Study on Betalains in Celosia cristata Linn. Callus Culture and

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Bioactive Constituents, Metabolites, and Functions

Study on Betalains in Celosia cristata Linn. Callus Culture and Identification of New Malonylated Amaranthins Kateryna Lystvan, Agnieszka Kumorkiewicz, Edward Szneler, and Slawomir Wybraniec J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01014 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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Journal of Agricultural and Food Chemistry 1

Study on Betalains in Celosia cristata Linn. Callus Culture and Identification of

2

New Malonylated Amaranthins

3 4

Kateryna Lystvan*,†, Agnieszka Kumorkiewicz‡, Edward Szneler§, Sławomir Wybraniec*,‡

5 6 7 8 9 10 11

†

Department of Genetic Engineering, Institute of Cell Biology and Genetic Engineering of National

Academy of Sciences of Ukraine (NASU), Academika Zabolotnoho, 148, 03143, Kyiv, Ukraine ‡

Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and

Technology, Cracow University of Technology, ul. Warszawska 24, Cracow 31-155, Poland §

Department of Chemistry, NMR Div, Jagiellonian University, ul. Ingardena 3, 31-007 Cracow,

Poland

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Corresponding authors *

Tel.: 380-44-526-7104, Fax: 380-44-526-7104, E-mail: [email protected]

*

Tel.: 48-12-628-3074, Fax: 48-12-628-2036, E-mail: [email protected] ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry 28 29

Abstract

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Betacyanins and betaxanthins were characterized and determined in an intensely pigmented red-

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coloured callus culture of Celosia cristata L. (Amaranthaceae). A new malonyl derivative, 6'-O-

33

malonyl-amaranthin (celoscristatin) was isolated and identified by spectroscopic and mass

34

spectrometric techniques. Its stereoisomer, 4'-O-malonyl-amaranthin (celoscristatin acyl-migrated)

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as well as its 15R diastereomer, were also detected in the callus as a result of malonyl group

36

migration in celoscristatin/isoceloscristatin, respectively. Amaranthin occurs in the callus as the

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major betacyanin, followed by celoscristatin, betanin, phyllocactin and other minor betacyanins.

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The effect of different carbon sources on the growth rates of Celosia callus as well as on betalains

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profiles in the callus cultures was studied. High dopamine content in callus culture was determined

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and compared with the content in C. cristata inflorescences. The dopamine-based betalain

41

(miraxanthin V) was detected as the main betaxanthin in the callus, however, at much lower

42

concentration level than the identified betacyanins. The studied callus culture of C. cristata can

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accumulate betalains in amounts which approach the quantities produced by most known high-

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yielding plant species.

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KEYWORDS: betacyanins; betalains; Celosia; malonylated derivatives; dopamine; callus culture

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INTRODUCTION

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Celosia cristata L. (Amaranthaceae), native to South America and very popular around the world,

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belongs to Celosia genus of edible and ornamental plants of about 60 species (family

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Amaranthaceae, order Caryophyllales).1 Numerous biological activities have been attributed to this

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species, namely antioxidant, hepatoprotective,2 - 4 immunostimulating2 as well as antiviral ones.5 Its

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inflorescences are coloured from yellow to violet due to betalain pigments content and exhibit an

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uncommon shape.1

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Growing interest in betalains is stimulated mainly by their chemopreventive and strong antioxidant

65

properties6-13 as well as their application as natural colorants in food industry.14,15 Reactive oxygen

66

and nitrogen species, responsible for chronic inflammation induced by biological, chemical and

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physical factors have been associated with an increased risk of human cancer as well as some other

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diseases such as atherosclerosis. Recent studies indicated that betalains can act as a very potent

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scavengers of inflammation factors and can improve various health conditions related to

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inflammation.16,17 There has been a growing interest in betacyanins (Figure 1) as potential

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chemopreventive agents capable of stopping tumor growth, indicating that the sources of

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betacyanins (including the root of Beta vulgaris L.) deserve increased attention in search of

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anticancer preparations.7,8,15,18-20

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Our studies on searching for new betacyanins in edible plant parts resulted in elucidation of several

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acylated pigment structures.21-23 Some of them were identified as intriguing malonylated

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betacyanins existing in isomeric versions following the acyl migration phenomenon.22,23 One of the

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most important betacyanins is betanidin 5-O-(6'-O-malonyl)-β-sophoroside (mammillarinin) which

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is endogenously present in fruits of Mammillaria species, frequently as a dominating pigment, as

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reported previously.23 Furthermore, except for its epimer, two other positional isomers of

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mammillarinin were identified as betanidin/isobetanidin 5-O-(4'-O-malonyl)-β-sophoroside which

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occur in the fruits as the acyl migration products.23 Our further efforts have been directed to



determination of betacyanin profiles in in vitro cultures as promising sources of new or rare

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betacyanins. One of them is a callus of Celosia cristata.

84

Previous studies1,11,24 demonstrated general betacyanin patterns in the Celosia genus. In C. cristata

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species, amaranthin and betanin (Figure 1) as well as celosianin I and celosianin II were identified11

86

whereas in C. plumosa L. only amaranthin was reported.1 The latter pigment, which is the most

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polar betacyanin to date, is the most characteristic pigment for the Amaranthaceae family.24

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It is known that in vitro cultures of various types (cultures of plant cells, tissues and organs) are

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considered as a novel source of biomass.25 In vitro cultures compared to wild or cultivated plants

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exhibit a large number of significant advantages. Stable and controlled growth conditions, the

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absence of pathogens, the ease to change external conditions, and relatively quick selection of

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highly productive cultures are among them. Moreover, new secondary metabolites, which are not

93

found in the corresponding intact plants, can be found in such cultures.25

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Several betalains have been produced by in vitro cultures of such plants as Myrtillocactus

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geometrizans Mart., Portulaca grandiflora Hook., Beta vulgaris L., Chenopodium rubrum L.,

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Chenopodium quinoa Willd and Phytolacca americana L., with the purpose of studying their

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biosynthesis and eventual commercial application.26,27,28 Studies on M. candida Scheidw. callus

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were performed to achieve cultures with higher pigmentation,29 however, no pigment identification

99

was accomplished.

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Recently, four betalains have been produced in cell cultures obtained from hypocotyls of C.

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argentea var. plumosa and maintained as two stable and differently colored (yellow and red) cell

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lines.30 However, qualitatively, no progress in biosynthetic production of novel betacyanins,

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especially the acylated ones, have been observed during the last decade. In vitro synthesis of novel

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betacyanins would be of high significance for further studies of their potential bioactivities.

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The callus culture of C. cristata which is cultivated in the Institute of Cell Biology and Genetic

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Engineering of NASU (National Academy of Sciences of Ukraine) produces significant amounts of

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betalains (Figure 2). In this contribution, the total betalain production and the detailed betacyanin

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and betaxanthin profiles of this callus culture have been investigated, including a dependence on ACS Paragon Plus Environment

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cultural media carbon sources.31,32 In addition, determination of dopamine as the precursor of

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dopamine-derived betalains in the callus culture was performed. These pigments (miraxanthin V24,33

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and 2-decarboxy-betanidin24 belonging to decarboxylated betalains35) were also detected in natural

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plant samples.24 Recently, their presence was also confirmed in the cell cultures of C. argentea var.

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plumosa.30

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MATERIALS AND METHODS

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Chemicals and Reference Compounds. Formic acid, LC-MS-grade acetonitrile and methanol,

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TMB ELISA Peroxidase Substrate (3,3',5,5'-tetramethylbenzidine) as well as dopamine

119

hydrochloride used as standard were obtained from Merck (Darmstadt, Germany). HPLC-grade

120

acetonitrile was obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO).

121

Deionized water for HPLC was purified using Milli-Q system (Millipore Corp., MA) or was

122

obtained from Merck (Darmstadt, Germany).

123

For structure confirmation (the retention time, absorption maximum and m/z value), completely

124

elucidated reference betacyanins (Figure 1) as well as their C-15 diastereomers (mostly by LC-

125

DAD-ESI-MS/MS and 2D-NMR) were derived from extracts of fruits, flowers or leaves of the

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following plants: betanidin 5-O-β-glucoside (betanin) from Beta vulgaris L.,14,15 betanidin 5-O-β-

127

glucuronosyl-glucoside (amaranthin) from Iresine herbstii35 and betanidin 5-O-(6'-O-malonyl)-β-

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glucoside (phyllocactin) from Hylocereus polyrhizus.21,22

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Plant Material. The initial long-term cultivated callus culture of Celosia cristata L. (Figure 2) was

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obtained from Germplasm bank of world flora of Institute of Cell Biology and Genetic Engineering

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of NAS of Ukraine, in which has been maintained for more than 10 years. The shoots of in vitro

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cultivated C. cristata plants were used to initiate the callus culture. During the first years of

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cultivation, the callus was characterized as yellowish-white cell mass. However, after the addition

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of casein hydrolysate to the cultural medium, the red pigments were being produced by this cell ACS Paragon Plus Environment


line. This ability of callus has remained for more than four years. The callus is a friable red-violet

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colored mass with areas of different color saturation, without any sign of organogenesis. The

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spontaneously appearing uncolored callus pieces were observed during the cultivation and were

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discarded. Light microscopy has shown that the callus consists of both round- and elongate-shaped

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cells with small amount of chloroplasts. Differently coloured inflorescences of C. cristata and C.

141

plumosa were purchased in a local market in summer 2016 and subsequently air-dried. The identity

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of this plant material was verified by Dr Mykyta Peregrym (O.V. Fomin Botanical Garden, Kyiv,

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Ukraine).

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Callus Culture Cultivation Conditions. The callus culture was cultivated on KC medium

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(Murashige and Skoog medium36 supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 1

147

mg/L), 6-benzylaminopurine (1 mg/L), casein hydrolysate (1 g/L), 30 g/l sucrose and solidified by

148

agar (8 g/l)) in Petri dishes. Cultures were maintained at 23±1 0C with a 16-hour photoperiod with

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subcultivation every two weeks. The growth index was determined for the evaluation of callus

150

growth rate. It was calculated as the callus fresh weight ratio at the end/at the beginning of the

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passage.

152 153

Cultivation of the C. cristata Callus Culture on Media with Different Carbon Sources.

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The callus culture of C. cristata was cultivated on medium which had the same salt and

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phytohormonal composition as above-mentioned KC medium. However, in order to form different

156

callus samples, instead of using sucrose as a source of carbon, 30 g/L of appropriate sugars

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(glucose, fructose, galactose, arabinose, rhamnose, maltose, xylose, lactose, sorbitol, and mannitol,

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respectively) were added to the medium. To prevent possible sugars degradation and the reduction

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of their amount in the nutrient medium during autoclaving, the corresponding sugar solutions were

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sterilized by filtering through bacterial filters with a pore size of 0.22 µm and added to the sterile

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medium before pouring them into Petri dishes. Before cultivating on sugar-enriched media, the C.

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cristata callus was cultured for 10 d on a KC medium that did not contain any sugars, so the callus ACS Paragon Plus Environment

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cells used their internal supply of simple and complex sugars. Cultivation on sugar-enriched media

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was carried out for 2 w, after which the callus was dried by lyophilization and the content of

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betalains was investigated.

166 167

Preparation of Extracts. For the study of total betalain content, the samples of air-dried (10-60

168

mg) or fresh (0.3-1 g) callus were homogenized manually with distilled water (2 x 0.5 mL). The

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homogenates were centrifuged at 14000 g for 10 min. The supernatant was used for

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spectrophotometric measurements or HPLC-DAD analyses without any further purification.

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For semipreparative pigment isolation, the C. cristata callus (150 g) was extracted three times with

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400 mL of water and subsequently filtered and concentrated according to a procedure of

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Kumorkiewicz and Wybraniec.37 Namely, the filtration was performed through a layer of

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0.063/0.200 mm silica gel (J.T. Baker, Deventer, Holland) to remove hydrocolloids and proteins to

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obtain a clear solution and subsequently through a 0.2 mm i.d. pore size filter (Millipore, Bedford,

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MA). The extract was concentrated using a freeze-drier. The pigment extract was purified by flash

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chromatography and preparative high-performance liquid chromatography.38 For the co-injection

178

experiments, the extracts of Beta vulgaris L. roots,14,15 Hylocereus polyrhizus fruits22 and Iresine

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herbstii leaves35 from previous studies were processed by a similar procedure.

180 181

Quantitation of the Total Concentration of Betalains in the Extracts of the Callus. The

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concentration of betacyanins and betaxanthins as well as the total concentration of betalains in the

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aqueous extracts of callus were determined by measuring of the extract optical density at the

184

different wavelengths (476, 538 and 600 nm) and calculating the concentrations by the method

185

proposed by Nilsson.39 All concentrations were expressed as mg/g dry (DW) or fresh (FW) weight

186

of callus. A spectrofluorometer Fluorat-02-Panorama (Lumex, St. Petersburg, Russian Federation)

187

used only in the spectrophotometric mode (absorption measurement) was utilized for the

188

quantitation of betalain-containing extracts.

189



Statistics. The growth index of callus and the content of dopamine and betalains as well as the

191

normalized concentration of betalains were expressed as the mean values of n (n=3÷100)

192

measurements ± standard error of the mean.

193 194

Analytical HPLC. For the study of dopamine content, a 10AVP HPLC system (Shimadzu Corp.,

195

Kyoto, Japan) equipped with a SPD-M10AVP photodiode array detector was used. Reversed phase

196

chromatography was performed with a 250 mm x 4.6 mm i.d., 5 µm, Zorbax Eclipse XDB-C18

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chromatographic column (Agilent, Santa Clara, CA) with a 20 mm x 3.9 mm i.d., 5 µm,

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Symmetry C8 Sentry Guard Cartridge (Waters, Milford, MA). For the separation, a gradient system

199

consisting of 1% aqueous formic acid (solvent A) and acetonitrile (solvent B) was used as follows:

200

0 min, 3% B; increasing linearly to 15 min, 30% B. The column was thermostated at 40 °C. The

201

injection volume was 10 µL, the flow rate 0.8 mL/min. Eluates were followed at λ 272 nm. The

202

identity of dopamine in the samples was confirmed by co-elution of the sample analyte with the

203

standard substance as well as by additional analyses by LC-DAD-ESI-MS.

204 205

Flash Chromatography and Semipreparative HPLC. For the concentration and purification of

206

the C. cristata callus pigments, a flash chromatography system consisting of a preparative HPLC

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system with LC-20AP pumps, UV/Vis SPD-20AV detector and LabSolutions 5.51 operating

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software (Shimadzu Corp.) equipped with a 250 mm x 50 mm i.d., 30 µm, C18 flash column

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(Interchim, Montluçon, France) was applied. For the separation, a gradient system consisting of

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0.2% aqueous formic acid (solvent A) and acetonitrile (solvent B) was used as follows: 0 min, 7%

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B; increasing linearly to 30 min, 40% B. The injection volume was 25 mL and the flow rate was 50

212

mL/min. The columns were thermostated at 25 °C and the detection was performed at 538 and 480

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nm. The yield of the purified extract fraction was ca. 300 mg per run.

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Further separation and isolation of pigments was performed on a 250 mm x 10 mm i.d., 10 µm,

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HPLC semipreparative column Luna C18(2) with a 10 mm x 10 mm i.d. guard column of the same

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material (Phenomenex, Torrance, CA) under the following gradient system consisting of 1% ACS Paragon Plus Environment

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aqueous formic acid (solvent A) and acetonitrile (solvent B) as follows: 0 min, 5% B; increasing

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linearly to 50 min, 25% B. The injection volume was 2 mL and the flow rate was 3 mL/min.

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Detection was generally performed at 538, 505, 480 and 310 nm with a PDA UV/Vis detector. The

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columns were thermostated at 25 °C. The eluates were pooled and preconcentrated under reduced

221

pressure at 25 °C and finally freeze-dried to obtain pure pigments. The yield of the purified

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celoscristatin 3 for the structural analysis was ca. 0.5 mg per run.

223 224

Chromatographic Analysis by LC-DAD-ESI-MS/MS System. For the chromatographic and low-

225

resolution mass spectrometric analyses of the all extracts and purified pigments, an LCMS-8030

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triple-quadrupole mass spectrometric system coupled to LC-20ADXR HPLC pumps controlled

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with LabSolutions software (Shimadzu, Japan) was used. For the aim of quantitation of the pigment

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profiles, due to the lack of betalainic standards, all the relevant chromatographic peaks of

229

betacyanins and betaxanthins were subjected to normalization based on peak areas in the selected

230

ion chromatograms from the MS detector. The samples were eluted through a 150 mm x 4.6 mm

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i.d., 5.0 µm, Kinetex C18 chromatographic column preceded by a guard column of the same

232

material (Phenomenex, Torrance, CA). The injection volume was 30 µL, and the flow rate was 0.5

233

mL/min. The column was thermostated at 40 ºC. The separation of the analytes was performed with

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a binary gradient elution. The mobile phases were: A – 2% formic acid in water, and B - pure

235

methanol. The gradient profile was: 0 min, 5% B; increasing linearly to 12 min, 30% B; increasing

236

linearly to 15 min, 80% B. Online UV/Vis spectra acquisition was performed using the PDA

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(photodiode-array detection) mode typically at 538, 505, 480 and 310 nm. The positive ion

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electrospray mass spectra were recorded on the LC-MS system which was controlled with

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LabSolutions software (electrospray voltage 4.5 kV; capillary 250 °C; sheath gas: N2), recording

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total ion chromatograms, mass spectra and ion chromatograms in selected ion monitoring mode

241

(SIM) as well as the fragmentation spectra. Argon was used as the collision gas for CID

242

experiments. The relative collision energies for MS/MS analyses were set at -35 V.

243



Chemical Degradation Analysis of Celoscristatin. For the identification of deacylated betacyanin

245

3 (600 µM), a modified procedure of Minale et al.40 consisting of alkaline hydrolysis in

246

deoxygenated 0.1 M NaOH for 10 min in an ice-bath was applied. Subsequent acidification of the

247

generated mixture with 0.1 M HCl resulted in recovery of an epimerized mixture of deacylated

248

betacyanins.

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For the confirmation of the main structural unit in the analyzed 6'-O-malonyl-amaranthin 3,

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enzymatic hydrolysis of purified 6'-O-malonyl-amaranthin 3 (300 µM) was performed in the

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presence of β-glucuronidase from Helix pomatia (Sigma-Aldrich) at pH 5.0 and 37 °C for 90 min.12

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The reaction progress was monitored by a direct injection of the mixture to the LC-DAD-MS

253

system without further purification.

254 255

Chromatographic Analysis with Detection by Ion-trap Time-of-flight System (LCMS-IT-

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TOF). The high resolution mass spectra (HRMS) of the unknown betacyanin, 3, as well as its

257

HRMS fragmentation pattern were analyzed using LCMS-IT-TOF mass spectrometer (Shimadzu)

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equipped with an electrospray (ESI) ion source and coupled to a Prominence HPLC (Shimadzu).

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Separation of compounds was carried on a 50 mm x 2.1 mm i.d., 1.9 µm Shim Pack GISS C18

260

column (Shimadzu). The injection volume was 2 µL, and the flow rate was 0.2 mL/min. The

261

column was thermostated at 40 ºC. The separation of the analytes was performed with a binary

262

gradient elution. The mobile phases were: A – 0.1% formic acid in water, and B - pure methanol.

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The gradient profile was: 0 min, 5% B; increasing linearly to 12 min, 30% B; increasing linearly to

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17 min, 80% B. Parameters of LCMS-IT-TOF spectrometer were set as follows: curved

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desolvation line (CDL) and heat block temperature 230 ˚C, nebulizing gas flow rate 1.5 L/min and

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capillary voltage 4.5 kV. All mass spectra, including fragmentation mass spectra, were recorded in

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the positive ion mode with mass range 100-2000 Da and collision energy between 12-50%,

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depending of the structure of each compound. The results of the HRMS experiments were studied

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using the Formula Predictor within the LCMS Solution software. Only empirical formulae with an

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mass error below 5 ppm were taken into account. ACS Paragon Plus Environment

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Journal of Agricultural and Food Chemistry

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NMR Experiments. The NMR spectra were recorded for 8 mg of celoscristatin sample on an

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Avance III 600 MHz instrument (Bruker Corp., Billerica, MA) in non-acidified D2O as well as in a

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mixture of H2O and D2O (90/10, v/v) at 300 K.11 The reference for the 1H chemical shifts was the

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residual solvent signal at δ=4.70 ppm (D2O) relative to TMS. All 1D (1H) and 2D NMR (gCOSY,

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gHSQC, gHMBC, g=gradient enhanced) measurements were performed using standard Bruker

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pulse sequences.

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RESULTS AND DISCUSSION

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Betalain and Dopamine Content in the Callus Culture of C. cristata. The growth rate of the

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long-term cultivated callus culture of C. cristata (Figure 2) producing the red-violet pigments was

283

determined. Due to the fact that the growth index was 2.9±0.9, the process rate can be considered as

284

fast and indicates the suitability of the chosen cultural media to the fast growth of the cell culture.

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The total pigment content in the callus was determined spectrophotometrically39 as 0.15 ± 0.01

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mg/g FW, from which ca. 73% were betacyanins (0.11± 0.01 mg/g FW of callus). This

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concentration is lower than the level of betalains accumulation in commercially used roots of red

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beetroot (up to 20 mg/g FW of betalains) but approaching the level in other sources of these

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pigments, e.g. fruits of Opuntia cacti (ca. 0.8 mg/g FW) and fruits of Hylocereus cacti (0.32-0.4

290

mg/g FW).14 In addition, a possibility of synthesis of novel betacyanins in the callus is still

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significant for any bioactivity studies.

292

Dopamine content in the callus culture was investigated and compared with the one in the

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inflorescenses of C. argentea var. plumosa and var. cristata, collected in nature, which were

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reported earlier as a source of dopamine.1

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In our study, dopamine in significant quantities was found only in 77% of the inflorescences

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samples (n = 39) whereas all investigated samples of callus (n = 80) contained this substance. High

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dopamine content (12.24 ± 0.89 mg/g of DW) was determined in the analyzed samples of air-dried ACS Paragon Plus Environment


callus cultures which was ca. two times higher than in the inflorescence samples (5.76 ± 0.83 mg/g

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of DW) (Table 1).

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The earlier data describing the presence of dopamine in Celosia species vary greatly. Schliemann et

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al.1 found dopamine in a large amount (6.3 mg/g FW) in yellow inflorescences of C. argentea var.

302

cristata. However, it was not detected at all by Guadarrama-Flores et al.30 in both inflorescences

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and hypocotyls of C. argentea. Instead, high dopamine content in yellow and red lines of C.

304

argentea callus culture was determined (17.7 and 19.6 mg/g DW, respectively), that is similar to

305

our results. Suspension culture of the C. argentea accumulated the catecholamine in even larger

306

quantities (ca. 30-40 mg/g DW).30

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In general, from betalain-synthesizing species, dopamine, was isolated for the first time in 1944

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from Hermidium (Mirabilis) alipes (Nyctaginaceae).41 To date, it has been found in more than 40

309

species of plants of different genera.42 However, large amounts of dopamine are present only in a

310

limited number of species. Green alga Ulvaria obscura (Ulvaceae) and legume Mucuna pruriens

311

(Fabaceae) are among them and contain dopamine in amounts of 4.4% DW43 and 0.5-1% DW44,

312

respectively. Most of other species which can produce dopamine, contain it at much smaller levels,

313

e.g., fruit peel of Cavendish banana Musa acuminata (0.1 mg/g) and fruit pulp of yellow banana

314

(0.042 mg/g FW).42 The callus culture investigated in this study contained 1.16 ± 0.07 mg/g FW of

315

dopamine, that is significantly higher than in most known dopamine-synthesizing plants.42

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The results presented in this section reveal that the studied callus culture of C. cristata can

317

accumulate betalains and dopamine in significant amounts, which are close to quantities, produced

318

by most of known high-yielding plant species, except for Beta vulgaris roots. The above data

319

suggest that the content of dopamine in inflorescences of Celosia species is greatly variable,

320

whereas its high content in the corresponding cell cultures is constant and characteristic for the

321

produced cultures.

322 323

LC-DAD-ESI-MS/MS and LCMS-IT-TOF Determination of Betacyanins. The detailed LC-

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DAD betacyanin profile in the analyzed Celosia cristata L. callus culture is presented in Figure 3 ACS Paragon Plus Environment

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and is listed in Table 2. Normalized concentrations (%) based on the peak areas in the selected ion

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LC-MS chromatograms of the main betalains are depicted in Table 3. The main two pigment pairs

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of the callus culture were readily identified in the LC-DAD-ESI-MS/MS chromatograms by their

328

characteristic pattern already observed in Iresine herbstii.11,34 The presence of the known structures

329

(Figure 1) of amaranthin 1 (betanidin 5-O-β-glucuronosylglucoside) and betanin 2 accompanied by

330

their C-15 isoforms (1' and 2') was confirmed by their wavelengths of maximum absorption in the

331

visible range λmax, their detected precursor ions at m/z 727 and 551, respectively (Table 2), as well

332

as by their retention times (co-elution with authentic standards isolated from Iresine herbstii leaves

333

(amaranthin)35 as well as from red beet extract (betanin).21,30,45

334

Other acylated betacyanins were detected by means of mass spectrometry and diode-array detection

335

coupled to HPLC. Two prominent chromatographic peaks of betacyanins, 3 and 5 (with their

336

respective isoforms, 3' and 5') were detected and identified as malonylated amaranthin (3) and

337

malonylated betanin (phyllocactin), 5. The precursor ion of 3/3' at m/z 813 suggested the presence

338

of malonylated amaranthin (813-727 = 86 Da). The presence of the fragmentation ion at m/z 637

339

after the detachment of the glucurunosyl moiety (Table 2 ) indicated the position of acylation at the

340

glucosyl ring (813-637 = 176 Da). Further HRMS determination of the exact m/z value of 3 in

341

conjunction with the HRMS fragmentation pattern of the precursor ion [M+H]+ (Table 4, Figure 4)

342

was performed by LCMS-IT-TOF and supported the identification of a malonylated amaranthin.

343

For this purpose, the exact m/z values of the characteristic fragments generated as a result of neutral

344

losses of glucuronosyl, malonyl and glucosyl as well as H2O and CO2 were measured which

345

matched the predicted values (Table 4). The betacyanin 3 has never been identified in any plant

346

material, therefore, additional NMR structure elucidation was performed. The trivial name of

347

celoscristatin is proposed for this new pigment, 3. The λmax of 536 nm determined for 1/1' and 3/3'

348

confirms the presence of the typical absorption bands for amaranthin and acylated amaranthin by

349

aliphatic acids.11,34



The presence of phyllocactin/isophyllocactin (5/5') in the callus was additionally confirmed by co-

351

elution of the sample with the authentic standard derived from Hylocereus polyrhizus fruits21,22 and

352

the spectral data ([M+H]+ m/z 637, λmax 536 nm).

353

Further inspection of the chromatograms and mass spectra revealed the positional stereoisomers of

354

3/3' and 5/5' formed as a result of malonyl group migration (4/4' and 6/6', respectively).

355

The phenomenon of acyl migration in betacyanins has been recently noticed22,23 and further studies

356

supported this finding.46 In this study, the interconversion between purified 3 and 4 as well as

357

between 3' and 4', was observed in alkaline solutions (pH 10) as well as in neutral solutions (at a

358

slower reaction rate). In each case, the resulting equilibrated pigment composition favours the 6'-O-

359

malonylated forms (3/3'). The formation of an intermediate strainless cyclic ortho-ester structure

360

between the glucosidic O-4' and O-6' hydroxyls, which was reported frequently in many cases of

361

acyl migration in acylated β-D-glucosides is responsible for the interconversions.46 Therefore, the

362

isomeric betacyanins 5/5' can be assigned as 4'-O-malonyl-amaranthin/isoamaranthin. Similarly, the

363

presence of 4'-O-malonylated stereoisomers 6/6' of phyllocactin/isophyllocactin 5/5' was confirmed

364

by co-elution with the authentic standards derived from Hylocereus polyrhizus.22,46

365

Betaxanthins were present in the callus at much lower levels (2-10% of betacyanins concentration).

366

The dopamine-based betaxanthin B31,18,19,24 was detected as the most prominent betaxanthin in the

367

callus and reached the highest relative peak area of 95.3% (Table 2). Other betaxanthins were found

368

at relatively low levels and were based on γ-aminobutyric acid (B1), proline (B2), valine (B4),

369

phenylalanine (B5), and tryptophan (B6). All these betaxanthins have already been identified in

370

other betalain producing plants.18,19,24

371 372

Alkaline Deacylation of Celoscristatin. The carbohydrate system as well as possible acylation

373

group was indicated by the alkaline deacylation of 3 and subsequent acidification of the resulting

374

mixture with HCl. The liberation of a mixture of amaranthin/isoamaranthin 1/1' analyzed

375

chromatographically suggested the presence of the 5-O-β-glucuronosyloglucosidic system in the


Page 14 of 38

Page 15 of 38


376

structure of 3. The difference between m/z of the analyzed 3 and 1 additionally indicated malonyl as

377

the acylating moiety.

378 379

Enzymatic Hydrolysis of Celoscristatin. For the confirmation of the main structural unit in the

380

analyzed 6'-O-malonyl-amaranthin 3, enzymatic hydrolysis of the newly identified and purified

381

pigment 3 by β-glucuronidase was performed40 in comparison to the known betacyanin, amaranthin,

382

in which the glucuronosyl moiety is hydrolytically detached. Acylation of the glucuronosyl ring

383

prevents betacyanins from the action of the enzyme.40 The results of the assay confirmed that the

384

hydrolysis of 3 had occurred and the deglucuronosylated betacyanin (phyllocactin 5) was detected

385

by LC-DAD-MS which indicated that the glucuronosyl ring is not acylated but the glucosyl

386

instead.. Additional comparison of the retention time of generated phyllocactin 5 with the

387

authenthic standard obtained from fruits of Hylocereus polyrhizus21,22 directly supported

388

malonylation of the substrate 3 at carbon C-6'.

389 390

NMR Structural Elucidation of Celoscristatin. The characteristic NMR signals of the aglycone

391

and glucose moieties confirmed the presence of a betanin derived compound.21-24,34,45 The

392

individual coupled 1H-spin systems of the aglycone (H-2, H-3ab, H-11, H-12; H-14ab, H-15) were

393

assigned in 1H NMR and gCOSY spectra. The three-spin system (H-15/H-14ab) showed easily

394

distinguishable cross-peaks in the gCOSY spectrum. Similarly, another spin system for H-2/H-3ab

395

was observable, indicating the presence of the carboxyl moiety at C-2. The doublets for the H-11

396

and H-12 protons were readily distinguishable by their low- and high-field shifts, respectively. A

397

broad signal for H-18 was detected by 1H NMR for freshly prepared solution of the pigment

398

avoiding the fast deuterium exchange.45 The dihydroindolic system was assigned by gHSQC

399

correlations of H-2, H-3ab, H-4 and H-7 with their respective carbons. The correlations of C-5 to H-

400

4/H-7, C-6 to H-4, C-8 to H-4, C-9 to H-7/H-3ab and C-10 to H-3ab (the dihydroindolic system)

401

as well as C-12 to H-14ab and H-18, C-13 to H-15 and H-18, C-17 to H-18, C-18 to H-12, C-19 to

402

H14ab, and C-20 to H-18 (the dihydropyridinic system) were determined by gHMBC in D2O but ACS Paragon Plus Environment


also in H2O/D2O (90/10, v/v) (necessary for obtaining the signals of exchangeable protons H-12 and

404

H-18) (Figure 5, Table 5).

405

The other 13C chemical shifts for carbons directly bound to protons were assigned by gHSQC

406

correlations. The presence of two anomeric protons H-1' and H-1'' indicating two sugar units by

407

their characteristic downfield shifts was readily observed. The gHMBC and gCOSY correlations

408

clearly ascertained the two sugar ring systems (Figure 5, Table 5). Detection of correlations

409

between H-2' and C-1'' as well as C-2' and H-1'' established the attachment position of the second

410

sugar moiety by gHMBC. The position of the glycosidic bond at the phenolic carbon C-5 was

411

readily confirmed by the gHMBC correlation with the anomeric proton H-1' and was earlier

412

indicated by the chemical shift difference between H-4 and H-7 of 0.09 ppm.41 The β-linkage

413

between the aglycone and glucopyranosyl moiety was denoted by the three-bond vicinal proton

414

coupling constant 3J1'-2' ~6-7 Hz. Similarly, the β-linkage was determined for the glucuronosyl

415

moiety. The presence of the glucuronosyl ring was finally indicated by the gHMBC detection of the

416

carboxyl carbon C-6'' at δ 177.2 ppm correlating with the protons H-5'' and H-4'' (Figure 5, Table

417

5).

418

A definitive evidence of the acyl moiety position was provided by the downfield chemical shift of

419

H-6'a/6'b protons. Further confirmation of this linkage position was obtained by the gHMBC

420

correlations (Figure 5) of C-1''' to H-6'a and H-6'b. Additional gHMBC and gHSQC experiments

421

performed in a mixture of H2O and D2O (90/10, v/v), which was frequently reported in betacyanins

422

(e.g. phyllocactin or mammillarinin),22,23 supported detection of the malonyl moiety with H-2'''a

423

and H-2'''b protons not being exchanged with deuterium. Above analysis completed the structure

424

identification of 3 as betanidin 6'-malonyl-5-O-(2'-O-β-D-glucuronosyl)-β-D-glucopyranoside)

425

which is the first reported malonylated amaranthin (celoscristatin).

426 427

Discussion of Betalain Profile in the Callus Culture. The revealed betacyanin profile exhibits

428

both similarities and differences to the patterns of pigments in inflorescences and cell cultures of

429

Celosia species which were reported earlier.1, 11, 30 ACS Paragon Plus Environment

Page 16 of 38

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430

The presence of amaranthin, frequently known as the major pigment, followed by betanin and their

431

diastereomers is typical for a variety of species of Amaranthaceae family.11 It was also shown that

432

amaranthin is the predominant betacyanin in C. argentea and C. cristata inflorescensces1,11 and was

433

found in callus and suspension cultures of C. argentea var. plumosa.30 The presence of betanin was

434

also reported for both of these intact plants11 and cell lines.30 The mentioned betacyanins have been

435

expectedly found in the studied callus and their total amount reached 78% of relative concentration

436

of all detected betacyanins.

437

The presence of the large amounts of dopamine in the investigated callus results in the production

438

of dopamine-based betaxanthin (B3) which reaches concentration comparable to betanin level.

439

Interestingly, the calli do not contain any dopamine-derived betacyanins, i.e. pigments which

440

contain 2-decarboxy-betanidin as a core structure. The obtained results differ from those of

441

dopamine-derived betacyanins determination in callus and suspension cultures of C. argentea var.

442

plumosa30 where their concentration in the calli was similar to the content of amaranthin (0.45-1.3

443

mg/g DW of 2-decarboxy-betanidin vs 1.3-5 mg/g DW of amaranthin). However, in the suspension

444

cultures the content of amaranthin was several times exceeded (ca. 2 mg/g DW of 2-decarboxy-

445

betanidin vs 0.5-0.6 mg/g DW of amaranthin). Presumably, in the callus developed in the current

446

study, dopamine is not involved in the processes of decarboxylated betacyanins biosynthesis and

447

only accumulates in the cells, reason for which remains unclear.

448

Similarly, the investigated callus cultures do not contain acylated derivatives characteristic for

449

intact Celosia plant, namely, p-coumaroyl- (celosianin I and isocelosianin I) and feruloyl-

450

(celosianin II and isocelosianin II) derivatives ,11 the amount of which can reach 40% of the total

451

betacyanins content.11 Instead, a distinctive feature of the studied in vitro cultures is a significant

452

amount of malonylated derivatives. Their content is higher than 20% of the total betacyanins

453

amount in the calli. Four of these derivatives (3/3' and 4/4') are the new betacyanins, not

454

characterized earlier in intact plants of Celosia species nor in other betalain-synthesizing ones. The

455

other pigments, namely 6'-O-malonyl-betanin/isobetanin (phyllocactin/isophyllocactin, 5/5') and



their isomers 6/6', have not been reported earlier for Celosia species, but were typically described

457

for the species of Cactaceae family.21,22

458

Malonylation along with glycosylation, hydroxycinnamates and glutathione conjugation, and

459

sulfonylation is a method of detoxification of harmful compounds including endobiotics (eg,

460

secondary metabolites) and xenobiotics.47 It should be noted that great differences exist between

461

growing conditions of intact plants versus in vitro cultures: type of nutrition (hetero- or

462

autotrophic), very different humidity level and content of carbon dioxide and ethylene, presence of

463

phytohormones, etc. All these factors can significantly affect plant biosynthesis and their influence

464

on the process of betacyanin acylation may be suggested as well. This is supported by initiation of

465

biosynthesis of significant amounts (6-15%) of the malonyl derivatives of 2-descarboxy-betanin and

466

betanin in hairy roots cultures of Beta vulgaris .48

467 468

Qualitative and Quantitative Composition Study of C. cristata Callus Betalains Depending on

469

the Carbon Source. Sugars can strongly influence the composition and the content of secondary

470

metabolites. There are reports describing the effects of various sugars on the total amounts of

471

betalains, e.g., in roots of red beet31 or sugar beet tumor cultures.26 However, to the best of our

472

knowledge, the number of such studies on betalain-synthesizing species is rather limited. There is

473

practically no work related to the investigations on changes in the detailed profiles of betalains in in

474

vitro cultures under the influence of such factors as sugars. Therefore, the effect of different carbon

475

sources on the growth rates of Celosia callus as well as on betalains biosynthesis in the callus

476

cultures has been studied.

477

As depicted in Figure 6, the Celosia callus culture was capable of growing on media with sucrose,

478

glucose, fructose and maltose. The other sugars, hexoses as galactose and rhamnose, pentoses as

479

arabinose and xylose, polyols as sorbitol and mannitol and disaccharide lactose failed to support the

480

growth of the callus. The study also revealed that the callus growth rate on the medium with glucose

481

was noticeably lower than on the media containing sucrose, maltose and fructose. However, this


Page 18 of 38

Page 19 of 38


482

callus produced betalains at the fastest rate (Figure 7). Similar effect of simple carbohydrates in

483

comparison with sucrose was observed for the transformed cells of sugar beet.26

484

The studies on the influence of different carbon sources on the composition of C. cristata callus

485

revealed that the betalainic profiles were similar in all of the tested media. The percentage content

486

of the main betacyanins present in the callus cultures is presented in Table 3. It indicates that the

487

qualitative and quantitative composition of the pigments do not depend on the type of carbohydrate

488

source. Furthermore, it was noticed that, in each case, the formation of betacyanins was

489

significantly higher (up to 10 times) than the production of betaxanthins.

490

In conclusion, from the practical point of view, the consistency of the betalainic profiles enables

491

control the callus growth rate by the change of the sugar types in the nutrient media or control the

492

total pigment production without the change of the pigment profiles. In addition, the changing of

493

the spectrum of synthesized pigments in the studied callus culture compared to corresponding intact

494

Celosia varieties as well as the possibility of generation of new betacyanins has been reported. The

495

investigated callus culture due to abundance and diversity of malonylated derivatives can become a

496

convenient object for study of biosynthetic processes of betacyanins acylation which is

497

characterized to date rather poorly. The biological activity of isolated malonylated betacyanins has

498

been not studied so far and the results obtained in this study enable convenient control of

499

comprehensive research on bioactivities of the produced pigments in the C. cristata callus at large

500

scale.

501 502

Acknowledgments

503 504

We are grateful to Germplasm bank of world flora of Institute of Cell Biology and Genetic

505

Engineering of NAS of Ukraine for providing an initial callus culture of Celosa cristata L. The

506

authors thank Beata Wileńska Ph.D., eng. and Bartłomiej Fedorczyk M.Sc. from Laboratory of

507

Biologically Active Compounds (Warsaw University) for the excellent technical assistance with

508

LCMS-IT-TOF experiments. ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry 509 510

References

511 512

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513

Phytochemistry 2001, 58, 159-165.

514

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515

antihepatotoxic polysaccharide isolated from Celosia argentea. Planta Med. 1997, 63, 216-219.

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(43) Van Alstyne, K.; Nelson, A.; Vyvyan, J.; Cancilla, D. Dopamine functions as an antiherbivore

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defense in the temperate green alga Ulvaria obscura. Oecologia 2006, 148, 304–311.

616

(44) Wichers, H.; Visser, J.; Huizing, H.; Pras, N. Occurrenceof L-DOPA and dopamine in plants

617

and cell cultures of Mucuna pruriens and effects of 2,4-D and NaCI on these compounds. Plant

618

Cell, Tissue Organ Cult. 1993, 33, 259-264.

619

(45) Strack, D.; Steglich, W.; Wray, V. Betalains. In Methods in Plant Biochemistry, Dey, P. M.;

620

Harborne, J. B.; Waterman, P. G., Eds.; Academic Press: London, UK, 1993; Vol. 8, pp 421-450.

621

(46) Wybraniec, S. Chromatographic investigation on acyl migration in betacyanins and their

622

decarboxylated derivatives. J. Chromatogr. B: Biomed. Sci. Appl. 2008, 861, 40-47.

623

(47) Sandermann, Jr, H. Plant metabolism of xenobiotics. Trends Biochem. Sci. 1992, 17, 82-84.

624

(48) Kobayashi, N.; Schmidt, J.; Wray, V.; Schliemann W. Formation and occurrence of dopamine-

625

derived betacyanins. Phytochemistry 2001, 56, 429-436.

626 627 628 629 630 631 632 633 634 635 636 637 638 639 640


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Page 25 of 38 641


List of figures

642 643

Figure 1. Chemical structures of betacyanins identified in the Celosia cristata L. callus culture.

644 645

Figure 2. The callus culture of Celosia cristata L.

646 647

Figure 3. The profile of betacyanins in the callus culture of Celosia cristata L. monitored by PDA

648

detector at 538 nm. The most abundant betaxanthin (miraxanthin V, B3) is indicated in the

649

chromatogram.

650 651

Figure 4. The HRMS fragmentation spectrum obtained by IT-TOF for celoscristatin 3 present in

652

Celosia cristata L. callus culture.

653 654

Figure 5. Important HMBC NMR correlations indicating the chromophoric structure and the

655

positions of the glycosidic bonds as well as the malonyl moiety in celoscristatin 3.

656 657

Figure 6. Growth rates of Celosia cristata L. callus on media contaning different sugars.

658 659

Figure 7. The total content (measured by spectrophotometry) of betacyanins and betaxanthins (mg/g

660

of dry weight) in Celosia cristata L. callus after two weeks of cultivation on nutrient media with the

661

addition of various sugars.

662 663 664 665 666 667



Page 26 of 38

Table 1. Content of Dopamine in Callus Culture of Celosia cristata and Inflorescences of C. cristata and C. plumosa. Content [mg/g] Inflorescences Callus culture

(violet)

dry weight fresh weight

12.24 ± 0.90

a

1.16 ± 0.07c

C. plumosa

C. cristata

(red) b

(orange) b

5.84 ± 2.12

5.83 ± 1.26

5.07 ± 1.70b

-

-

-

The data are expressed as mean ± standard error of the mean. The means are an average of n samples: for a n = 30, b n = 10, c n = 50; “-” – not studied


Page 27 of 38


Table 2. Chromatographic, Spectrophotometric and Low-Resolution Mass-Spectrometric (TripleQuadrupole) Data of the Analyzed Betaxanthin and Betacyanin Pigments in the Celosia cristata L. Callus Culture. No.

Rt [min]

Compound

m/z m/z from MS/MS λmax [nm] [M+H]+ of [M+H]+

Normalized concentration [%]a

Betaxanthins B1 γ-Aminobutyric acid-betaxanthin

7.7

462

297

8.3

-

b

309

B3 Dopamine-betaxanthin (miraxanthin V)

11.8

457

347

B4 Valine-betaxanthin

12.2

466

311

B5 Phenylalanine-betaxanthin

16.2

-

b

359

B6 Tryptophan-betaxanthin

16.4

472

398

Betanidin 5-O-β-glucuronosylglucoside (amaranthin)

7.3

536

727

551; 389

58.1

± 2.2

1' Isobetanidin 5-O-β-glucuronosylglucoside (isoamaranthin)

8.3

536

727

551; 389

4.8

± 1.4

8.9

535

551

389

8.1

± 0.5 ± 1.9

B2 Proline-betaxanthin (indicaxanthin)

-

1.1

± 0.23

1.3

± 0.19

95.3

± 2.5

0.67 ± 0.04 0.74 ± 0.03 0.89 ± 0.05

Betacyanins 1

2

Betanidin 5-O-β-glucoside (betanin)

3

6'-O-Malonyl-amaranthin (celoscristatin)

10.5

536

813

727; 637; 551; 389 15.1

2' Isobetanidin 5-O-β-glucoside (isobetanin)

10.7

535

551

389

0.82 ± 0.05

4'-O-Malonyl-amaranthin

10.9

536

813

727; 637; 551; 389

2.2

± 0.1

3' 6'-O-Malonyl-isoamaranthin (isoceloscristatin)

11.4

536

813

727; 637; 551; 389

1.7

± 0.2

4' 4'-O-Malonyl-isoamaranthin

11.7

536

813

727; 637; 551; 389

0.18 ± 0.04

4

5

6'-O-Malonyl-betanin (phyllocactin)

12.4

536

637

551; 389

6.9

± 0.4

6

4'-O-Malonyl-betanin

13.0

536

637

551; 389

0.9

± 0.02

13.6

536

637

551; 389

1.1

± 0.1

5' 6'-O-Malonyl-isobetanin (isophyllocactin) a

14.2 536 637 551; 389 6' 4'-O-Malonyl-betanin The data are expressed as mean ± standard error of the mean. b the λmax could not be observed.


0.10 ± 0.02


Table 3. Normalized Concentrations Based on Peak Areas in Chromatograms (Low-Resolution Mass-Spectrometric (Triple-Quadrupole)) Data of the Main Betacyanins of Celosia cristata L. Callus Culture Cultivated on Media with Different Carbon Sources Normalized concentration [%] in mediuma sucrose maltose glucose fructose 727 63.0 ± 2.5 64.8 ± 2.4 66.2 ± 1.6 64.0 ± 1.5 Amaranthin 727 10.4 ± 1.4 7.0 ± 0.6 8.3 ± 1.0 8.2 ± 0.9 Isoamaranthin 551 4.8 ± 0.5 5.2 ± 0.7 3.8 ± 0.2 5.1 ± 0.7 Betanin 813 16.4 ± 1.9 17.2 ± 1.7 16.8 ± 0.8 18.0 ± 1.3 Celoscristatin 813 1.9 ± 0.3 1.5 ± 0.2 1.6 ± 0.2 1.6 ± 0.1 Isoceloscristatin 637 2.7 ± 0.4 3.5 ± 0.6 2.5 ± 0.3 2.4 ± 0.3 Phyllocactin a The data are expressed as mean ± standard error of the mean. Compound

m/z


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Table 4. High-resolution Mass Spectrometric Data Obtained by IT-TOF for Celoscristatin 3 Present in Celosia cristata L. Callus Culture as well as for Its Fragmentation Ions Formed in the Course of Neutral Losses. Celoscristatin fragmentation a

ions

Molecular

[M+H]+

[M+H]+

Error

Error

formula

observed

predicted

[mDa]

[ppm]

813.1822

813.1832

-1.0

-1.23

---

Precursor ion [celoscristatin+H]+ C33H37N2O22

a

MS2 ions

nl: - H2O

C33H35N2O21

795.1715

795.1727

-1.2

-1.51

619

nl: - CO2

C32H37N2O20

769.1920

769.1934

-1.4

-1.82

593

nl: - Mal

C30H35N2O19

727.1805

727.1829

-2.4

-3.30

551; 389

nl: - Gluc

C27H29N2O16

637.1497

637.1512

-1.5

-2.35

593; 551; 389

nl: - Gluc/ CO2

C26H29N2O14

593.1599

593.1613

-1.4

-2.36

345

nl: - Gluc/ Mal

C24H27N2O13

551.1511

551.1508

0.3

0.54

389

nl: - Gluc/ Mal/ Glc

C18H17N2O8

389.0972

389.0979

-0.7

-1.80

345

nl – neutral losses from [celoscristatin+H]+



Table 5. The NMR Data for Celoscristatin 3 Isolated from the Celosia cristata L. Callus Culture. No.

1

2 3a/b

4.92, dd, 3.4; 10.3 3.09, dd, 16.6; 10.2 3.58, dd, 3.3; 16.4 7.05, s

4 5 6 7 8 9 10 11 12 13 14a/b 15 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′a/b 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 1′′′ 2′′′a/b 3′′′ a 1

641

H NMRa

6.94, s

8.25, d, 8.8 5.78, d, 8.8 3.21, bs 3.34, bs 4.37, bt, 7.2 6.25, bs

5.19, d, 6.7 3.85 (overlap) 3.76 (overlap) 3.64, (overlap) 3.79, (overlap) 4.40, dd, 11.7; 2.1 4.47, dd, 11.9; 5.1 4.87, d, 6.3 3.36, (overlap) 3.53, (overlap) 3.47, (overlap) 3.73, (overlap)

3.38, s

H NMR δ [ppm], mult, J [Hz];

13

C NMRb, c 66.8 34.5 114.1 145.8 148.8 100.4 139.3 126.0 180.1 145.5 107.7 165.9 28.4 55.4 152.6 106.7 181.5 165.5 103.7 84.0 78.0 71.8 76.2 64.9 105.4 76.2 78.2 74.6 78.2 177.2 173.5 44.7 177.6 b 13

C NMR δ [ppm];

c 13

C chemical shifts were derived from gHSQC and gHMBC;


Page 30 of 38

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Figure 1.



Figure 2.


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Figure 3.



Figure 4.


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Figure 5.



Figure 6.


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Figure 7.



TOC Graphic


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Study on Betalains in Celosia cristata Linn. Callus Culture and

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