making these compounds useful in cosmetic formulations.

83 Vol. 26, No. 2: 73–98 Czech J. Food Sci.

HO HO OH OH HO 13´ HO HO OH OH OCH3 O O HO O HO O OCH 3 4´

OCH3 HO coupling HO coupling HO

H3CO OCH3 H3CO OCH3 H3CO OCH3 OH OH OH isoflavilium cation substituted isoflavilium cation santalin A

H2O methylation

OCH3 HO H OH

HO O OH HO O O O O

OCH3 HO HO HO

H3CO OCH3 H3CO OCH3 H3CO OCH3 OH OH OH santalin AC santalin B

Figure 42 also yellow-green) lipophilic pigments that oc- five carbon atoms. Almost all other carotenoids are cur in all organisms capable of photosynthesis formally derived from this basic non-cyclic C40H56 including green algae, higher plants, and also hydrocarbon by subsequent hydrogenation, oxida- some bacteria. The coloration of many flowers, tion, ring formation (cyclisation), shifting of double fruits, vegetables, and grains is caused by caro- bonds and/or methyl groups, chain shortening, and tenoid-containing chromoplasts that are usually combinations of these reactions. devoid of chlorophylls. Unlike chlorophylls and Carotenoids can be classified into carotenes many other plant pigments, they are also found in (hydrocarbons without additional O-containing various animals and animal products (milk, butter, groups) and xanthophylls (carotenoids contain- egg yolk, salmon, shrimp) though they are always ing oxygen functions). The most frequently found of the plant origin (Sandmann 1994). O-containing functions are hydroxyl groups (mon- All carotenoids are terpenoids (tetraterpenoids) ols, diols, and polyols), epoxy-(5,6-epoxides and as they contain 40 carbon atoms in eight C5 isoprene 5,8-epoxides), methoxy-, formyl-, oxo-, carboxy-, units linked in a regular head to tail manner, except and ester groups. Some xanthophylls can be bound in the centre of the molecule where the order is in- to proteins and sugars. verted tail to tail, so that molecule is symmetrical. The two methyl groups near the centre are separated 9.1 Carotenes by six carbon atoms and the other methyl groups by The biosynthesis of carotenoids involves a series 5 4 of steps, i.e. the formation of the key building stone mevalonic acid (a product of acetic acid 6 3 metabolism) which gives rise to isopentenyl di- 7 O 2 phosphate, the common C5 precursor of all natu- 8 1 ral isoprenoids, the formation of geranylgeranyl 1H-isochromene diphosphate, the formation of acyclic phytoene, 1H-isochromene cyclisation to form alicyclic carotenes, and the Figure 43 formation of xanthophylls.

84 Czech J. Food Sci. Vol. 26, No. 2: 73–98

5 CO2 O CH3 O CH3 O OO methylation enzym e+ 5 enzym e O O O O O O H3CS HO S O O H3C acetyl-CoA malonyl-CoA (ACP) O S O S enzyme enzyme enzyme-bound unreduced hexaketide condensation H2O enolisation HS-enzym e reduction H O CH3 CH3 hydroxylation HO O CH3 O O HO O OH O O O H C H3C condensation 3 H3C O O O O S monascusone A enzyme

OO dehydration enzyme H3C S H3C H2O 3-oxohexanoyl-enzyme O O HO CH H H 3 CH CH3 H3C condensation 3 HO O O O HO O O O O H C H3C 3 O H C O 3 O hexaketide chromophore monascin OO enzyme H3C S 3-oxooctanoyl-enzyme dehydration

H3C H C HS-enzym e 3 H3C O H CH3 O O O O H H O CH3 dehydration CH3 H C O O 3 O O O O O rubropunctatin H C 3 O H3C O ankaflavin

Figure 44 Through the condensation of two molecules compound prephytoene diphosphate (Figure 47) of geranylgeranyl diphosphate (electrophilic (IUBMB 2007). addition giving tertiary cation), catalysed by For the phytoene formation, a proton is lost from phytoene synthase (EC 2.5.1.32), phytoene C-15 generating a new double bond in the centre of (15-cis-7,8,11,12,7',8’,11',12'-octahydro-ψ,ψ-caro- the molecule, and thus a short conjugated chain is tene), the first carotene, is formed via a cyclopropyl developed. In fungi and higher plants, 15-cis-phy-

H3C

O O H H CH3 CH3 O O NH NH O O

H3C O H3C O

rubropunctamine monascorubramine

Figure 45

85 Vol. 26, No. 2: 73–98 Czech J. Food Sci.

COOH

O COOH HS-CoA O CoA S O OH + OH HO HO OH EC 2.3.1.133 HO OH 4-coumaroyl-CoA shikimic acid 4-coumaroyl 5-O -shikimic acid

malonyl-CoA NADPH + H +2 O 4-coumaroyl-CoA hydrolysis EC 1.14.13.36

NADP + H2O2O

3 HS-CoA + 2 CO2 C2O COOH

OO O

O OH OH HO OH HO OH bisdemethoxycurcumin (keto form) caffeoyl 5-O -shikimic acid

HS-CoA

EC 2.3.1.133

shikimic acid

O OH O CoA S

HO OH HO OH bisdemethoxycurcumin (enol form) caffeoyl-CoA

SAM oxidation methylation malonyl-CoA EC 2.1.1.104 4-coumaroyl-CoA hydrolysis SAH oxidation O OH methylation O OCH3 CoA S

HO OH HO

OCH3 demethoxycurcumin feruloyl-CoA

oxidation 3 HS-CoA + 2 CO2 methylation malonyl-CoA feruloyl-CoA hydrolysis O OH

H3CO OCH3

HO OH curcumin Figure 46

86 Czech J. Food Sci. Vol. 26, No. 2: 73–98

CH3

CH3 CH3 CH3 CH3

OPP geranylgeranyl diphosphate (allylic cation)

CH3 EC 2.5.1.32 PP OPP CH3 H H C H CH3 R 3 H OPP R CH3 CH prephytoene diphosphate R CH3 3 R

H3C PP CH 3 20 20´ 15 15´ H H3C CH3 geranylgeranyl diphosphate 13 13´ H3C H 19 19´ 11 14 14´ 11´ R H3C CH3 9 12 12´ 9´ CH3 18 7 10 10´ 7´ 18´ R H C CH 3 5 8 8´ 5´ 3 17 17´ 3 6 6´ 3´ H3C CH3 1 4 4´ 1´ 16 CH3 16´ 2 R = 2´ H C CH 3 phytoene 3 CH3 CH3 CH3

cis/trans-isomerisation

CH3 CH3 CH3 CH3

CH3 H3C

phytofluene CH3 CH3 CH3 CH3

dehydrogenation CH3 CH3 CH3 CH3

CH3 H3C ]-carotene CH3 CH3 CH3 CH3 AH2 + O2

EC 1.14.99.30

A + 2 CH3 CH3 CH3 CH3 H2O

CH3 H3C

neurosporene CH3 CH3 CH3 CH3

AH2 + O2

EC 1.14.99.30

A + 2 CH3 CH3 CH3 CH3 H2O

CH3 H3C

lycopene CH3 CH3 CH3 CH3

Figure 47

87 Vol. 26, No. 2: 73–98 Czech J. Food Sci. toene is generally found, the 15-trans-isomer to be involved in fungi and plants, probably at occurring in some bacteria. Their occurrence the phytoene level. As a result, plants accumulate depends on the stereochemistry of the hydrogen small amounts of the cis-carotenoids. removal from C-15 (Gross 1991). Then, through Acyclic carotenes, such as phytoene, phytoflu- the dehydrogenation sequence, the chromophore ene, ζ-carotene, and neurosporene, generally oc- is extended by two double bonds alternately from cur in small amounts. In nature, they accompany either side so that phytoene is converted into lyco- the major carotenes and xanthophylls, e.g. in the pene (thirteen double bonds, eleven of which are oil obtained from palm Elaeis guineensis, Are- conjugated) through stepwise dehydrogenation via caceae (Tai & Choo 2000). Lycopene, however, phytofluene, ζ-carotene, and neurosporene (caro- is the major red pigment of e.g. ripe tomatoes tene 7,8-desaturase, EC 1.14.99.30) (IUBMB 2007). (Solanum lycopersicum, syn. Lycopersicon escu- Since all the carotenes have all-trans configuration, lentum, Solanaceae). The majority of carotenes a cis/trans-isomerisation of the central double has (and xanthophylls) found in nature are cyclic24

H CCH H C CH 3 3 3 3 H

H i ii H CH3 CH2 iii \-carotene type H

i ii iii cyclisations

16 17

H3C CH3 H3C CH3 H3C CH3 7 2 1 6 8 18 3 5 CH 4 CH3 2 CH3 E-carotene type J-carotene type H-carotene type

hydroxylation hydroxylation hydroxylation enol/keto tautomerisation

H3C CH3 H H3C CH3 H3C CH3 H3C CH3 epoxidation O H HO CH3 HO CH3 CH3 HO CH3 O 3-hydroxy-5,6-epoxide type 3-hydroxy-E -carotene type 3-oxo-E -carotene type 3-hydroxy-D -carotene type

rearrangement epoxidation

7 16 O 8 17 H C H3C CH3 H3C CH3 H3C CH3 3 6 rearrangement H3C CH3 1 5 18 O OH 2 4 H 3 HO CH3 HO CH3 HO CH3 OH OH allene type 3-hydroxy-5,6-epoxide type 3,6-dihydroxy intermediate N-carotene type (cyclopentyl ketone) Figure 48

24Carotenoids with cyclic ends are integral constituents of plants, algae, and cyanobacteria photosynthetic reaction centers. Higher plant chloroplasts typically accumulate lutein, β-carotene, violaxanthin, and neoxanthin in the thylakoid membrane-bound photosystems. β-Carotene is generally found in the reaction center where it plays a critical photoprotective role by quenching triplet chlorophyll and singlet oxygen, and can undergo a rapid degradation during photooxidation. In the chromoplasts of ripening fruits, flower petals, and the chloroplasts of senescing leaves, carotenoids are found in membranes or in oil bodies or other structures within the stroma.

88 Czech J. Food Sci. Vol. 26, No. 2: 73–98 at their termini and loose, as a consequence, the this ring is formed at just one terminus, then the terminal double bond(s). The cyclisation of one or result is β,ψ-carotene (γ-carotene); if the ε-ring is both end groups of the linear all-trans-lycopene formed at just one terminus of the molecule, then (ψ-carotene type) into carotenes with β-rings ε,ψ-carotene (δ-carotene) forms. If at one terminus (β-carotene type), ε-rings (ε-carotene type) or the β-ring and at the second terminus the ε-ring are not very common γ-rings (γ-carotene type) is an formed, then the product is β,ε-carotene (known as important branch-point in the biosynthesis of α-carotene). Carotene with two ε-rings (ε-carotene) carotenoids. The cyclisation reaction are catalysed is usually found in trace amounts. by lycopene β-, ε- or γ-cyclases (EC 5.5.1.-) that 25 are not yet well characterised (Figure 48). The 9.2 Xanthophylls reaction sequences and the enzymes involved are schematically shown in Figure 49. Carotenes are oxygenated in various ways (hy- The best-known carotene produced from lycopene droxylation, epoxydation, etc.) as a late step in the (ψ,ψ-carotene) is β,β-carotene (known as β-caro- biosynthesis to form xanthophylls which repre- tene), the major pigment of carrot (Daucus carota, sent most of the carotenoid pigments in plants. Apiaceae), which has two β-rings (Figure 50). If The reaction mechanisms are given in Figure 48

D-zeacarotene

EC 5.5.1.- A + H2O AH2 + O2

H,H-carotene-3-ol H-carotene neurosporene E-zeacarotene EC 1.14.99.- EC 5.5.1.- A + H O 2 AH2 + O2 EC 1.14.99.- EC 5.5.1.- EC 1.14.99.30

AH2 + O2 A + H2O

lactucaxanthin G-carotene lycopene J-carotene E-carotene EC 5.5.1.- EC 5.5.1.- EC 5.5.1.- AH + O AH2 + O2 2 2 A + H O EC 5.5.1.- 2 EC 1.14.99.-

A + H2O NADP + H2O NADPH + H + O2 A + H2O AH2 + O2 EC 1.14.99.-

zeinoxanthin D-carotene canthaxanthin echinenone E-cryptoxanthin EC 1.14.99.- EC 1.14.99.-

AH + O AH2 + O2 NADPH + H + O2 AH2 + O2 2 2

EC 1.14.99.- EC 1.14.99.- EC 1.14.99.- EC 1.14.99.- A + H OAH + O A + H OAH + O 2 2 2 2 2 2 A + H2O NADP + H2O A + H2O A + H2O

astaxanthin adonirubin 3-hydroxy- lutein echinenone zeaxanthin EC 1.14.99.- EC 1.14.99.- L-dehydroascorbic acid NADPH + H + O2

EC 1.10.99.3 EC 1.14.13.90

L-ascorbic acid NADP + H2O L-ascorbic acid L-dehydroascorbic acid EC 1.10.99.3

neoxanthin violaxanthin antheraxanthin EC 5.3.99.9 NADP + H2O NADPH + H + O2 EC 5.3.99.8 EC 1.14.13.90 EC 5.3.99.8

capsorubin capsanthin

Figure 49

25Lycopene ε-cyclase (EC 5.5.1.-) also catalyses the formation of α-zeacarotene (one ε-ring) from neurosporene, while under the catalysis of lycopene β-cyclase (EC 5.5.1.-) β-zeacarotene (one β-ring) is formed (Figures 51 and 52). Both zeacarotenes occur e.g. in palm oil as the minor pigments (Tai & Choo 2000).

89 Vol. 26, No. 2: 73–98 Czech J. Food Sci.

H3C CH3 CH3 CH3

CH3

CH3 CH3 CH3 CH3 CH3 D-zeacarotene

CH CH H3C CH3 3 3

CH3

CH3 CH3 CH3 CH3 CH3 E-zeacarotene

H3C H3C CH3 CH3 CH3

CH3 CH3 H3C CH3 CH3 D-carotene

H3C H3C CH3 CH3 CH3

CH3 CH3 H3C CH3 CH3 E-carotene

H3C CH3 CH3 CH3

CH3

CH3 CH3 CH3 CH3 CH3 J-carotene

H3C CH3 CH3 CH3

CH3

CH3 CH3 CH3 CH3 CH3 G-carotene

H3C CH CH H3C CH3 3 3

H C CH CH3 CH3 3 3 CH3 H-carotene

Figure 50 and the sequences leading to the most prominent Zeinoxanthin (β,ε-carotene-3-ol), for example, is xanthophylls are schematically shown in Figure 49. formed from α-carotene by hydroxylation of the The enzymes envolved include NADPH-depend- β-ring under the action the NADPH-dependent ent β-ring hydroxylase (EC 1.14.99.-), NADPH-de- β-ring hydroxylase (EC 1.14.99.-). Lutein (β,ε-ca- pendent ε-ring hydroxylase (EC 1.14.99.-), β-ring rotene-3,3'-diol) is a product of zeinoxanthin carotene 3-hydroxylase using reduced substrates hydroxylation using NADPH-dependent ε-ring

AH2 (EC 1.14.99.-), β-ring carotenoid 4-ketolase hydroxylase (EC 1.14.99.-). β-Cryptoxanthin (EC 1.14.99.-) using reduced substrates AH2, vio- (β,β-carotene-3-ol) forms from β-carotene by laxanthin de-epoxidase (EC 1.10.99.3), zeaxanthin hydroxylation under the catalysis of β-carotene epoxidase (antheraxanthin epoxidase, EC 1.14.13.90), 3-hydroxylase (EC 1.14.99.). Zeaxanthin (β,β-caro- capsanthin-capsorubin synthase (EC 5.3.99.8), and ne- tene-3,3'-diol is a dihydroxyderivative of β-carotene oxanthin synthase (EC 5.3.99.9). Structures of the most formed from β-cryptoxanthin by the action of the important xanthophylls are given in Figure 51. same enzyme. Echinenone (β,β-carotene-4-one) Hydroxylation plays an important role in the is formed from β-carotene under the catalysis of oxygenation of carotenes and is catalysed by hy- β-ring carotenoid 4-ketolase (EC 1.14.99.-). droxylases acting specifically onto the individual Lettuce (Lactuca sativa, Asteraceae) is a rare rings (e.g. on β-rings or ε-rings etc.) (Figure 48). example of plants known to accumulate substan-

90 Czech J. Food Sci. Vol. 26, No. 2: 73–98

H3C H3C CH3 CH3 CH3 tial amounts of lactucaxanthin. Zeaxanthin is the yellow pigment of corn (Zea mays, Poaceae) and H C CH CH3 CH3 3 3 many other plant materials, astaxanthin (β,β-ca- HO CH3 zeinoxanthin

H3C OH rotene-3,3'-dihydroxy-4,4'-dione) or (3S,3S')-asta- CH CH H3C CH3 3 3 xanthin is commonly found in marine animals and is responsible for the pink to red coloration of CH CH H3C CH3 lutein 3 3 HO CH3 crustaceans, shellfish, and some fish (e.g. salmon). H3C CH CH H3C CH3 3 3 These animals are unable to synthesise carotenoids and astaxanthin is produced by the modification of

CH3 CH3 H3CCH3 HO CH3 Ecryptoxanthin β-carotene obtained from the diet (Velíšek 2002).

H3C OH Crustaceans (e.g. shrimps, prawns, and crabs) have CH CH H3C CH3 3 3 astaxanthin covalently bound to proteins (carotenoproteins) forming dark blue-red and green-red CH CH H3C CH3 zeaxanthin 3 3 HO CH3 pigments. Denaturation and hydrolysis of proteins H3C 26 CH CH H3C CH3 3 3 on cooking releases free astaxanthin. Antheraxanthin and violaxanthin are the most H C CH CH3 CH3 3 3 CH3 echinenone O important representatives of epoxides derived from O H3C β-carotene. The light-dependent interconversion of H C CH CH3 CH3 3 3 these xanthophylls is due to the violaxanthin cycle

CH3 CH3 H3C CH3 (xanthophyll cycle), which involves deepoxidation CH canthaxanthin 3 O O of violaxanthin in the light and reepoxidation of the H3C CH CH H3C CH3 3 3 zeaxanthin formed in the dark, via antheraxanthin. Violaxanthin further becomes the precursor of the H C CH CH3 CH3 3 3 HO CH3 adonirubin O allene xanthophyl neoxanthin. In photosynthetic O H3C OH tissues, carotenoids generally occur in their stable H C CH CH3 CH3 3 3 trans-configuration. For neoxanthin, however, it would appear that the 9’-cis isomer – 9'-cis-neo- CH3 CH3 H3C CH3 HO CH3 astaxanthin xanthin, is favored in photosynthesising tissues, O H3C OH CH CH H3C CH3 3 3 while trans-neoxanthin preferentially accumulates

O in non-photosynthetic plant tissues or plants grown H C CH antheraxanthin CH3 CH3 3 3 HO CH3 in the dark or under low light conditions.

H3C OH The biosynthesis of cyclopentyl ketones with the H3C CH3 CH3 CH3 O κ-ring (ketoxanthophylls) involves a pinacolic rear- O H C CH CH3 CH3 3 3 rangement of the 3-hydroxy-5,6-epoxy end-group. HO CH violaxanthin 3 Thus antheraxanthin is the precursor of capsanthin, H CH3 CH3 H3CCH3 H3C CH3 the major red pigment of the fruit of the genus O CH3 CH3 Capsicum (Solanaceae), both the sweet bell peper H C OH OH 3 27 HO neoxanthin OH CH3 (C. annuum) and the hot type chili (C. frutescens).

H C CH CH3 CH3 H3C 3 3 CH3 26 CH3 The crab Homarus gammarus contains, e.g. a blue O CH3 CH3 OH carotenoprotein α-crustacyanin. The protein (320 kDa) HO CH3 capsanthin composes of 16 subunits each of which contains one

CH3 CH3 H3C CH3 molecule of astaxanthin (Zagalaski 1994). O CH3 O 27 H3C The fruit of Capsicum plants have a variety of names H C CH3 CH3 CH3 3 capsorubin depending on the place and type. They are commonly called chili pepper, red or green pepper, or just pepper in OH H3C OH CH CH H3C CH3 3 3 Britain and the US; the large mild form is called bell pepper in the US, capsicum in Australian English and Indian CH3 CH3 H3C CH3 HO CH3 lactucaxanthin English, and paprika in some other countries (although paprika can also refer to the powdered spice made from Figure 51 various capsicum fruit).

91 Vol. 26, No. 2: 73–98 Czech J. Food Sci.

Violaxanthin is the precursor of the minor pigment some other carotenoids is vitamin A1 (all-trans-re- capsorubin and β-cryptoxanthin-5,6-epoxide be- tinol) (Velíšek & Cejpek 2007a). Among the im- comes the precursor of cryptocapsin. portant catabolic products of carotenoids, called diapocarotenoids, is the food colour annatto and 9.1.3 Apocarotenoids the coloring principal of the spice saffron. Annatto from the seeds of the tropical shrub Cleavage of carotenes and xanthophylls leads to Bixa orellana (Bixaceae) is used as an orange food various fragments clasified as apocarotenoids that colour28 for a variety of foods (margarines, cheeses, exert a number of biological functions. The best- desserts, breakfast cereals, liquers, etc.). It is also known apocarotene produced from β-carotene and still used in the cosmetic industry for the body care

CH3 CH3 CH3 CH3

CH3 H3C

lycopene CH3 CH3 CH3 CH3

2 O2

EC 1.13.12.- O CH3

2 CH3 CH3 6-methylhept-5-ene-2-one CH3 CH3 O O

bixin aldehyde CH3 CH3

NAD

EC 1.2.1.-

NADH + H CH3 CH3 OH O O

OH norbixin CH3 CH3

SAM

EC 2.1.1.-

SAH CH3 CH3 OCH3 O O

OH bixin CH3 CH3 SAM

EC 2.1.1.-

SAH CH3 CH3 OCH3 O O

H3CO bixin dimethyl ester CH3 CH3

Figure 52

28Processing is primarily done by abrading away bixin in a suspending agent (water, vegetable oil), although solvent processing is now also employed. Abrasion may be followed by aqueous alkaline hydrolysis with simultaneous production of all-trans-bixin, norbixin, and trans-norbixin. Annatto is usually marketed as an extract of the annatto seed, containing amounts of the active pigments that can vary from less than 1% to over 85% (Tennant & O’Callaghan 2005).

92 Czech J. Food Sci. Vol. 26, No. 2: 73–98 products. The pigments are found on the surface soluble apocarotenoid norbixin (also known as of the seeds where they accumulate in a resinous, 9’-cis-norbixin) and the oil-soluble apocarotenoid oily substance. This covering contains a variety monomethyl ester bixin (9’-cis-bixin) (Jako et al. of pigments including bixin dimethyl ester and 2002; BIOCYC 2007). a variety of apocarotenoids such as all-trans-bi- The biosynthesis of bixin (Bouvier et al. 2003a, xin called trans-bixin and all-trans-norbixin (free b; BIOCYC 2007) starts with all-trans-lycopene dicarboxylic acid); however, the two main con- which is split by lycopene cleavage dioxygenase stituents of industrial relevance are the water- (EC 1.13.12.-) to two apocarotenoids, methyl-(4-

H3C OH H3C CH3 CH3 CH3

H C CH CH3 CH3 3 3 HO CH3 zeaxanthin 2 O2 H C CH EC 1.14.99.- 3 3 O 2 HO CH 3 + 2 H2O A H2A hydroxy-E-cyclocitral CH3 CH3 CH3 CH3 OH O O O O EC 1.2.99.3 crocetin dialdehyde CH3 CH3 OH crocetin CH3 CH3 UDP-D-Glc OH OH EC 2.4.1.- OH O UDP UDP-D-Glc UDP CH CH O 3 3 CH3 CH3 OH HO O O HO O HO O CH CH EC 2.4.1.- O 3 3 O CH3 CH3 O O OH crocetin diglucosyl ester OH crocetin monoglucosyl ester HO HO UDP-D-Glc UDP-D-Glc OH OH OH OH EC 2.4.1.- EC 2.4.1.- OH UDP UDP-D-Glc UDP UDP O HO O CH3 CH3 HO HO OH O CH3 CH3 O O EC 2.4.1.- O O OH O CH3 CH3 O O O OH HO O CH CH crocetin gentiobiosylglucosyl ester O 3 3 OH OH HO O OH crocetin monogentiobiosyl ester HO OH OH HO OH UDP-D-Glc UDP-D-Glc OH OH OH EC 2.4.1.- EC 2.4.1.- OH OH UDP UDP O OH O O HO O CH3 CH3 O HO O O O OH CH CH O 3 3 HO O OH OH crocetin digentiobiosyl ester (crocin) HO OH

Figure 53

29Picrocrocin is largely responsible for the bitter taste of this spice, whereas safranal is the main constituent of its aroma. These two compounds are produced early in the degradation pathway of zeaxanthin and the volatile safranal is produced non-enzymatically by the action of heat. Traditional drying methods have been developed to generate saffron aroma (Carmona et al. 2006).

93 Vol. 26, No. 2: 73–98 Czech J. Food Sci. methylpent-3-en-1-yl)ketone (6-methylhept-5-ene- cosyl ester and crocetin diglucosyl ester (crocetin

2-one) and the residual C32 aldehyde called bixin 8,8'-glucosyltransferase, EC 2.4.1.-). The crocetin aldehyde (Figure 52). Bixin aldehyde dehydroge- monoglucosyl ester is transformed to crocetin nase (EC 1.2.1.-) oxidises bixin aldehyde to the monogentianobiosyl ester (glucosyltransferase, corresponding dicarboxylic acid norbixin, which EC 2.4.1.-) which is further glucosylated form- is methylated in two steps using SAM (norbixin ing crocetin gentiobiosylglucosyl ester (croce- methyltransferase, EC 2.1.1.-), first to bixin and tin monogentiobiosyl ester glucosyltransferase, finally to bixin dimethyl ester. EC 2.4.1.-). Crocetin diglucosyl ester gives the The colour as well as the bitter taste and fragrance same glycoside in a reaction catalysed by gluco- in stigmas of saffron (Crocus sativus, Iridaceae)29, syltransferase (EC 2.4.1.-). Final glucosylation of this extremely expensive spice, are due in large part crocetin gentiobiosylglucosyl ester yields crocin to the products of degradation of the xanthophyll (crocetin digentianobiosyl ester) (Côté et al. 2001; zeaxanthin. The colour is mainly due to a number Moraga et al. 2004). of apocarotenoid glycosides derived from crocetin. Crocetin is a water-insoluble aglycone, which, 9.2 Iridoids through sequential glucosylation, is converted to the soluble crocin (also known as α-crocin). Crocin Gardenia jasminoides (syn. G. augusta, Rubiace- also occurs in the fruit of Gardenia jasmonoides ae), known as common gardenia, cape jasmine or (Rubiaceae). cape jessamine, is a fragrant flower growing in In the biosynthetic pathway leading to croce- Southern China, Taiwan and Japan. Gardenia fruits tin (Figure 53), zeaxanthin is split by zeaxanthin are used within the traditional chinese medicine cleavage dioxygenase (EC 1.14.99.-) to hydroxy- and as a yellow dye for clothes and food (such as Korean mung bean hwangpomuk β-cyclocitral and two molecules of C20 dialdehyde the jelly called ). The (crocetin dialdehyde) which is oxidised by alde- fruits contain three major pigment types, iridoid hyde dehydrogenase (EC 1.2.99.3) to crocetin. pigments, crocins, and flavonoids that have been Crocetin is then converted to crocetin monoglu- used as food colorants.30

hydrolysis hydroxylation CH CH3 CH3 CH3 3 oxidation CH3 glucosylation OPP cyclisation O oxidation O H H OH O D-Glc H H H H O O O O O O O H3CCH3 H3C

geranyl diphosphate 8-epiiridodial (keto form) 8-epiiridotrial (keto form) 8-epiiridotrial (hemiacetal form) boschnaloside

oxidation

HO OH OH OH OH CH3 H H D-Glc H H H O D-Glc O O D-Glc O D-Glc O D-Glc H H H H H O O O O O O O O O O hydroxylation esterification oxidation oxidation OCH3 OCH3 OH gardenoside geniposide geniposidic acid tarennoside

Figure 54

30The production of food colorants from gardenia is being investigated at the present time. Most procedures involve extraction of the fruit with water, treatment with enzymes having β-glucosidase activity, and reaction with primary amines from either amino acids or proteins. Manipulation of the reaction conditions such as time, pH, temperature, oxygen content, etc. enables a series of colorants to be produced that vary from yellow to green, red, violet, and blue (Hendry& Houghton 1992).

94 Czech J. Food Sci. Vol. 26, No. 2: 73–98

O O CH3 CH3 OH HO H3C H3C HO OH H C CH 3 OH HO 3

HO CH3 H3C OH H3C CH3 OH CH3 CH3 OH HO OH O (+)-gossypol (-)-gossypol O

Figure 55

The major gardenia pigments, gardenoside and pigment occurring in immature flower buds geniposide, are biosynthesised from the iridodial and seeds of the cotton plant (e.g. Gossypium isomer (1R,2S,5R,8S)-iridodial known as 8-epi- hirsutum and other G. species, Malvaceae) that iridodial. The biosynthesis proceeds via 8-epiiri- functions for defense against pathogens and her- dotrial, and 8-epi-7-deoxyloganic acid and leads bivores. Cotton plant is important commercially to boschnaloside, tarennoside, geniposidic acid, for its soft fibre cotton that grows around the geniposide, and gardenoside (Figure 54). Alterna- seeds, for the cottonseed oil, and other products. tively, geniposidic acid may be also formed from Cottonseed oil is used in salad and cooking oils loganic acid and gardenoside may be produced from and, after hydrogenation, in shortenings and 7-deoxyloganic acid. Oxidation and dehydration margarine. The cake, or meal, remaining after of loganic acid leads to one of the minor gardenia the oil extraction is used in poultry and live- pigments called geniposidic acid (Sampaio-San- stock feeds. Gossypol occurs naturally in the tos & Kaplan 2001; Collu et al. 2001). plant in two forms referred to as (+)-gossypol and (–)-gossypol (Figure 55) (Dewick 2002) in 9.3 Miscellaneous terpenoids the ratio of 3:2 (Liu et al. 2005; Benedict et al. 2006). (–)-Gossypol can be toxic to nonruminant Gossypol or 2,2'-bis(8-formyl-1,6,7-trihydroxy- animals (when cottonseed serves as feed) and 5-isopropyl-3-methyl)naphthalene is a yellow even to human beings.31

CH3 OH CH3 OH CH3 O OH

H3C H3C H3C H3C OH H

H3CCH3 H3C CH3 H3C CH3 H3C CH3

G-cadinene 8-hydroxy-(+)- G -cadinene trans-8-hydroxy-calamenene desoxyhemigossypol

oxidation

H3CCH3 O O O OH O OH HO CH3 O H + e OH OH OH OH OH HO OH H3C OH H3C OH H3C OH O H3C OH H3CCH3 H3CCH3 H3CCH3 H3CCH3 gossypol resonance form of free radical free radical hemigossypol

Figure 56

31The toxic symptoms are associated with the (–)-isomer, which permeates cells and is active as a male contraceptive, altering sperm maturation, spermatozoid motility, and inactivation of sperm enzymes. In addition to its contraceptive properties, gossypol has also long been known to possess anti-malarial properties. Other researchers are investigating the anti-cancer properties of gossypol (Shidaifat et al. 1996; Coutinho 2002).

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Corresponding author: Prof. Ing. Jan Velíšek, DrSc., Vysoká škola chemicko-technologická v Praze, Fakulta potravinářské a biochemické technologie, Ústav chemie a analýzy potravin, Technická 5, 166 28 Praha 6, Česká republika tel.: + 420 220 443 177, fax: + 420 233 339 990, e-mail: [email protected]