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Chapter 2.

REVIEW OF LITERATURE

Review of literature

13

2.1. Mutual exclusiveness of betalains and anthocyanins

Caryophyllales, earlier known as Centrospermae, is a natural assemblage of families

having distinct morphological characteristics such as free-central (sometimes basal)

placentation, perisperm, and curved embryos (Bittrich, 1993; APG, 2009). Plants under

Caryophyllales have unique type (P3) of plastid in sieve element (Behnke, 1999). P3 type of

plastids exhibit a peripheral ring of filaments surrounding a globular or angular protein crystal

(Cronquist and Thorne, 1994). Chemosystematic marker studies have shown that all families

in Caryophyllales order, except Caryophyllaceae and Molluginaceae, produce betalain

pigments instead of anthocyanins (Waterman, 2007; Brockington et al., 2011). Anthocyanins

are present in almost all plants. Thus, these two hydrophilic pigment groups are mutually

exclusive. To date, there is no knowledge of any specific advantage of betalains biosynthesis

over anthocyanins in these plants. Based on poor monophyletic lineage among betalains

accumulating plants (Cuénoud et al., 2002), the most acceptable explanation of mutual

exclusiveness of these pigments may be that both the pigments coexisted in pre-historic plants

(Clement and Mabry, 1996). But, due to selective expression, anthocyanidin synthase (ANS)

activity was lost in plants of 16 families under the Caryophyllales order (Grotewold, 2006).

Although anthocyanin biosynthetic genes dihydroflavonol 4-reductase (DFR) and ANS are

present in betalains producing plants (Shimada et al., 2005; Shimada et al., 2007), their

promoters are different from those of anthocyanin-producing plants (Shimada et al., 2007).

Based on L-5,6-dihydroxyphenylalanine (DOPA) dioxygenase (DODA), a key enzyme in

betalains biosynthesis, homology analysis it was apparent that betalains producing plants and

non-betalains producing plants had differences in amino acid sequence at catalytic site

(Christinet et al., 2004). The DODA sequences in plants are completely different from that of

fungi, though they do the same catalytic function indicating evolutionary convergence. Mutual

exclusiveness of anthocyanins and betalains could be understood through reciprocal

experiments involving genes of betalain biosynthesis pathway enzymes to examine their

expression and activity in anthocyanin-pigmented taxa (Brockington et al., 2011). Further to

this, Harris et al. (2012) successfully induced betalains production in anthocyanin producing

cell culture of Solanum tuberosum and petals of Antirrhinum majus through introduction of

Portulaca grandiflora and A. muscaria DOPA dioxygenase (DODA) gene constructs and

feeding of L-DOPA. This indicated that components of betalains biosynthesis are active in

plants that accumulate anthocyanin.

Molecular phylogeny studies reported that Dilleniaceae and Santalales are closely

related to Caryophyllales (Hoot et al., 1999; Soltis et al., 1999; Soltis et al., 2000). Recently,

pigment analysis confirmed that Santalum album (Sri Harsha et al., 2013), a member of

Santalaceae, accumulates anthocyanins. This calls for inclusion of more families under

Caryophylalles order (Cuénoud et al., 2002). In support of this, certain anthocyanin producing


14

taxa, Molluginaceae (e.g., Limeum) have been predicted to accumulate both anthocyanins and

betalains (Cuénoud et al., 2002), however so far there is no biochemical evidence (Clement et

al., 1994). In line with molecular phylogeny findings of Caryophyllales, three more families

may be included under core Caryophyllales (Fig. 2.1), members of which accumulate betalains

instead of anthocyanins.

Figure 2.1. Mutually exclusive families accumulating betalain and anthocyanin pigments.

Modified from Brockington et al., 2011. *Newly elevated to family based on

recommendations by Cuenoud et al., 2002.

2.2. Occurrence

Betalains accumulate in leaf, stem, root, fruit, inflorescence/flower, petiole, bract and

seed grains. A list of betalains-containing plants and their parts is provided in Table 2.1. Till

date, pigment profile of about 20 genera has been investigated. As a result, about 100 betalains

have been structurally characterised including few pigments from fungal sources. There are

many more genera to be explored for their pigment content. Only few betalains sources such

as red beet, dragon fruit, cactus pear, and amaranth have been extensively studied with respect

to pigment extraction and/or purification for food uses, and biological activities of the extracts

(Stintzing and Carle, 2004).

Bladder cells of epidermal layer in leaves of Mesembryantheum chrystalinum

accumulate betalains (Ibdah, 2002). In cultured red beet cells (Schliemann et al., 1999),

betacyanins and betaxanthins accumulate in different cell layers. In cactus stem, betacyanins

accumulate in outer layers of chlorenchyma (Mosco, 2012). The hydrophilic nature of


15

Table 2.1. Betalains content of various plants.

Family/

total no. of

genera#

Species Common name/

pigmented part

Betalains

content

(mg/100 g fw)

Reference

Chenopodiaceae Beta vulgaris Red beet/ root 40–200 Stintzing and

Carle, 2007

B. vulgaris L. ssp.

cicla [L.]

Alef.

Swiss Chard/

Petiole

4–8 Stintzing and

Carle, 2008

Chenopodium

rubrum

Red goosefoot/

flower

NA NA

Chenopodium

formosanum

Djulis/seed

grain

94 Tsai et al.,

2010

Chenopodium

quinoa

Quinoa/grain NA NA

Suaeda salsa Seepweed/leaf 250 Wang et al.,

2006

Cactaceae/ 98 Hylocereus

polyrhizus

Pitaya/fruit 31–41 Vaillant et al.,

2005; Shea,

2012

H. purpusii, H.

costaricensis,

H. undatus, H. sp.

(hybrids)

Red flesh fruit

species

Wybraniec and

Mizrahi, 2005

Opuntia ficus-india Cactus pear/fruit 17–860* Stintzing and

Carle, 2008

Myrtillocactus

geometrizans

Garambullo

tree/fruit

214* Reynoso et al.,

1997

Opuntia matudae Xoconostle/fruit 10.6–20.2* Guzmán-

Maldonado et

al., 2010

Mammillaria sp. /fruit 1.6–18.1 Wybraniec and

Nowak-

Wydra, 2007

Schlumbergera sp. Christmas

cactus/flower

163 Kobayashi et

al., 2000


16

Selenicereus

megalanthus

Yellow

Pitaya/fruit

NA Kugler et al.,

2006

Amaranthaceae/

166

Amarathus sp. Amaranth/seed/

flower

46–199 Cai et al.,

2005

Celosia sp.

Iresine sp.

Common

cockscomb/

Inflorescence

92.1–621.5 Cai et al.,

2005

Gomphrena

globosa

Inflorescence 7.6–55.7 Kugler et al.,

2007

Portulacaceae/31 Portulaca

grandiflora

Moss

rose/petal/stem

12.5–17.8* Trezzini and

Zrÿd, 1991a

Aizoaceae/127 Mesembryanthemu-

m crystallinum

Ice plant/flower,

leaf

3.2 Vogt et al.,

1999

Dorotheanthus

bellidiformis

Livingstone

daisy/flower

NA NA

Lampranthus

productus

Ice plant/flower 66−417 Kugler et al.,

2007; Gandía-

Herrero et al.,

2005

Glottiphylum

oligocarpum

/flower 135 Gandía-

Herrero et al.,

2005

Glottiphylum

pigmaeum

/flower 138 Gandía-

Herrero et al.,

2005

Nyctaginaceae/

31

Bougainvillea sp. Bougainvillea/

bract

259.4–551.8* Kugler et al.,

2006

Mirabilis jalapa Four o’clocks/

flower

6.5 Piattelli et al.,

1965c

Boerhavia erecta Erect spiderling/

bark

185.5 Stintzing et

al., 2004

Basellaceae/4 Basella alba Malabar

spinach/fruit

36.1 Lin et al.,

2010

Basella rubra Spinach

vine/fruit

NA Glässgen et

al., 1993

Ullucus tuberosus Ulluco/tuber 7 Svenson et al.,


17

2008

Phytolaccaceae/

15

Rivina humilis Pigeonberry/

fruit

350 (1700*) Khan et al.,

2012

Phytolacca

decandra

Pokeweed/fruit 1400* Forni et al.,

1983

Phytolacca

americana

Pokeberry/ fruit 0.18 Neamtu et al.,

1979

*Dry weight basis. NA- not available. List updated from Refs. Moreno et al., 2008; Pavokovic

and Krsnik-Rasol, 2011. Total number of genera has been adapted (Bittrich, 1993; Cuenoud et

al., 2002; Mabberley, 1997).

betalains and their stability in slightly acidic pH implies that betalains are localised in

vacuoles. Few studies involving tyrosine feeding suggested that synthesis of betalains takes

place in cytoplasm and probably the pigments are imported to vacuoles for storage. This claim

has been proved following detection of cytoplasmic DODA (Christinet et al., 2004), and

cytochrome P450 that produces cyclo-DOPA required for betalains biosynthesis (Hatlestad et

al., 2012).

In the last one and half decades, some new sources of betalains have been reported viz.,

Rivina humilis (Khan et al., 2012), Basella alba (Lin et al., 2010), Chenopodium formosanum

(Tsai et al., 2010), Suaeda salsa (Wang et al., 2006), and Ullucus tuberosus (Svenson et al.,

2008).

2.3. Structure identification

The most important difference between the sub-categories of betalains viz. betacyanins

and betaxanthins is their spectral characteristics owing to their chemical structures. In the early

years (till late 1970s) of structure elucidation of betalains, electrophoretic migration,

chromatography (paper, TLC, gel) spectral characteristics such as infrared spectrum,

absorption in visible range (max) were mainly used. Synthesis, enzymatic hydrolysis,

functional group tests and H-NMR spectroscopy provided the confirmation of the structures.

Of late, advanced analytical instruments such as GC, HPLC, LC-MS, 2-D 13

C-NMR are

routinely employed for structure elucidation of betalains (Strack et al., 2003; Stintzing et al.,

2006; Wybraniec and Nowak-Wydra, 2007; Wybraniec et al., 2007; Nemzer et al., 2011).

Betalamic acid had been identified as naturally occurring core structure of betalains

(Kimler et al., 1971). It has a chiral center at C-15 (Fig. 1.3). The oxygen is replaced by

nitrogen in an aldimine bond with cyclo-DOPA to form betanidin. C-5/6 of betanidin is

glucosylated (β-14 linkage) to form various groups of betacyanins. These betacyanins show

variations in the position of their sugar moiety (e.g., 5-O-β-D-Glucose, 6-O-β-D-Glucose) and


18

acyl groups (e.g., feruloyl, p-coumaric acid) that form ester bond with the sugar at many

positions (e.g., 6-O, 2-O). There are betacyanins with disaccharide and trisaccharide

substitutions (Piattelli and Imperato, 1970b; Imperato, 1975b). This shows that betacyanins are

structurally complex set of pigments. To further complicate it, 5-O and 6-O substitutions have

been observed to produce marked difference in chromatic strength and also max (Heuer et al.,

1992). To simplify, based on their side chains, betacyanins have been classified into various

groups such as betanin, amaranthin, bougainvillein and gomphrenin. Betalamic acid shows

aldimine conjugation with amines (e.g., glutamine) and amino acids (e.g., tyrosine). Table 2.2

presents a comprehensive list of betalains structurally unambiguously identified till date.

Table 2.2. Comprehensive list of betalains identified till date.

Betacyanin Residue (attached to

betalamic acid)

Source Reference

Aglycone

Betanidin cyclo-DOPA Beta vulgaris L. Wyler and

Dreiding, 1959;

Wilcox et al.,

1965

Isobetanidin cyclo-DOPA Beta vulgaris L. Wyler and

Dreiding, 1959;

Wilcox et al.,

1965

2-Descarboxy betanidin

Decarboxylated cyclo-DOPA

Carpobrotus acinaciformis L.

Piattelli and Impellizzeri, 1970

Glycone

(Betanin group)

Betanin cyclo-DOPA-5-O-Glc Beta vulgaris L. Wyler and Dreiding, 1957

Isobetanin cyclo-DOPA-5-O-Glc Beta vulgaris L. Wyler and

Dreiding, 1957

Phyllocactin cyclo-DOPA-5-O(6-O-malonyl)-Glc

Phyllocactus hybridus Piattelli and Minale, 1964

Isophyllocactin cyclo-DOPA-5-O(6-O-malonyl)-Glc

Phyllocactus hybridus Piattelli and Minale, 1964

Lampranthin I cyclo-DOPA-5-O[6-O-(E)-p-coumaroyl]-Glc

Lampranthus sociorum Piattelli and Impellizzeri, 1969

Isolampranthin I cyclo-DOPA-5-O[6-O-(E)-p-coumaroyl]-Glc

Lampranthus sociorum Piattelli and Impellizzeri, 1969

Lampranthin II cyclo-DOPA-5-O[6-O-

(E)-feruloyl]-Glc

Lampranthus sociorum Piattelli and

Impellizzeri, 1969

Isolampranthin II cyclo-DOPA-5-O(6-O-

(E)-feruloyl)-Glc

Lampranthus sociorum Piattelli and

Impellizzeri, 1969


19

Table 2.2. continued...

Rivinianin cyclo-DOPA-5-O(3-sulphate)-Glc

Rivina humilis L. Imperato,

1975a

Neobetanin* cyclo-DOPA-5-O-Glc Beta vulgaris subsp. vulgaris var. conditiva

Alard et al., 1985

Betanidin 5-O(5-

O-E-feruloyl-2-O-

apiosyl)-glc

cyclo-DOPA-5-O(5-O-

E-feruloyl-2-O-

apiosyl)-Glc

Phytolacca americana

L.

Schliemann

et al., 1996

Betanidin 5-O(5-

O-E-feruloyl-2-O-apiosyl)-glc

cyclo-DOPA-5-O(5-O-

E-feruloyl-2-O-apiosyl)-Glc


L.

Schliemann

et al., 1996

Prebetanin cyclo-DOPA-5-O(6-O-

sulphate)-Glc


L.

Schliemann

et al., 1996

Isoprebetanin cyclo-DOPA-5-O(6-O-

sulphate)-Glc


L.

Schliemann

et al., 1996

2-Apiosyl phyllocactin

cyclo-DOPA-5-O(2-O-

apiosyl-6-O-malonyl)-

Glc

Schlumbergerabuckleyi Kobayashi et

al., 2000

2-Apiosyl

isophyllocactin

cyclo-DOPA-5-O(2-O-

apiosyl-6-O-malonyl)-Glc


al., 2000

Betanidin 5-O[2-

O(5-O-E- feruloyl)-apiosyl-

6-O-malonyl)]-glc

cyclo-DOPA-5-O[2-

O(5-O-E-feruloyl)-

apiosyl-6-O-malonyl)]-Glc


al., 2000

2-Descarboxy betanin

cyclo-DOPA-5-O-Glc Beta vulgaris L. Kobayashi et al., 2001

6-Malonyl-2-descarboxy betanin

2-descarboxy-cyclo-

DOPA-5-O(6-malonyl)-Glc

Beta vulgaris L. Kobayashi et

al., 2001

Hylocerenin cyclo-DOPA-5-O[6-

O(3-hydroxy-3-methylglutaryl)]-Glc

Hylocereus polyrhizus Wybraniec et

al., 2001

Isohylocerenin cyclo-DOPA-5-O[6-

O(3-hydroxy-3-methylglutaryl)]-Glc

Hylocereus polyrhizus Wybraniec et

al., 2001

Mammillarinin cyclo-DOPA-5-O(6-O-malonyl)-β-sophoroside

Mammillaria sp. Wybraniec

and Nowak-Wydra, 2007

Glycone

(Amaranthin

group)

Iresinin I cyclo-DOPA-5-O[6-

O(3-hydroxy-3- methylglutaric acid)]-

Glc

Iresine herbstii Minale et al.,

1966

Suaedin cyclo-DOPA-5-O[2-O(citryl)-GlcU]-Glc

Suaeda fruticosa Forsk. Piattelli and Imperato,

1971

Celosianin I cyclo-DOPA-5-O[2-O(p-coumaroyl)-GlcU]-

Glc

Celosia cristata Steglich and

Strack, 1990


20


Celosianin II cyclo-DOPA-5-O[2-O(feruloyl)-GlcU]-Glc

Celosia cristata Steglich and

Strack, 1990

Amaranthin cyclo-DOPA-5-O(2-O-GlcU)-Glc

Amaranthus cruentus Strack et al., 1993

Isoamaranthin cyclo-DOPA-5-O(2-O-GlcU)-Glc

Amaranthus cruentus Cai et al., 2005

Isocelosianin I cyclo-DOPA-5-O[2-O(p-coumaroyl)-GlcU]-

Glc

Celosia cristata Cai et al., 2005

Isocelosianin II cyclo-DOPA-5-O[2-O(feruloyl)-GlcU]-Glc

Celosia cristata Cai et al.,

2005

Isoiresinin I cyclo-DOPA-5-O[6-O(3-hydroxy-3-

methylglutaric acid)]-

Glc

Iresine herbstii Cai et al.,

2005

Glycone

(Bougainvillein)

Bougainvillein-r I cyclo-DOPA-5-O-

sophoroside

Bougainvillea glabra

var. “Mrs. Butt”

Piattelli and

Imperato,

1970a

Isobougainvillein-r I cyclo-DOPA-5-O-

sophoroside



Piattelli and

Imperato,

1970a

Bougainvillein-r II cyclo-DOPA-5-O(p-

coumaroyl)-sophoroside



Piattelli and

Imperato,

1970a

Isobougainvillein-r

II

cyclo-DOPA-5-O-

sophoroside



Piattelli and

Imperato,

1970a

Bougainvillein-v cyclo-DOPA-6-O-

sophoroside


var. sanderiana

Piattelli and

Imperato,

1970b

Isobougainvillein-v cyclo-DOPA-6-O-

sophoroside


var. sanderiana

Piattelli and

Imperato,

1970b

Betanidin 6-O(2-

glucosylrutinoside)

cyclo-DOPA-6-O(2-

glucosyl-6-O-

rhamnose)-Glc

Bougainvillea glabra Imperato,

1975b


21


Isobetanidin 6-O(2-

glucosylrutinoside)

cyclo-DOPA-6-O(2-

glucosyl-6-O-

rhamnose)-Glc

Bougainvillea glabra Imperato,

1975b

Betanidin 6-O(6-O-

E-caffeoyl)-

sophoroside

cyclo-DOPA-6-O(6-O-

E-caffeoyl)-sophoroside

Bougainvillea glabra Heuer et al.,

1994

Betanidin 6-O(6-O-

E-p-coumaroyl)-

sophoroside

cyclo-DOPA-6-O(6-O-

E-p-coumaroyl)-

sophoroside


1994

Betanidin 6-O(6-O-

E-p-coumaroyl)-

sophoroside

cyclo-DOPA-6-O(6-O-

E-p-coumaroyl)-

sophoroside


1994

Betanidin 6-O{2-O-

sophorosyl[(6-O-E-

caffeoyl)-(6-O-E-p-

coumaroyl)]}-

sophoroside

cyclo-DOPA-6-O{2-O-

sophorosyl[(6-O-E-

caffeoyl)-( 6-O-E-p-

coumaroyl)]}-

sophoroside


1994

Betanidin 6-O{2-O-

glucosyl[(6-O-E-

caffeoyl)-(6-O-E-p-

coumaroyl)]}-

sophoroside

cyclo-DOPA-6-O{2-O-

glucosyl[(6-O-E-

caffeoyl)-( 6-O-E-p-

coumaroyl)]}-

sophoroside


1994

Betanidin 6-O[(2-O-

glucosyl)(6,6-di-O-

E-p-coumaroyl)]-

sophoroside

cyclo-DOPA-6-O[(2-O-

glucosyl)(6,6-di-O-E-

p-coumaroyl)]-

sophoroside


1994

Betanidin 6-O(6,6-

di-O-E-p-

coumaroyl)]-

sophoroside

cyclo-DOPA-6-O(6,6-

di-O-E-p-coumaroyl)]-

sophoroside


1994


22


Glycone

(Gomphrenin

group)

Gomphrenin I cyclo-DOPA-6-O-Glc Gomphrena globosa

L.

Minale et al.,

1967

Isogomphrenin I cyclo-DOPA-6-O-Glc Gomphrena globosa

L.

Minale et al.,

1967

Gomphrenin II cyclo-DOPA-6-O(6-O-

E-4-coumaroyl)-Glc

Gomphrena globosa

L.

Heuer et al.,

1992

Isogomphrenin II cyclo-DOPA-6-O(6-O-

E-4-coumaroyl)-Glc

Gomphrena globosa

L.

Heuer et al.,

1992

Gomphrenin III cyclo-DOPA-6-O(6-O-

E-feruloyl)-Glc

Gomphrena globosa

L.

Heuer et al.,

1992

Isogomphrenin III cyclo-DOPA-6-O(6-O-

E-feruloyl)-Glc

Gomphrena globosa

L.

Heuer et al.,

1992

Sinapoyl-

gomphrenin I#

NA Gomphrena globosa

L.

Kugler et al.,

2007

Sinapoyl-

isogomphrenin I#

NA Gomphrena globosa

L.

Kugler et al.,

2007

Betacyanin-like

fungal pigments

Muscapurpurin cyclo-Stizolobic acid Amanita muscaria Musso, 1979

Muscarubrin Pyrroline 2-carboxylic

acid

Amanita muscaria Stintzing and

Schliemann,

2007

Plant based

betaxanthins

Betalamic acid NA Beta vulgaris L. Wyler et al.,

1963

Indicaxanthin Proline Opuntia ficus indica

L.

Piattelli et al.,

1964

Portulacaxanthin I Hydroxyproline Portulaca

grandiflora

Piattelli et al.,

1965b

Vulgaxanthin I Glutamine Beta vulgaris L. Piattelli et al.,

1965a


23


Vulgaxanthin II Glutamic acid Beta vulgaris L. Piattelli et al.,

1965a

Miraxanthin I Methionine sulphoxide Mirabilis jalapa L. Piattelli et al.,

1965c

Miraxanthin II Aspartic acid Mirabilis jalapa L. Piattelli et al.,

1965c

Miraxanthin III Tyramine Mirabilis jalapa L. Piattelli et al.,

1965c

Miraxanthin IV not characterised Mirabilis jalapa L. Piattelli et al.,

1965c

Miraxanthin V Dopamine Mirabilis jalapa L. Piattelli et al.,

1965c

Miraxanthin VI not characterised Mirabilis jalapa L. Piattelli et al.,

1965c

Dopaxanthin II DOPA Glottiphyllum

longum (Haw.)

N.E.Br.

Impellizzeri et

al., 1973

Vulgaxanthin IV Leucine Beta vulgaris L. Piattelli, 1976

Humilixanthin 5-hydroxynorvaline Rivina humilis L. Strack et al.,

1987

Portulacaxanthin II Tyrosine Portulaca

grandiflora

Trezzini and

Zryd, 1991a

Portulacaxanthin III Glycine Portulaca

grandiflora

Trezzini and

Zryd, 1991a

Vulgaxanthin III Asparagine Beta vulgaris L. Trezzini and

Zryd, 1991b

Valine-Bx Valine Beta vulgaris L.

subsp. Cicla (L.)

Alef. Cv. Bright

Lights

Kugler et al.,

2004

Threonine-Bx Threonine Beta vulgaris L.

subsp. cicla (L.)

Alef. Cv. Bright

Lights

Kugler et al.,

2004


24


Ethanolamine-Bx Ethanolamine Beta vulgaris L.

subsp. cicla (L.)

Alef. Cv. Bright

Lights

Kugler et al.,

2004

Serine-Bx Serine Beta vulgaris L.

subsp. cicla (L.)

Alef. Cv. Bright

Lights

Kugler et al.,

2004

Phenylalanine-Bx Phenylalanine Beta vulgaris L.

subsp. cicla (L.)

Alef. Cv. Bright

Lights

Kugler et al.,

2004

-Aminobutyric

acid-Bx

-Aminobutyric acid Beta vulgaris L.

subsp. cicla (L.)

Alef. Cv. Bright

Lights

Kugler et al.,

2004

Isoleucine-Bx Isoleucine Beta vulgaris L.

subsp. cicla (L.)

Alef. Cv. Bright

Lights

Kugler et al.,

2004

Alanine-Bx Alanine Beta vulgaris L.

subsp. cicla (L.)

Alef. Cv. Bright

Lights

Kugler et al.,

2004

3-methoxytyramine-

Bx

3-methoxytyramine Celosia

cristata/plumosa

Cai et al.,

2005

Tryptophan-Bx Tryptophan Celosia

cristata/plumosa

Cai et al.,

2005

Methionine-Bx Methionine Opuntia sp. Stintzing et

al., 2005

Dopaxanthin I DOPA Bougainvillea sp. Kugler et al.,

2007

Arginine-Bx Arginine Gomphrena globosa Kugler et al.,

2007


25


NA- not available.

*Neobetanin is yellow pigment showing spectral characteristics similar to betaxanthins, but

does not have C15 chiral centre unlike all other betanins or isobetanins. NA- not applicable,

Lysine-Bx Lysine Gomphrena globosa Kugler et al.,

2007

Putrescine-Bx Putrescine Bougainvillea sp. Kugler et al.,

2007

Proline isomer-Bx Proline isomer Opuntia sp. Castellanos-

Santiago and

Yahia, 2008

Valine isomer-Bx Valine isomer Opuntia sp. Castellanos-

Santiago and

Yahia, 2008

Phenethylamine-Bx Phenethylamine Opuntia sp. Castellanos-

Santiago and

Yahia, 2008

Methylated arginine-

Bx

Methylated arginine Amaranthus tricolor

L.

Biswas et al.,

2012

Fungal

betaxanthins

Muscaflavin§ NA Hygrocybe sp. von Ardenne

et al., 1974

Muscaaurin I Ibotenic acid Amanita muscaria Musso, 1979

Muscaaurin II Stizolobic acid Amanita muscaria Musso, 1979

Muscaaurin III Mixture of vulgaxanthin

I, miraxanthin III, 2-

aminoadipic acid-Bx

Amanita muscaria Musso, 1979

Muscaaurin IV Mixture of vulgaxanthin

I, miraxanthin III


Muscaaurin V Mixture of vulgaxanthin

II, vulgaxanthin IV,

indicaxanthin, valine-Bx


Muscaaurin VI Mixture of vulgaxanthin

II, indicaxanthin,


Muscaaurin VII Histidine Amanita muscaria Musso, 1979


26

Glc- β-D-glucose, GlcU- β-D-glucoronic acid, DOPA- 3,4-dihydroxyphenylalanine, (E)- trans,

sophoroside- (2-O-β-glucosyl)-β-glucoside,

#Tentatively identified compounds based on absorbance and mass spectra. Glc- β-D-glucose,

GlcU- β-D-glucoronic acid, DOPA- 3,4-dihydroxyphenylalanine, (E)- trans, sophoroside- (2-

O-β-D-glucosyl)-β-D-glucoside, rutinoside- glucose 6-O-rhamnose

§Betalamic acid-like compound that can form aldimine bond with amino acids to produce

yellow pigments called as hygroaurins, which are similar to betaxanthins.

2.4. Biosynthesis of betalains

2.4.1. Biosynthesis pathway

Betalains biosynthesis pathway was elucidated by Impellizzeri and Piattelli (1972) and

updated by Strack et al. (2003). More recently, Gandia-Herrero and Garcia-Carmona (2013)

have shed light on the advances in betalains biosynthesis in the last decade. Betalains are

synthesised from tyrosine. It starts with massive accumulation of tyrosine (Kishima et al.,

1991), which is hydroxylated by tyrosine hydroxylase activity of plant tyrosinase, producing

DOPA. Till recently, it was believed that diphenol/DOPA oxidase (DO) activity of tyrosinase

catalyse conversion of DOPA (o-diphenol) to o-quinone, which forms cyclo-DOPA through

molecular rearrangements. However, a recent report pointed towards involvement of a

cytochrome P450, CYP76AD1, in formation of cyclo-DOPA (Hatlestad et al., 2012). The next

step involves aromatic ring cleavage of DOPA catalysed by DODA, followed by molecular

rearrangement to form betalamic acid, chromophore of all betalains. DODA was first

identified in the Amanita muscaria (Hinz et al., 1997). The plant enzyme DODA was cloned

from Portulaca grandiflora (Christinet et al., 2004). This enzyme showed no obvious

sequence or structural similarity with that of A. muscaria. Plant DODA displayed regiospecific

extradiol 4,5-dioxygenase (Christinet et al., 2004), different from the 2,3- and 4,5 dioxygenase

activity of the A. muscaria (Hinz et al., 1997). DOPA level before and during betalains

biosynthesis in Portulaca petals had been found to be moderate (Kishima et al., 1991).

However, in the currently accepted biosynthesis pathway of betalains elucidated by

Impellizzeri and Piattelli (1972) and updated by Strack et al. (2003), Grotewold (2006),

Pavokovic and Krsnik-Rasol (2011), DOPA is required in the following unconnected but

essential steps (Fig. 2.2), 1) DODA acts on DOPA and gives rise to 4,5-seco-DOPA which is

non-enzymatically rearranged to give betalamic acid, the core structure of betalains, 2)

CYP76AD1, a cytochrome P450, converts DOPA to dopaquinone which is cyclised to cyclo-

DOPA, 3) DOPA decarboxylase acts on DOPA to form dopamine, which can give rise to

betacyanins, 4) DOPA condenses with betalamic acid to form dopaxanthin, a betaxanthin.

Betacyanins are red-violet pigments comprising sub-groups such as betanins, amaranthins,


27

gomphrenins, 2-descarboxy-betanins. The betalamic acid condenses with cyclo-DOPA to form

betanidin, which is glucosylated by betanidin-5-O-glucosyltransferase (BGT) to produce

betanin. More recently, it has been proposed that the origin of betanin is cyclo-DOPA, which

is glucosylated by UDP-glucose:cyclo-DOPA-5-O-glucosyltransferase (cDGT), followed by

condensation with betalamic acid (Sasaki et al., 2005). It appears that in both cases, cyclo-

DOPA is provided by CYP76AD1 (Hatlestad et al., 2012). Betanin may be further acylated in

some plants to form lampranthins in the presence of hydroxycinnamoyltransferases (Strack et

al., 2003). In the presence of betanidin-6-O-glucosyltransferase, betanidin gets glucosylated to

form gomphrenins (Strack et al., 2003). Dopamine is oxidised by DO and cyclised to 2-

descarboxy-cyclo-DOPA, which enters into aldimine formation reaction with betalamic acid

to form 2-descarboxy-betanidin.

Betalamic acid condenses with an amino acid (e.g., Ser, Val, Leu, Iso, and Phe) or amino acid

derivative (e.g., 3-methoxytyramine) to form the yellow to orange betaxanthins. In the whole

biosynthesis scheme, tyrosinase and DODA are key enzymes. Gandia-Herrero et al. (2005)

proposed another pathway, which considered the fact that cellular reducing agent ascorbic acid

reduces o-quinone to o-diphenols i.e., DOPA. It appears that in presence of ascorbic acid,

tyrosinase (EC 1.14.18.1) uses betaxanthins as substrates as shown in Fig. 2.3. Thus, DOPA

remains available for DODA to produce betalamic acid. Considering all the evidences

reported so far, betanidin could be formed in two different ways, in the absence of ascorbic

acid (Fig. 2.2), and presence of ascorbic acid (Gandia-Herrero et al., 2005) (Fig. 2.3). The

recent report of CYP76AD1 involved in cyclo-DOPA formation (Hatlestad et al., 2012)

supports the above claim that tyrosinase does not use L-DOPA to form cyclo-DOPA.

2.4.2. Tyrosinase

Tyrosinase is binucleated copper containing PPO enzyme with dual functions of

monophenolase activity (EC 1.14.18.1) and diphenol oxidase (DO, EC 1.10.3.1). Plant

tyrosinase has been reported to play catalytic role in biosynthesis of pigments such as

betalains, aurones, and nordihydroguaiaretic acid (Strack and Schliemann, 2001). The enzyme

involved in betalains biosynthesis was first isolated and characterised from Portulaca

grandiflora (Steiner et al., 1999). The enzyme was found to exhibit dual functions which the

researchers could not separate from each other. However, Yamamoto et al (2001) isolated and

purified tyrosine hydroxylase (monophenolase activity) (TOH) independent of DO activity

from P. grandiflora. It was characterised as coenzyme (pterin compounds) dependent enzyme.

This report pointed to the possibility that, indeed, TOH (EC 1.14.18.1) and DO (EC 1.10.3.1)

could be separated and have independent mechanisms of expression/regulation. Concurring

with this assumption, a recent report described a new cytochrome P450 that produces cyclo-

DOPA (Hatlestad et al., 2012). This implies that DO activity is not involved in formation of

cyclo-DOPA.


28

Figure 2.2. Schematic diagram showing pathways through which DOPA is consumed to

accomplish betalains biosynthesis. Revised from Gandia-Herrero and Garcia-Carmona (2013).

2.4.3. Regulation of betalains biosynthesis

The gene coding DODA has been cloned from P. grandiflora (Christinet et al., 2004),

M. jalapa (Sasaki et al., 2009) and beetroot (Gandía-Herrero and García-Carmona, 2012).

From all these studies, it is known that DODA is a cytoplasmic protein (monomer, 30-32

kDa). Transcriptional regulation of DODA was evident in A. muscaria (Hinz et al., 1997), and

Phytolacca americana (Takahashi et al., 2009). The promoter region of DODA in P.

Americana contained regulatory genes MYB and bHLH responsive elements.


29

Figure 2.3. Schematic diagram showing role of tyrosinase and fusion of the pathways of

biosynthesis of betacyanins and betaxanthins (Gandia-Herrero et al., 2005).

MYB, basic helix–loop–helix (bHLH) and WD40 repeats (WDRs) are transcription factors

(TFs) that regulate flavonoid biosynthesis (Hichri et al., 2011). MYB-type transcription

factors have been also reported to involve in betalains biosynthesis in red beet (Alan et al.,

2011). Involvement of similar type of TFs (say, MYB) in regulation of anthocyanins and

betalains biosynthesis may give us important information on mutual exclusiveness of

anthocyanins and betalains, mainly because, promoter domains for anthocyanins biosynthetic

genes are present in betalains producing plants (Shimada et al., 2007).

Environmental factors such as sunlight, UV light, low red:far red light ratio, salt and

abiotic stress promote betalains accumulation, whereas blue light and phenyl ammonia lyase

(PAL) inhibitor 2-aminoindan 2-phosphonic acid (AIP) may inhibit the biosynthesis (Vogt et

al., 1999). Suaeda salsa seedlings showed enhanced accumulation of betacyanins in dark

(Wang et al., 2007b; Wang and Wang, 2007), whereas most other plants accumulate betalains

relatively higher in light (Alan et al., 2011; Cao et al., 2012). From these reports, it appears

that cryptochrome 2, phytochrome, Ca2+

signalling cascade, cytochrome P450, transcription

factors, etc are involved in betalains biosynthesis (discussed in the next section).

2.5. Ecophysiological factors influencing betalains accumulation

2.5.1. Physical and stress factors

Plant pigments such as anthocyanins, betalains, carotenoids are accumulated in plant

parts when chlorophyll level goes down following action of chlorophyllase enzyme (Brady,


30

1987). Consequent to this, chloroplast is changed to chromoplast to facilitate pigment

accumulation (Looney and Patterson, 1967). Vacuolar pigments anthocyanins and betalains

also accumulate following chlorophyll degradation (Tucker and Grierson, 1987). Change in

pigment content during ontogeny enables plants to adapt to environmental condition, various

stress, and damages (Lichtenthaler, 1996). In this way, many factors may directly or indirectly

affect the accumulation of these secondary metabolites. Light is one of the important factors

that affect betalains biosynthesis. Sunlight promotes biosynthesis of dihydropyridine moiety,

which is precursor of betalamic acid (de Nicola et al., 1975). Soon research focus shifted on

effect of different wavelengths of light on betalains biosynthesis. Involvement of interaction of

phytochrome and a blue light responsive cryptochrome (Kochhar et al., 1981) was proposed

for accumulation of amaranthin in Amaranthus caudatus var. viridis seedlings. Cockburn et al.

(1996) supported this by showing that phytochrome can participate in signal transduction

pathway that leads to accumulation of betacyanin in Mesembryanthemum crystallinum. The

study also revealed that low red:far red light ratio and salt stress synergistically affected CAM

and pigmentation. As a protective mechanism against radiation-induced stress, UV light

enhances accumulation of betalains (Ibdah, 2002; Vogt et al., 1999; Cockburn et al., 1996).

Blue light was observed to be more efficient in inducing betalains accumulation than UV light

in cultured cells of Portulaca (Gallardo et al., 1999; Kishima et al., 1995). In contrast, blue

light was shown to degrade betacyanins and inhibit TOH possibly through cryptochrome 2

protein in S. salsa seedlings (Wang and Tao, 2006). Recently, some interesting evidences have

come up on darkness, low temperature and high salinity resulted in enhanced betacyanin

accumulation in halophyte S. salsa seedlings (Wang et al., 2006). It was also noted that

darkness during germination is one of the most important environmental factors controlling

betacyanin accumulation. Some more studies have demonstrated that Ca2+

, Ca2+

-regulated ion

channels, and calmodulin (Lichtenthaler, 1996) might be responsible for dark-induced

betacyanin accumulation. In general, calcium has been established as a ubiquitous signalling

molecule in plant owing to changes in its intracellular level in response to various stimuli.

Among the possible reasons of increase in betacyanin accumulation after Ca2+

treatment may

be through direct/indirect regulation of BGT, a type of UDP-glucose: flavonoid O-glucosyl

transferase, which is reportedly regulated by Ca2+

(Cheng et al., 1994). Further inference that

can be drawn from these observations may be that Ca2+

level also regulates directly or

indirectly tyrosinase activity (Wang and Wang, 2007).

One of the enzymes required for betacyanin/betanin biosynthesis is BGT, reported from

Dorotheanthus bellidiformis (Heuer et al., 1996) and red beet (Sepúlveda-Jiménez et al.,

2005). In red beet, BGT expression was induced by wounding, bacterial infection, oxidative

stress (Sepúlveda-Jiménez et al., 2005). This was followed by increase in betanin

accumulation. Other researchers have also reported earlier about the involvement of reactive


31

oxygen species in betacyanin accumulation (Wang et al., 2007a) in S. salsa seedlings and

leaves watered with H2O2.

2.5.2. Elicitors

For betalains production, in vitro cultures have been extensively studied (reviewed by

Georgiev et al., 2008). Effect of growth regulators, hormones, biotic and abiotic elicitors in

enhancement of betalains accumulation in vitro have been reviewed (Georgiev et al., 2008).

All these studies, so far, have not resulted in any commercial scale success. Since betalains are

water soluble, there is big challenge in separation of the pigments from the in vitro production

medium. Another reason may be the poor stability of betalains. In addition, production cost is

also a constrain. In view of this, betalains extracted from plant parts can bridge the gap

between demand of natural colours and supply. Mass propagation technique such as

micropropagation could be used for transferring plants from laboratory to field. There have

been reports of superior performance of tissue culture derived plants showing not only

uniform pigment content but better growth characteristics also compared to seedling grown

plants (Khan, 2006). This indicates that lab-to-field plants may play a vital role in ensuring

sustainable production of natural colours. In this context, model plants may be explored for

possible use as alternative sources of betalains. If need be, the colour spectrum and level of

betalains in the model plant should be improved through pigment elicitation, selection of

variety, and innovative farming systems.

Elicitation in intact plants may be important to translate the findings in vitro. However,

there are problems of poor reproducibility owing to seasonal variation and other

environmental factors. In an attempt to enhance betalains level, Amaranthus mangostanus

seedlings were treated with methyl jasmonic acid (MeJA), salicylic acid (SA), H2O2, and

ethylene to elicit pigment production in light and dark conditions (Cao et al., 2012). The

response was relatively higher in light on treatment with MeJA (>10 M), and ethylene (>1.0

mM) compared to untreated group. SA and H2O2 did not produce significant elicitation.

Molecules like jasmonic acid (JA), SA, H2O2, and ethylene are endogenously produced

signalling molecules. Exogenous application of these compounds at elevated concentration

exerts stress, which induce or mediate signal transduction pathways leading to secondary

metabolite production (reviewed by Zhao et al., 2005; Vasconsuelo and Boland, 2007). For

example, MeJA is known to elicit production of indol glucosinolates, -thujaplicin,

benzophenanthridines, isoflavones, soyasaponin, polyamines, taxol, flavonoids including

anthocyanins and resveratrol, etc (for detailed table see Zhao et al., 2005). It acts through

induction of JA signalling pathway. However, there may be other mechanisms such as

induction of other signalling cascades through Ca2+

, SA, H2O2, etc. Enhancement of betalains

production on treatment with MeJA has been reported in red beet hairy root culture (Suresh et


32

al., 2004). More studies on use of elicitors on intact plants need to be carried out to ascertain

optimum conditions for reproducible results.

2.6. Stability

After the approval of red beet as food colourant, it has been used for colouring foods

such as desserts, confectioneries, dry mixes, dairy and meat products. It was estimated that

less than 50 mg betanin/kg can produce the desired colour (Delgado-Vargas et al., 2000).

However, these pigments have poor stability because of which it may not be able to replace

synthetic dyes (Ozela, 2004). There are many factors that affect betalains stability (Fig. 2.4).

Figure 2.4. Factors affecting betalains stability (A) and sites prone to deglycosylation (blue

bracket), decarboxylation (green) and dehydrogenation (red) in betacyanins (B). Adapted with

modifications from references, Herbach et al., 2006b and Stintzing and Carle, 2007. PPO-

polyphenol oxidase, POD-peroxidase.


33

2.6.1. Factors affecting betalains stability

2.6.1.1. Structure, concentration and composition

Structurally different betalains viz. betacyanins and betaxanthins have been compared

for their stability at room temperature and when subjected to high temperature in acidic pH.

Betacaynins, specially, betanin was found to be more stable than betaxanthins, specially,

vulgaxanthin I in both the conditions (Sapers and Hornstein, 1979; Singer and von Elbe,

1980). At pH 7, vulgaxanthin I was observed to be more stable (Savolainen and Kuusi, 1978).

It was reported that glucosylated betalains were comparatively more stable than their aglycons

due to higher oxidation-reduction potential (von Elbe and Attoe, 1985) but, further

glucosylation did not increase stability (Huang and von Elbe, 1986). On the other hand,

esterification (mostly on glucosylated betalains) with aliphatic acids may increase stability

(Reynoso et al., 1997; Barrera et al., 1998). Position of esterification seems to be important

determinant of stability in case aromatic acids are involved (Heuer et al., 1994; Schliemann

and Strack, 1998). Conversely, betanin was observed to be more stable than acylated

betacyanins (Herbach et al., 2006a).

Concentration as well as matrix components have been also observed to enhance

betalains stability (Merin et al., 1987; Moßhammer et al., 2005; Moßhammer et al., 2007).

2.6.1.2. pH

Many reports have observed that betalains in juice/crude extracts are stable at pH 5

(Han et al., 1998; Castellar et al., 2003; El Gharras et al., 2008; Harivaindaran et al., 2008;

Woo et al., 2011). However, studies on purified betalains have shown that betacyanins are

stable in acidic pH (4-6) (Huang and von Elbe, 1985, 1987; Castellar et al., 2003; Vaillant et

al., 2005), whereas betaxanthins are relatively more stable at pH 7 or slightly above (Cai et al.,

2001). Betalamic acid also exhibits higher stability at alkaline pH (Kimler et al., 1971). It was

observed that alkaline pH causes breakdown of aldimine linkage, whereas in acidic

environment favours condensation of betalamic acid and the amine group (Schwartz and von

Elbe, 1983). Also, acidic pH causes dehydrogenation (Mabry et al., 1967) and C15

isomerisation (Wyler and Dreiding, 1984).

2.6.1.3. Water activity (aw)

Cleavage of aldimine bond of betalains depends upon aw value (Herbach et al., 2006b).

It is understood that water activity affects mobility of reactants and oxygen solubility

(Delgado-Vargas et al., 2000). aw value less than 0.63 has been observed to improve betalains

stability (Kearsley and Katsaboxakis, 1980). Hence, different processing techniques such as

spray drying (Cai and Corke, 2000) and concentration (Castellar et al., 2006) have been

employed to enhance pigment stability by reducing aw value.

2.6.1.4. Light

Exposure to light including UV destabilises betalains (von Elbe et al., 1974; Attoe


34

and von Elbe, 1981; Cai et al., 1998) owing to excitation of electrons in the chromophore

resulting in decreased activation energy or increased reactivity (Jackman and Smith, 1996).

Addition of antioxidants such as isoascorbic acid and ascorbic acid was effective in

counteracting destruction of betalains during light exposure (Bilyk et al., 1981; Herbach et al.,

2006a). Apparently, light-induced destruction of betalains is dependent on oxygen, as there

was no significant degradation in anaerobic conditions (Attoe and von Elbe, 1981; Huang and

von Elbe, 1986), and also, temperature because above 40C there was no impact of

illumination, whereas below 25C there was significant detrimental effect of betalains (Attoe

and von Elbe 1981; Huang and von Elbe 1986).

2.6.1.5. Oxygen and other oxidants

In the presence of oxygen, betanin and betanidin stability decreases probably due to

superoxide radicals that can destabilise the core structure (Wyler et al., 1963; Pasch and von

Elbe, 1979; Czapski, 1985). Oxygen-induced betalains degradation kinetics has been worked

out (Attoe and von Elbe, 1982, 1984, 1985; von Elbe and Attoe, 1985). Apart from

degradation, oxygen content also inhibits recovery of pigments after degradation (von Elbe et

al., 1974; Huang and von Elbe, 1987). Hydrogen peroxide has been also known to accelerate

betalains degradation (Wasserman and Guilfoy, 1984), which could be counteracted by

addition of antioxidants (Altamirano et al., 1992; Han et al., 1998). It appears that

betaxanthins are more prone to chemical oxidation induced by hydrogen peroxide (Wasserman

and Guilfoy, 1984) than betacyanins. In inert atmosphere, there was no significant degradation

of betalains (Attoe and von Elbe, 1982; von Elbe and Attoe, 1985).

2.6.1.6. Antioxidants

Antioxidants such as ascorbic acid and isoascorbic acid have been regularly used as

betalains stabilisers (Reynoso et al., 1997; Han et al., 1998; Herbach et al., 2006a). Among the

two antioxidants, there is controversy on which one is more efficient stabiliser (Bilyk and

Howard, 1982; Attoe and von Elbe, 1985; Barrera et al., 1998; Herbach et al., 2006a).

However, at high concentration ascorbic acid acts as pro-oxidant (Pasch and von Elbe, 1979).

Attoe and von Elbe (1985) showed that phenolic compounds do not stabilise betalains.

2.6.1.7. Temperature

Thermal degradation of betalains depends upon the duration of exposure and

temperature (Saguy et al., 1978), and the degradation follows first order kinetics (Saguy et al.,

1978; Herbach et al., 2004). At high temperature (>40), betalains degrade fast (von Elbe et al.,

1974; Saguy et al., 1978), whereas at low temperature (<10) their degradation slows down

(Cai et al., 1998). At elevated temperature, the pH optimum for betalains stability increases

slightly (Havlikova et al., 1983). Nemzer et al. (2011) characterised the degradation products

of betalains during various processing techniques that involve exposure to high temperature.

They reported that among many changes, it was noteworthy that betanin concentration varied


35

widely in products obtained through spray drying, freeze drying and air drying. Woo et al.

(2011) proposed the optimum temperature for processing of betalains containing juice as

90C. It has been observed that after thermal degradation, betalains can regenerate in the

presence of antioxidants such as ascorbic acid and isoascorbic acid at low temperature (Han et

al., 1998; Herbach et al., 2006a). The regeneration is due to condensation of betalamic acid

and amine group (Huang and von Elbe, 1985).

2.6.1.8. Metals

Many metal ions including Fe2+

, Fe3+

, Cu+, Cu

2+, Sn

2+, Al

3+, Cr

3+, Hg

2+ bleach betalains

colour (Herbach et al., 2006b) by forming metal-pigment complex resulting in

hypochromic/bathochromic shift in the absorption spectrum of the pigment. The spectral shift

could be successfully reversed by addition of EDTA which chelates metal ion. The

concentration of metal ion that induces colour bleaching appears to depend upon the matrix

(Czapski, 1990). In the study, it was also observed that purified compounds were bleached by

even 3 times less than the concentration of metal ion that can bleach the colour in juice.

2.6.1.9. Decolourising enzymes

Both membrane bound and cell wall bound peroxidase in beet root have been implicated

in betalains degradation (Wasserman and Guilfoy, 1984), and betacyanins are more prone to

degradation by peroxidase than betaxanthins (Wasserman and Guilfoy, 1984). Peroxidase

breaks down betanin to betalamic acid and oxidized cyclo-DOPA-5-O-glucoside polymers

(Martinez-Parra and Munoz, 2001). Polyphenol oxidases have been also associated with

betalains degradation (Lee and Smith, 1979). Presence of a betanin oxidase was reported in

beet root which can breakdown betanin to 2-hydroxy-2 hydro-betalamic acid and cyclo-

DOPA-5-O-glucoside (Zakharova et al., 1987). The role of decarboxylase in betalains

breakdown has been documented (Manchali et al., 2013).

2.7. Stabilisation

It is important to stabilize betalains before we subject them to commercial applications.

Among the various ways of stabilization of betalains, copigmentation, complexation and

encapsulation are novel and emerging techniques.

2.7.1. Complex formation

Change in colour absorption such as bathochromic and hypochromic shift has been

observed indicating complex formation of betalains (specially betanin) with metal ions such as

Cu2+

(Kuusi et al., 1977; Butera et al., 2002), Cu+, Hg

2+ (Kuusi et al., 1977), Fe

2+, Ni

2+, Co

2+

(Sporna-Kucab et al., 2011). It was confirmed by addition of metal-chelator EDTA, which

could reverse the spectral changes. These complexes reduce colour stability of betalains as in

case of Fe2+

, Cu2+

(Sporna-Kucab et al., 2011; Pasch and von Elbe, 1979; Chapter 5 of this

thesis), and Ni2+

, Co2+

(Sporna-Kucab et al., 2011). On the other hand, -cyclodextrin (-CD)-

betanin complex renders protection to colour (Hamburg and Hamburg, 1991). The authors


36

attributed it to adsorption of free water by -CD. CDs have been reported to improve betalains

stability in the absence of light and O2 (Drunkler et al., 2006). Interestingly, in a recent report,

it has been shown that -CD forms 1:1 inclusion complex with betanin (Norasiha et al., 2009).

The authors confirmed this through IR spectroscopy and XRD evidences of 1:1 inclusion

complex, which was clearly different from 1:1 physical mixture. α, β, γ-CD are

polysaccharides containing six to eight D-glucopyranose residues, connected by α-

1,4glycosidic bonds. These macromolecules are arranged in toroidal shape with hydrophobic

central cavity and hydrophilic outer surface having exposed hydroxyl groups. An inclusion

complex may form when a drug/biomolecule sits in the cavity of CD (reviewed by Stella and

He, 2008) (Fig. 2.5), both in solid and solution phase. The review concluded that among the

CDs, modified CDs such as hydroxypropyl--CD (HP--CD) and sulfobutylether--CD

(SBE--CD) are devoid of any toxic effects in vivo. Hence, HP--CD and SBE--CD may be

used for stabilizing betalains for food and other applications. In addition, a recent report

provided evidence that addition of CD improves global sensorial quality without reducing

the aroma of the sample/extract (Andreu-Sevilla et al., 2011).

Recently, inorganic matrices have been used as stabilizing agent of betalains. Lima et

al. (2009) observed that adsorption of betalains in alumina stabilized the pigment for more

than twenty months. They attributed it to acid-base interaction. Another group of researchers

proposed ceramic particles (TEOS) for stabilization of betalains against UV-light, and

temperature to increase its application (Estévez et al., n.d.).

Figure 2.5. Drug-cyclodextrin binding in equilibrium to form 1:1 inclusion complex (adapted

from Stella and He, 2008).

Many of the phenolic acids such as catechin, quercetin, chlorogenic acid, and butylated

hydroxyanisole, tested so far, have been found to have either no effect, or reduce stability of

betanin (Attoe and Von Elbe, 1985). Contrastingly, stabilization of betalains in the presence of

catechin has been proposed in a patent (Tsai and Hsiao, 2010). It was claimed that catechin

stabilized betalains in solution, and after heat treatment during regeneration of the pigments a

relatively stable schiff-base complex, betalains-catechin, is formed.


37

2.7.2. Copigmentation

Copigmentation phenomenon was first described by Willstatter and Zollinger (1916)

with respect to grape pigment, oenin (later named as malvidin 3-glucoside). Its hue turned to

bluer red after addition of tannin or gallic acid. Later, Maud and Robert (1931) reported

different colour hues of the same pigment in different plant parts. Copigmentation takes place

owing to direct or weak interactions between anthocyanins and other naturally occurring

compounds. The interactions result in a wide range of colours. Already documented

copigments are metals, phenols, alkaloids, amino acids, organic acids, nucleotides,

polysaccharides, metals, and anthocyanins themselves (Mazza and Brouillard, 1990).

Copigmentation phenomenon enhances colour intensity and stability of anthocyanins. Increase

in colour intensity is attributed to interaction of a copigment with an anthocyanin resulting in

bathochromic shift in its absorption spectrum and concomitant hyperchromaticity

(Mazzaracchio et al., 2004). The nature of interactions may be complex formation or a weak

interaction of a copigment with an anthocyanin producing all these favourable effects. In case

of betalains, antioxidants such as ascorbic acid, and isoascorbic acid have been known to

remove O2 from the solution and decrease polarity at C11 position, which is prone to

nucleophilic attack (Herbach et al., 2006b).

2.7.3. Encapsulation

It is known that addition of polysaccharides such as pectin, guar gum increases pigment

stability by reducing hygroscopicity (Lejeune et al., 1983). One of the preliminary studies in

this area was to encapsulate amaranth betacyanins (Cai and Corke, 2000) using maltodextrin

(MDE, 10-25 dextrose equivalent), and native/modified starch as coating agent. Feed mixture

contained 20% total solids. It was observed that higher inlet temperature (>180C) resulted in

drying loss (>4%) of pigment. The authors claimed that MDE of mixed dextrose equivalent

(DE) had superior storage stability of encapsulated pigments. The loss was 10-16% in four

months, however in another study, encapsulation with MDE (10 DE) of Opuntia lasiacantha

Pfeiffer (red prickly pear) betanin extract had only 14% loss in 6 months (Díaz et al., 2006).

Stability of encapsulated red beet betalains was independent of MDE concentration (Azeredo

et al., 2007). This study reported degradation of only 10% betalains during six months of

storage. Saenz et al. (2009) reported superior stability of indicaxanthin in encapsulated extract

compared to betacyanin at 60C. Following this, Gandia-Herrero et al. (2010) encapsulated

purified indicaxanthin with MDE (20%, w/v) to produce stabilised pigment with

uncompromised colour intensity. They reported that encapsulated pigment did not lose

significantly during storage in dark at 4C and 20C upto six months. From all these reports, it

appears that stability depends upon the pigment source specially in case of encapsulation.

Also, these reports indicate the suitability of stabilization through encapsulation for the

hygroscopic and poorly stable betalains to widen commercial applications. Pietrzkowski and


38

Thresher (2010) patented red beet dried extract containing formulation which was free flowing

having 5% (w/w) betalains, with increased stable and water solubility. In encapsulated

betalains formulation, Chik et al. (2011) included certain additives that can confer better

functionality. Betalains from certain less known leafy vegetables and medicinal plants such as

Basella alba can be extracted with naturally containing gummy substances, which may act as

intrinsic agent for stability as well as value-addition. The gummy substances in B. alba are

known to contribute in detoxification, digestion (USDA SR23, 2010) and hematological

parameters (Bamidele et al., 2010).

After reviewing the reports on encapsulation of betalains, the optimum conditions for

spray drying may result in a yield of encapsulated pigment anywhere between 90-98%

containing less than 8% moisture. It appears that the yield and moisture content depend upon

inlet temperature. In case of carotenoids such as -carotene and bixin, respective yields of

62% (Desobry et al., 1997; Loksuwan, 2007), and 54% (Barbosa et al., 2005) have been

reported, whereas anthocyanins encapsulation yields 80%-97% (Ferreira et al., 2009), which

was 65%-93% in presence of certain additives such as acacia gum and tricalcium phosphate

(Nayak and Rastogi, 2010). It was reported that betalains recovery after spray-drying was

improved when xanthan gum was added in encapsulating agent (such as MDE) (Ravichandran

et al., 2012). Interestingly, freeze-drying improved betalains recovery after encapsulation

(Ravichandran et al., 2012).

2.8. Dietary safety

Safety is an essential aspect of a phytochemical for human consumption. In a report

released by FAO/WHO Expert Committee (FAO and WHO, 1974), regarding safety of beet

root pigment it was noted “There is no information available on the metabolism of this

naturally occurring betanin. The available long-term and reproduction studies are inadequate

because only a few parameters were examined and many other essential observations have not

been reported. No specific information is available on embryotoxicity including teratogenicity.

This colour is, however, a normal constituent of food. Although the primary criteria are the

same for evaluating the safety of food colours whether of natural or synthetic origin,

consideration must be given to the quantities of food colour ingested as a result of

technological use relative to its ingestion as an ingredient of food. This and the availability of

an adequate specification permits evaluation in the absence of a full range of toxicological

investigations”. Following this, absorption, excretion, metabolism and cardiovascular effects

of beet root extract was studied (Krantz and Wahlstrom, 1980). From the study, it was

established that oral ingestion of betanin resulted in poor absorption, and its metabolism take

place in the gut. Also, it was shown that betanin transiently increased blood pressure and heart

rate, which was blocked in the presence of specific adrenergic and cholinergic blockers.

Absence of genotoxicity (Haveland-Smith, 1981), mutagenicity and short-term toxicity of beet


39

pigments on S. typhimurium (Zampini et al., 2011) and rats have been observed (von Elbe and

Schwartz, 1981). It was also reported that beet pigments were unable to initiate or promote

hepatocarcinogenesis in rats (Schwartz et al., 1983). Betanin could inhibit IgE and IgG

production, suggesting lack of allergic response (Pourrat et al., 1987; Kuramoto et al., 1996).

Towards sustainable production, betalains extract from hairy roots of red beet were assessed

for their safety in rats (Khan, 2006). The results showed that hairy root derived betalains did

not produce any recognisable toxicity. However, it was reported that beet red induced weak

mutagenicity (in Ames test) similar to erythorbic acid and chlorine dioxide (Ishidate et al.,

1984). Beeturia had been a concern which is due to consumption of beet pigments. It is the

phenomenon of excretion of coloured urine after red beet consumption. Recently in a

systematic review (Mitchell, 2001), it was observed that beeturia was independent of an

individual’s physiological constitution, and it was not under polymorphic genetic control.

Beeturia was directly related with quantity of consumption, coingestion with certain organic

acids including ascorbic acid and oxalic acid, and rate of gastric emptying. Hence, beeturia

was considered as just an idiosyncratic response to food. It means that beeturia is not a

physiological dysfunction.

For assessment of dietary safety, bioavailability and biological activity, beet root has

been used as source of the pigments, in most of the reports. As a result, red beet is well

accepted as red food colourant, specially betanin, denoted as E-162 in the European Union

(Downham & Collins, 2000) and 73.40 in the chapter 21 of the Code of Federal Regulations

(CFR) section of the Food and Drug Administration (FDA) in the USA (Griffiths, 2005). The

pigment finds applications in foods such as yoghurt, confectionery, ice creams, syrups,

sausages and processed meats. Betalains research is picking up, as a result many new sources

with wide colour spectrum have been reported. In order to put them to use in foods, their

safety aspects were documented (Krifa et al., 1987; Reynoso et al., 1999; Sembries et al.,

2006; Khan et al., 2011a; Hor et al., 2012; Klewicka et al., 2012). Safety assessment is

essential in view of the presence of structurally different betalains in these new sources and

advancement in processing technology. While P. americana (pokeweed) berries have been

known to contain toxic saponins because of which they have not been commercially exploited

(Forni et al., 1983), Celosia argentea var. cristata contains high level of dopamine (41.15

µM/g fresh weight) (Schliemann et al., 2001), that may be toxic for human consumption.

Recently pulsed electric fields (Zvitov et al., 2003; Fincan et al., 2004; Shynkaryk et al., 2008;

López et al., 2009; Loginova et al., 2011; Kannan, 2011), microwave coupled with enzyme

preatment (Moussa-Ayoub et al., 2011), enzymes (Chethana et al., 2007), gamma-radiation

pretreatment (Nayak et al., 2006), and aqueous two-phase (Krifa et al., 1987) have been used

for betalains extraction. For concentration of betalains, fermentation technology (Fincan et al.,

2004; Castellar et al., 2008), convective drying (Gokhale and Lele, 2012), and some other


40

novel methods of processing have been reported (Abeysekere et al., 1990; Thimmaraju et al.,

2003; Rudrappa et al., 2004; Vaz et al., 2005). Fermentation derived filtrate and pulsed

electric fields assisted extraction products have been reported to be safe (Krifa et al., 1987;

Klewicka et al., 2012). Studies on safety, biological activity, bioavailability of the betalains

produced using the different processing methods need to be conducted. Interestingly, some of

the processing technologies have been shown to exhibit superior biological activity (Krifa et

al., 1987; Kannan, 2011; Kim et al., 2007; Ravichandran et al., 2011; Lee et al., 2012) owing

to enhanced extraction of antioxidants including betalains. However, some reports (Shynkaryk

et al., 2008; Abeysekere et al., 1990; Rudrappa et al., 2004) seem to contradict the beneficial

effects of these processing methods.

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