review of literature

Chapter 2.

REVIEW OF LITERATURE

Review of literature 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 13

Review of literature 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

14

Review of literature Table 2.1. Betalains content of various plants. Family/

Species

total no. of

Common name/

Betalains

pigmented part

content

genera# Chenopodiaceae

Reference

(mg/100 g fw) Beta vulgaris

Red beet/ root

40–200

Stintzing and Carle, 2007

B. vulgaris L. ssp.

Swiss Chard/

cicla [L.]

Petiole

4–8


Alef. Chenopodium

Red goosefoot/

NA

NA

rubrum

flower

Chenopodium

Djulis/seed

94

Tsai et al.,

formosanum

grain

Chenopodium

Quinoa/grain

NA

NA

Seepweed/leaf

250

Wang et al.,

2010

quinoa Suaeda salsa

2006 Cactaceae/ 98

Hylocereus

Pitaya/fruit

31–41

polyrhizus

Vaillant et al., 2005; Shea, 2012

H.

purpusii,

H. Red flesh fruit

costaricensis,

Wybraniec and

species

Mizrahi, 2005

H. undatus, H. sp. (hybrids) Opuntia ficus-india

Cactus pear/fruit

17–860*


Myrtillocactus

Garambullo

geometrizans

tree/fruit

Opuntia matudae

Xoconostle/fruit

214*

Reynoso et al., 1997

10.6–20.2*

GuzmánMaldonado et al., 2010

Mammillaria sp.

/fruit

1.6–18.1

Wybraniec and NowakWydra, 2007

Schlumbergera sp.

Christmas cactus/flower 15

163

Kobayashi et al., 2000

Review of literature

Amaranthaceae/

Selenicereus

Yellow

megalanthus

Pitaya/fruit

Amarathus sp.

Amaranth/seed/

166

NA

2006 46–199

flower Celosia sp.

Common

Iresine sp.

cockscomb/

Kugler et al.,

Cai et al., 2005

92.1–621.5

Cai et al., 2005

Inflorescence Gomphrena

Inflorescence

7.6–55.7

globosa Portulacaceae/31

Aizoaceae/127

Kugler et al., 2007

Portulaca

Moss

12.5–17.8*

Trezzini and

grandiflora

rose/petal/stem

Zrÿd, 1991a

Mesembryanthemu-

Ice plant/flower,  3.2

Vogt et al.,

m crystallinum

leaf

1999

Dorotheanthus

Livingstone

bellidiformis

daisy/flower

Lampranthus

Ice plant/flower

NA

NA

66−417

Kugler et al.,

productus

2007; GandíaHerrero et al., 2005

Glottiphylum

/flower

135

oligocarpum

GandíaHerrero et al., 2005

Glottiphylum

/flower

138

pigmaeum

GandíaHerrero et al., 2005

Nyctaginaceae/

Bougainvillea sp.

31

Bougainvillea/

259.4–551.8*

bract Mirabilis jalapa

Four o’clocks/

2006 6.5

flower Boerhavia erecta

Erect spiderling/

Basella alba

Malabar

185.5

Spinach

36.1

Ulluco/tuber 16

Lin et al., 2010

NA

vine/fruit Ullucus tuberosus

Stintzing et al., 2004

spinach/fruit Basella rubra

Piattelli et al., 1965c

bark Basellaceae/4

Kugler et al.,

Glässgen et al., 1993

7

Svenson et al.,

Review of literature 2008 Phytolaccaceae/

Rivina humilis

15

Pigeonberry/

350 (1700*)

fruit Phytolacca

Pokeweed/fruit

2012 1400*

decandra Phytolacca

Khan et al.,

Forni et al., 1983

Pokeberry/ fruit

americana

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-14 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 17

Review of literature 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

Source

Reference

Beta vulgaris L.

Wyler

betalamic acid) Aglycone Betanidin

cyclo-DOPA

and

Dreiding, Wilcox

1959; et

al.,

1965 Isobetanidin

cyclo-DOPA

Beta vulgaris L.

Wyler

and

Dreiding, Wilcox

1959; et

al.,

1965 2-Descarboxy betanidin Glycone (Betanin group) Betanin

Decarboxylated cycloDOPA

Carpobrotus acinaciformis L.

Piattelli and Impellizzeri, 1970

cyclo-DOPA-5-O-Glc

Beta vulgaris L.

Isobetanin

cyclo-DOPA-5-O-Glc

Beta vulgaris L.

Phyllocactin

cyclo-DOPA-5-O(6-Omalonyl)-Glc cyclo-DOPA-5-O(6-Omalonyl)-Glc cyclo-DOPA-5-O[6-O(E)-p-coumaroyl]-Glc cyclo-DOPA-5-O[6-O(E)-p-coumaroyl]-Glc cyclo-DOPA-5-O[6-O(E)-feruloyl]-Glc cyclo-DOPA-5-O(6-O(E)-feruloyl)-Glc

Phyllocactus hybridus

Wyler and Dreiding, 1957 Wyler and Dreiding, 1957 Piattelli and Minale, 1964 Piattelli and Minale, 1964 Piattelli and Impellizzeri, 1969 Piattelli and Impellizzeri, 1969 Piattelli and Impellizzeri, 1969 Piattelli and Impellizzeri, 1969

Isophyllocactin Lampranthin I Isolampranthin I Lampranthin II Isolampranthin II

Phyllocactus hybridus Lampranthus sociorum Lampranthus sociorum Lampranthus sociorum Lampranthus sociorum

18

Review of literature Table 2.2. continued... Rivinianin Neobetanin* Betanidin 5-O(5O-E-feruloyl-2-Oapiosyl)-glc Betanidin 5-O(5O-E-feruloyl-2-Oapiosyl)-glc Prebetanin Isoprebetanin 2-Apiosyl phyllocactin 2-Apiosyl isophyllocactin Betanidin 5-O[2O(5-O-Eferuloyl)-apiosyl6-O-malonyl)]-glc 2-Descarboxy betanin 6-Malonyl-2descarboxy betanin Hylocerenin

Isohylocerenin

Mammillarinin

Glycone (Amaranthin group) Iresinin I

Suaedin

Celosianin I

cyclo-DOPA-5-O(3sulphate)-Glc cyclo-DOPA-5-O-Glc cyclo-DOPA-5-O(5-OE-feruloyl-2-Oapiosyl)-Glc cyclo-DOPA-5-O(5-OE-feruloyl-2-Oapiosyl)-Glc cyclo-DOPA-5-O(6-Osulphate)-Glc cyclo-DOPA-5-O(6-Osulphate)-Glc cyclo-DOPA-5-O(2-Oapiosyl-6-O-malonyl)Glc cyclo-DOPA-5-O(2-Oapiosyl-6-O-malonyl)Glc cyclo-DOPA-5-O[2O(5-O-E-feruloyl)apiosyl-6-O-malonyl)]Glc cyclo-DOPA-5-O-Glc 2-descarboxy-cycloDOPA-5-O(6-malonyl)Glc cyclo-DOPA-5-O[6O(3-hydroxy-3methylglutaryl)]-Glc cyclo-DOPA-5-O[6O(3-hydroxy-3methylglutaryl)]-Glc cyclo-DOPA-5-O(6-Omalonyl)-β-sophoroside

Rivina humilis L.

Imperato, 1975a Beta vulgaris subsp. Alard et al., vulgaris var. conditiva 1985 Phytolacca americana Schliemann L. et al., 1996 Phytolacca L.

americana

Phytolacca americana L. Phytolacca americana L. Schlumbergerabuckleyi

19

Schliemann et al., 1996 Schliemann et al., 1996 Kobayashi et al., 2000

Schlumbergerabuckleyi Kobayashi et al., 2000 Schlumbergerabuckleyi Kobayashi et al., 2000

Beta vulgaris L. Beta vulgaris L.

Kobayashi et al., 2001 Kobayashi et al., 2001

Hylocereus polyrhizus

Wybraniec et al., 2001

Hylocereus polyrhizus

Wybraniec et al., 2001

Mammillaria sp.

Wybraniec and NowakWydra, 2007

Iresine herbstii cyclo-DOPA-5-O[6O(3-hydroxy-3methylglutaric acid)]Glc Suaeda fruticosa Forsk. cyclo-DOPA-5-O[2O(citryl)-GlcU]-Glc cyclo-DOPA-5-O[2O(p-coumaroyl)-GlcU]Glc

Schliemann et al., 1996

Celosia cristata

Minale et al., 1966

Piattelli and Imperato, 1971 Steglich and Strack, 1990

Review of literature Table 2.2. continued... Celosianin II Amaranthin Isoamaranthin Isocelosianin I

Isocelosianin II Isoiresinin I

cyclo-DOPA-5-O[2O(feruloyl)-GlcU]-Glc cyclo-DOPA-5-O(2-OGlcU)-Glc cyclo-DOPA-5-O(2-OGlcU)-Glc cyclo-DOPA-5-O[2O(p-coumaroyl)-GlcU]Glc cyclo-DOPA-5-O[2O(feruloyl)-GlcU]-Glc cyclo-DOPA-5-O[6O(3-hydroxy-3methylglutaric acid)]Glc

Celosia cristata

cyclo-DOPA-5-Osophoroside

Amaranthus cruentus Amaranthus cruentus Celosia cristata

Celosia cristata

Steglich and Strack, 1990 Strack et al., 1993 Cai et al., 2005 Cai et al., 2005 Cai et 2005 Cai et 2005

al.,

Bougainvillea glabra

Piattelli

and

var. “Mrs. Butt”

Imperato,

Iresine herbstii

al.,

Glycone (Bougainvillein) Bougainvillein-r I

1970a Isobougainvillein-r I

cyclo-DOPA-5-O-


Piattelli

sophoroside


Imperato,

and

1970a Bougainvillein-r II

cyclo-DOPA-5-O(p-


Piattelli

coumaroyl)-sophoroside


Imperato,

and

1970a Isobougainvillein-r

cyclo-DOPA-5-O-


Piattelli

II

sophoroside


Imperato,

and

1970a Bougainvillein-v

cyclo-DOPA-6-O-


Piattelli

sophoroside

var. sanderiana

Imperato,

and

1970b Isobougainvillein-v

cyclo-DOPA-6-O-


Piattelli

sophoroside

var. sanderiana

Imperato, 1970b

Betanidin 6-O(2-

cyclo-DOPA-6-O(2-

glucosylrutinoside)

glucosyl-6-O-


Imperato, 1975b

rhamnose)-Glc

20

and

Review of literature Table 2.2. continued... Isobetanidin 6-O(2-

cyclo-DOPA-6-O(2-

glucosylrutinoside)

glucosyl-6-O-


Imperato, 1975b

rhamnose)-Glc Betanidin 6-O(6-O-

cyclo-DOPA-6-O(6-O-

E-caffeoyl)-

E-caffeoyl)-sophoroside


Heuer et al., 1994

sophoroside Betanidin 6-O(6-O-

cyclo-DOPA-6-O(6-O-

E-p-coumaroyl)-

E-p-coumaroyl)-

sophoroside

sophoroside

Betanidin 6-O(6-O-


E-p-coumaroyl)-

E-p-coumaroyl)-

sophoroside

sophoroside

Betanidin 6-O{2-O-

cyclo-DOPA-6-O{2-O-

sophorosyl[(6-O-E-

sophorosyl[(6-O-E-

caffeoyl)-(6-O-E-p-

caffeoyl)-( 6-O-E-p-

coumaroyl)]}-

coumaroyl)]}-

sophoroside

sophoroside

Betanidin 6-O{2-O-

cyclo-DOPA-6-O{2-O-

glucosyl[(6-O-E-

glucosyl[(6-O-E-

caffeoyl)-(6-O-E-p-

caffeoyl)-( 6-O-E-p-

coumaroyl)]}-

coumaroyl)]}-

sophoroside

sophoroside

Betanidin 6-O[(2-O-

cyclo-DOPA-6-O[(2-O-

glucosyl)(6,6-di-O-

glucosyl)(6,6-di-O-E-

E-p-coumaroyl)]-

p-coumaroyl)]-

sophoroside

sophoroside

Betanidin 6-O(6,6-

cyclo-DOPA-6-O(6,6-

di-O-E-p-

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

coumaroyl)]-

sophoroside


Heuer et al., 1994


Heuer et al., 1994


Heuer et al., 1994


Heuer et al., 1994

sophoroside

21


Heuer et al., 1994


Heuer et al., 1994

Review of literature Table 2.2. continued... Glycone (Gomphrenin group) Gomphrenin I

cyclo-DOPA-6-O-Glc

Isogomphrenin I

Gomphrenin II

Isogomphrenin II

Gomphrenin III

Isogomphrenin III

Sinapoyl-

Gomphrena globosa

Minale et al.,

L.

1967

Gomphrena globosa

Minale et al.,

L.

1967


Gomphrena globosa

Heuer et al.,

E-4-coumaroyl)-Glc

L.

1992


Gomphrena globosa

Heuer et al.,

E-4-coumaroyl)-Glc

L.

1992


Gomphrena globosa

Heuer et al.,

E-feruloyl)-Glc

L.

1992


Gomphrena globosa

Heuer et al.,

E-feruloyl)-Glc

L.

1992

NA

Gomphrena globosa

Kugler et al.,

L.

2007

Gomphrena globosa

Kugler et al.,

L.

2007

Amanita muscaria

Musso, 1979

cyclo-DOPA-6-O-Glc

gomphrenin I# Sinapoyl-

NA #

isogomphrenin I

Betacyanin-like fungal pigments Muscapurpurin

cyclo-Stizolobic acid

Muscarubrin

Pyrroline

2-carboxylic Amanita muscaria

acid

Stintzing

and

Schliemann, 2007

Plant

based

betaxanthins Betalamic acid

NA

Beta vulgaris L.

Wyler et al., 1963

Indicaxanthin

Portulacaxanthin I

Vulgaxanthin I

Proline

Opuntia ficus indica Piattelli et al.,

Hydroxyproline

Glutamine

L.

1964

Portulaca

Piattelli et al.,

grandiflora

1965b

Beta vulgaris L.

Piattelli et al., 1965a

22

Review of literature Table 2.2. continued... Vulgaxanthin II

Glutamic acid

Beta vulgaris L.

Piattelli et al., 1965a

Miraxanthin I

Methionine sulphoxide

Mirabilis jalapa L.


Miraxanthin II

Aspartic acid

Mirabilis jalapa L.


Miraxanthin III

Tyramine

Mirabilis jalapa L.


Miraxanthin IV

not characterised

Mirabilis jalapa L.


Miraxanthin V

Dopamine

Mirabilis jalapa L.


Miraxanthin VI

not characterised

Mirabilis jalapa L.


Dopaxanthin II

DOPA

Glottiphyllum longum

Impellizzeri et (Haw.) al., 1973

N.E.Br. Vulgaxanthin IV

Leucine

Beta vulgaris L.

Piattelli, 1976

Humilixanthin

5-hydroxynorvaline

Rivina humilis L.

Strack et al., 1987

Portulacaxanthin II

Portulacaxanthin III

Vulgaxanthin III

Tyrosine

Glycine

Asparagine

Portulaca

Trezzini

and

grandiflora

Zryd, 1991a

Portulaca

Trezzini

grandiflora

Zryd, 1991a

Beta vulgaris L.

Trezzini

and

and

Zryd, 1991b Valine-Bx

Valine

Beta vulgaris L.

Kugler et al.,

subsp. Cicla (L.)

2004

Alef. Cv. Bright Lights Threonine-Bx

Threonine

Beta vulgaris L.

Kugler et al.,

subsp. cicla (L.)

2004

Alef. Cv. Bright Lights

23

Review of literature Table 2.2. continued... Ethanolamine-Bx

Ethanolamine

Beta vulgaris L.

Kugler et al.,

subsp. cicla (L.)

2004

Alef. Cv. Bright Lights Serine-Bx

Serine

Beta vulgaris L.

Kugler et al.,

subsp. cicla (L.)

2004

Alef. Cv. Bright Lights Phenylalanine-Bx

Phenylalanine

Beta vulgaris L.

Kugler et al.,

subsp. cicla (L.)

2004

Alef. Cv. Bright Lights -Aminobutyric

-Aminobutyric acid

acid-Bx

Beta vulgaris L.

Kugler et al.,

subsp. cicla (L.)

2004

Alef. Cv. Bright Lights Isoleucine-Bx

Isoleucine

Beta vulgaris L.

Kugler et al.,

subsp. cicla (L.)

2004

Alef. Cv. Bright Lights Alanine-Bx

Alanine

Beta vulgaris L.

Kugler et al.,

subsp. cicla (L.)

2004

Alef. Cv. Bright Lights 3-methoxytyramine-

3-methoxytyramine

Bx Tryptophan-Bx

Methionine-Bx

Tryptophan

Methionine

Celosia

Cai

et

al.,

cristata/plumosa

2005

Celosia

Cai

et

al.,

cristata/plumosa

2005

Opuntia sp.

Stintzing

et

al., 2005 Dopaxanthin I

DOPA

Bougainvillea sp.

Kugler et al., 2007

Arginine-Bx

Arginine

Gomphrena globosa

Kugler et al., 2007

24

Review of literature Table 2.2. continued... 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.

CastellanosSantiago

and

Yahia, 2008 Valine isomer-Bx

Valine isomer

Opuntia sp.

CastellanosSantiago

and

Yahia, 2008 Phenethylamine-Bx

Phenethylamine

Opuntia sp.

CastellanosSantiago

and

Yahia, 2008 Methylated arginine- Methylated arginine

Amaranthus tricolor Biswas et al.,

Bx

L.

2012

Hygrocybe sp.

von

Fungal betaxanthins Muscaflavin§

NA

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

Amanita muscaria

Musso, 1979

Amanita muscaria

Musso, 1979

Amanita muscaria

Musso, 1979

Amanita muscaria

Musso, 1979

Amanita muscaria

Musso, 1979

I, miraxanthin III, 2aminoadipic acid-Bx 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

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, 25

Review of literature 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 cycloDOPA, 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,

26

Review of literature 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, cycloDOPA 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 2descarboxy-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 cycloDOPA (Hatlestad et al., 2012). This implies that DO activity is not involved in formation of cyclo-DOPA. 27


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. 28


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, Ca 2+ 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, 29

Review of literature 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 Ca 2+ (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 30

Review of literature 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), H 2O2, 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 Ca 2+, SA, H2O2, etc. Enhancement of betalains production on treatment with MeJA has been reported in red beet hairy root culture (Suresh et

31

Review of literature 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. PPOpolyphenol oxidase, POD-peroxidase. 32

Review of literature 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 a w 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 a w value. 2.6.1.4. Light Exposure to light including UV destabilises betalains (von Elbe et al., 1974; Attoe 33

Review of literature 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 40C there was no impact of illumination, whereas below 25C 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 (180C) 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 60C. 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 4C and 20C 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 37

Review of literature 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 38

Review of literature 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 39

Review of literature 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.

40