Thermal Degradation of Major Gomphrenin Pigments in the Fruit Juice

Generation of decarboxylated and dehydrogenated gomphrenins during heating of Basella alba L. fruit juice containing high levels of betacyanin pigment...
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Thermal Degradation of Major Gomphrenin Pigments in the Fruit Juice of Basella Alba L. (Malabar Spinach) Agnieszka Kumorkiewicz, and Slawomir Wybraniec J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02357 • Publication Date (Web): 27 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Thermal Degradation of Major Gomphrenin Pigments in the Fruit Juice of

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Basella Alba L. (Malabar Spinach)

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Agnieszka Kumorkiewicz, Sławomir Wybraniec*

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

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

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*

Corresponding author. Tel.: +48-12-628-3074; fax: +48-12-628-2036.

E-mail address: [email protected] (S. Wybraniec).

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Abstract

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Generation of decarboxylated and dehydrogenated gomphrenins during heating of Basella

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alba L. fruit juice which contains high levels of betacyanin pigments was monitored by LC-

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DAD-ESI-MS/MS. The presence of principal decarboxylation products, 2-, 17- and 2,17-

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decarboxy-gomphrenins, their diastereomers as well as minor levels of their dehydrogenated

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derivatives are reported. In addition, determination of molecular masses of decarboxylated

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gomphrenins by high-resolution mass spectrometry (LCMS-IT-TOF) was performed.

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Enzymatic deglucosylation of decarboxylated and dehydrogenated gomphrenins resulted in

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generation of betanidin diagnostic derivatives for further identification process. In addition,

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experiments were conducted to prove that the position of glucosylation of the chromophoric

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part of betacyanins (betanidin part) has decisive influence on different chromatographic

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properties of their decarboxylated derivatives.

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KEYWORDS: decarboxylated and dehydrogenated gomphrenin; betacyanins; betalains;

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betanidin; Basella alba; Malabar spinach; plant pigments

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INTRODUCTION

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Basella alba L. (Basellaceae), frequently known as Malabar spinach (as well as Indian

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spinach, Ceylon spinach, vine spinach, or climbing spinach), is a succulent, branched, smooth,

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perennial twining herbaceous vine that can reach several meters in length.1-4 The stem of

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Basella alba is green, but the stem, leaves, and petioles of the cultivar Basella alba 'Rubra' are

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red-violet. The fruits are fleshy, stalkless, ovoid or spherical, 5 to 6 mm long, and purple

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when mature; they contain betacyanins as the major pigments.1

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The leaves of Basella alba are similar to spinach in that they can be prepared and consumed

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for potential health benefit, which makes them attractive for common diets.2 Promising

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applications of various parts of the plant for disease treatment and healing effects in humans

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have been reported.1 The plant, especially the leaves and stem, has been explored for its

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medicinal properties in ancient Indian and Chinese traditional medicine practices to treat

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constipation, as a diuretic, and as an anti-inflammatory agent.2-4 However, the purple fruits of

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Basella alba and Basella alba 'Rubra', rich in betacyanins and other bioactive

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phytochemicals, have not been investigated as food preparations and deserve exploration for

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possible medicinal and functional food applications. Recently, some anticarcinogenic

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activities of the fruit juice have been noted.3 The pigment-rich fruit extract was also tested as

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a natural colorant for ice cream.5

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Betacyanins are water-soluble plant pigments that are a subgroup of betalains, which are

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found in most families of the Caryophyllales6; they are extensively used in the food industry

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as food colorants.7 In addition, betacyanins have chemopreventive characteristics and strong

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antioxidant properties.8-18 Recent research has focused on new structures and derivatives of

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betacyanins, as well as their influence on health. These pigments are present mostly in plant

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fruits, flowers, and roots, as well as in tissues exposed to stress.6,7,16

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Despite the potential benefits and uses of betacyanins, systematic research of their activities is

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lacking. The 6-O-glycoside of betanidin is an extremely important betacyanin (Figure 1) and

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is present in high concentration in fruits of Basella alba L. and in leaves of its variety Basella

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alba var. rubra. According to our recent studies, because of the presence of the phenolic

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group at carbon C-6 in gomphrenin I, the only possible quinonoid intermediate during

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oxidation of gomphrenin I is a dopachromic derivative.19-21 Therefore, gomphrenin I enables a

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unique possibility to observe reaction pathways complementary to betanin reaction routes,

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which is important for understanding the mechanism of betacyanin oxidation. This technique

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may also reveal other pro-health activities and chemical properties. To date, the highest

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antioxidant activity among betacyanins has been attributed to gomphrenin I.14

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A series of reports have been published that detail several new groups of betacyanin

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degradation products, especially decarboxylated derivatives, in preparations subjected to

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thermal processing.22-26 Presumably, new gomphrenin derivatives should also have promising

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pro-health activities and important potentials for studies of betacyanin oxidation mechanism.

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For this reason, our research focused on the identification of colored degradation products in

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heated fruit juice of Basella alba L. We established the first tentative structures formed by

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decarboxylation and dehydrogenation of the main pigment present in the juice (gomphrenin I)

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and its diastereomer by means of liquid chromatography coupled to diode array detection and

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electrospray ionization tandem mass spectrometry (LC-DAD-ESI-MS/MS). To aid the

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identification process, we performed enzymatic deglucosylation of the degradation products,

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which resulted in the formation of diagnostic betanidin derivatives. For the identification, we

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deglucosylated a series of already known betanin-based standards.

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MATERIALS AND METHODS

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Reagents. Formic acid, LC-MS grade methanol,water as well as almond β-glucosidase were

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obtained from Sigma Chemical Co. (St. Louis, MO, USA).

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Preparation of juice from Basella alba fruits. Basella alba L. fruits were collected in a

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greenhouse of University of Agriculture in Cracow. In order to obtain the juice, 100 g of the

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fruits were manually squeezed and 20 mL of obtained liquid was centrifuged and filtered

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through a 0.2 mm i.d. pore size filter and then threefold diluted with water for storage at -20

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ºC (typically for few weeks) before the subsequent experiments. For semipreparative

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isolation of gomphrenin/isogomphrenin 1/1', the juice was first filtered through a bed (10 cm

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height x 2 cm i.d.) of 0.063/0.200 mm silica (J.T. Baker, Deventer, Holland) to remove

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hydrocolloids and proteins to obtain a clear solution and subsequently through a 0.2 mm i.d.

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pore size filter. This solution was purified by semipreparative liquid chromatography.

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Heating experiments on Basella alba fruit juice and isolated gomphrenins. 3 mL of

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Basella alba fruit juice was diluted three times with water, acidified with 50 µL of glacial

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acetic acid and heated at 85 ºC in a water bath for 40 min according to previous studies. 22, 23

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200 µL aliquots of the heated samples were collected for LC-DAD-ESI-MS/MS analysis

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every 5 min. For additional heating experiments of previously isolated single diastereomers of

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gomphrenin, 2- and 17-decarboxy-gomphrenin (for recognition of elution order of 4/4' and

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5/5'), their 1 mL solutions (100 µM) were acidified with 10 µL of glacial acetic acid and

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heated at 85 ºC in a water bath for 10-20 min. 100 µL aliquots of the heated samples were

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collected every 5 min and analyzed by LC-DAD-ESI-MS/MS.

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Semi-synthesis of gomphrenin derivatives. For the comparative hydrolysis experiments

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with single purified gomphrenin derivatives, thermal decarboxylation and dehydrogenation of

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gomphrenin and isogomphrenin in triple diluted 200 mL of Basella alba fruit juice was

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performed as described for heating experiments. Heating of gomphrenin and isogomphrenin

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present in the juice within 10-20 min resulted in production of derivatives (Figure 1) differing

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in decarboxylation position (2/2', 3, 4/4' and 5/5') as described in detail in Results and

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Discussion section (Fig. 1) (Table 1). Similarly, prolonged heating to 30-40 minutes

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generated increased levels of dehydrogenated derivatives 2,17-bidecarboxy-2,3-dehydro-

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neogomphrenin 9 and 2-decarboxy-2,3-dehydro-neogomphrenin 11. For the comparative

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studies, the analogous betanin-derived compounds isolated previously by high-speed

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countercurrent chromatography and/or HPLC were prepared.19,27

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Deglucosylation of gomphrenin- and betanin-based derivatives. β-glucosidase hydrolysis

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for deglucosylation of previously isolated single diastereomers of selected gomphrenin

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derivatives as well as their corresponding betanin derivatives were performed in solutions

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containing 25 mM acetate buffer (pH 5), almond β-glucosidase (15 units/mL) as well as 20-50

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µM pigment at 30 °C for 30 min. For the chromatographic analyses, 20 µL samples of

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reaction mixtures were injected directly into the LC-DAD-MS/MS system without further

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purification.

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Semipreparative chromatography. For the isolation of gomphrenin/isogomphrenin from

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the juice of Basella alba L. as well as gomphrenin-based derivatives obtained by heating of

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diluted Basella alba fruit juice, a flash chromatography system (preparative HPLC system

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

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

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(Interchim, France) was applied. Further separation and isolation of pigments was performed

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on an HPLC semipreparative column Luna C18(2) 250 x 10 mm i.d., 10 µm (Phenomenex,

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Torrance, CA, USA) with a 10 mm x 10 mm i.d. guard column of the same material

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(Phenomenex, Torrance, CA USA) under the following gradient system (System 1): 6% A in

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B at 0 min; gradient to 20% A in B at 30 min. (A, acetonitrile; B, 1% (v/v) HCOOH in H2O).

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The injection volume was 2 mL and the flow rate was 3 mL/min. Detection was performed at

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538, 505, 480 and 440 nm with a PDA UV/Vis detector; column temp 30 °C. The eluates

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were pooled, and concentrated under reduced pressure at 25 °C and finally freeze-dried to

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obtain pure pigments.

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Chromatographic analysis by LC-DAD-ESI-MS/MS system. For the chromatographic and

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mass spectrometric analyses, an LCMS-8030 mass spectrometric system (Schimadzu, Kyoto,

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Japan) coupled to LC-20ADXR HPLC pumps, an injector model SIL-20ACXR, and a PDA

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detector (photo diode array) model SPD-M20A, all controlled with LabSolutions software

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version 5.60 SP1 (Schimadzu, Japan) was used. The samples were eluted through a 150 mm x

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4.6 mm i.d., 5.0 µm, Kinetex C18 chromatographic column preceded by a guard column of

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the same material (Phenomenex, Torrance, CA, USA). The injection volume was 20 µL, and

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the flow rate was 0.5 mL/min. The column was thermostated at 40 ºC. The separation of the

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analytes was performed with a binary gradient elution. The mobile phases were: A - 2 %

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formic acid in water, and B - pure methanol. The gradient profile was: (t [min], % B), (0, 5),

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(12, 70), (15, 80), (19, 80). The full range PDA signal was recorded, and chromatograms at

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538, 505, 490 and 440 nm were individually displayed. Positive ion electrospray mass spectra

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were recorded on the LC-MS system which was controlled with LabSolutions software. The

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ionisation electrospray source operated in positive mode (ESI+), at an electrospray voltage of

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4.5 kV and capillary temperature at 250 ºC, using N2 as a gas for the spray, recording total ion

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chromatograms, mass spectra and ion chromatograms in selected ion monitoring mode (SIM)

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as well as the fragmentation spectra. Argon was used as the collision gas for the collision-

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induced dissociation (CID) experiments. The relative collision energies for MS/MS analyses

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were set at -35 V.

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Chromatographic analysis with detection by ion-trap time-of-flight system (LCMS-IT-

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TOF). All mass spectra were analyzed using LCMS-IT-TOF mass spectrometer (Shimadzu)

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equipped with an electrospray (ESI) ion source and coupled to the HPLC Prominence

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(Shimadzu). Separation of compounds was carried on a 50 mm x 2.1 mm i.d., 1.9 µm Shim

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Pack GISS C18 column (Shimadzu). The injection volume was 2 µL, and the flow rate was

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

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performed with a binary gradient elution. The mobile phases were: A – 0.1 % formic acid in

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water, and B - pure methanol. The gradient profile was: (t [min], % B), (0, 5), (12, 30), (17,

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80), (19, 80). 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

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

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recorded in the positive ion mode with mass range 100-2000 Da and collision energy between

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12 - 50 % depending on the compound`s structure. The results of high resolution mass

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spectrometry experiments (HRMS) were studied using the Formula Predictor within the

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LCMS Solution software. Only empirical formulae with an mass error below 5 ppm were

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taken into account.

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RESULTS AND DISCUSSION

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The chromatogram in Figure 2A depicts a typical betacyanin profile in Basella alba fruit

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juice. The dominant presence of the known gomphrenin 1 and its isoform 1' with minute

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quantities of gomphrenin II and III (acylated gomphrenins) is well known.28 For simplicity,

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we will refer to the main pigment as “gomphrenin” instead of “gomphrenin I” and

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gomphrenins II and III will be named “acylated gomphrenins.” This is justified by the unique

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importance of gomphrenin, which we believe is comparable to another basic betacyanin:

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betanin; both pigments are basic O-glucosylated positional isomers. Additionally, the series of

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gomphrenin derivatives presumably have important pro-health properties and will be

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frequently studied. Therefore, deriving names from the “gomphrenin” root is also justified for

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simplification of the nomenclature of the multitude of the derivatives.

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The resulting high-performance liquid chromatography (HPLC) chromatograms of

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decarboxylation/dehydrogenation products formed during the heating experiments are

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depicted in Figure 2B-C and their LC-DAD-MS fingerprints are presented in Table 1. For the

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most prominent decarboxylated gomphrenins, additional results from high-resolution liquid

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chromatography coupled with ion-trap and time-of-flight (LCMS-IT-TOF) analyses are listed

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in Table 2.

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Generation of monodecarboxy-gomphrenins during heating of Basella alba L. fruit

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juice. The aqueous solutions acidified by acetic acid were heated for 10 to 15 minutes and

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pigment profiles were obtained; the profiles were similar to the profiles of early products of

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Beta vulgaris L. root heating, but the retention times were shifted.22,24,26 This reflects the

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similar conditions of the experiments (aqueous solutions acidified by acetic acid at similar

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temperatures) to those applied previously for heating of Beta vulgaris L. juice.22 The

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temperature (85˚C) of the heating process was high enough for monitoring changes in the

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compositions of the resulting mixtures. The main resulting chromatographic peaks

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corresponded to well-separated compounds 2 and 2', as well as a slightly resolved pair 4/4'

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(Figure 2B). Additionally, lower signals for compound 3 and another slightly resolved pair

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5/5' were detected. All the detected compounds (Figure 1) were less polar than their

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corresponding precursors (gomphrenin/isogomphrenin 1/1'). Further interpretation of the LC-

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DAD and LC-MS/MS spectra revealed that the main products appeared to be mono-

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decarboxylated derivatives with absorption maxima at λmax 506 nm for 2/2' and λmax 534 nm

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for 4/4' (Table 1) with characteristic pseudomolecular ions with m/z 507 due to loss of CO2

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from the corresponding precursors (gomphrenin/isogomphrenin 1/1'). In the collision-induced

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fragmentation experiments, the daughter ion spectra displayed fragments of [M+H]+ at m/z

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345 in each case, which represented the decarboxylated aglycone (betanidin) part of the

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molecules. Additionally, LC-IT-TOF analyses yielding m/z 507.1603, 507.1613, 507.1619,

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and 507.1601 (C23H26N2O11, calculated mass: 507.1609) supported the identification of

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decarboxylated gomphrenins 2/2' and 4/4' (Table 2).

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Previous absorption data indicated that betanin has a characteristic absorption of

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approximately λmax 506 nm.22,24 Therefore, it was possible to conclude that the pair 2 and 2'

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were 17-decarboxy-gomphrenin and 17-decarboxy-isogomphrenin, respectively. The slightly

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higher retention times of 2 and 2' compared to their corresponding precursors 1 and 1' support

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this assumption.

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For 4/4', the only possible pair of diastereomers were 2-decarboxylated derivatives. This

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conclusion was supported by an absorption maximum (λmax 533 nm) and chromatographic

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retention (not completely resolved pair of peaks) that were similar to those of betanin thermal

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degradation products (2-decarboxy-betanin/-isobetanin) in heated red beet juice,22,24,26 as well

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as in endogenously present dopamine-derived 2-decarboxy-betacyanins in hairy roots of Beta

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vulgaris and Carpobrotus acinaciformis.29,30 Therefore, the pair 4 and 4' was assigned to 2-

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decarboxy-gomphrenin and 2-decarboxy-isogomphrenin, respectively. Interestingly, these

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pigments were much better separated in reversed-phase HPLC, especially at low

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concentrations of formic acid in the eluent, than their corresponding betanin derivatives.22,23

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The elution order of 4 and 4' on the C18 HPLC column was established by analysis of

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decarboxylation products of previously isolated gomphrenin/isogomphrenin (1/1'), assuming

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that isomerization is less strong than decarboxylation. This procedure was previously

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successfully applied in experiments of 2-decarboxy-betanin/-isobetanin elution order

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recognition in which the reversed order was revealed compared to the precursor pair

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(betanin/isobetanin) and the pair of 17-decarboxy-betanin/-isobetanin.22

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Figure 3 represents the results of the experiment in which, rather unexpectedly, the elution

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order for 4 and 4' appeared the same as for 1 and 1' (the form 4 is eluted earlier than the

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isoform 4'). This finding is in contrast to betanin-based 2-decarboxylated derivatives.22 In

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addition, during the experiment, equal quantities of both forms of 17-decarboxy-gomphrenin/-

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isogomphrenin (2/2') were generated, irrespective of the starting epimer of gomphrenin (1/1')

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resembling the generation profile of the corresponding 17-decarboxy-betanin/-isobetanin.22

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The mechanism of epimerization at carbon C-15 in betanidin had already been explained by

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Dunkelblum et al.31 Further experiments based on a method of hydrolysis and cross-

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recondensation of selected mixtures of betacyanins are needed to confirm the elution order of

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4 and 4'.32

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Further analysis of the data indicated the presence of a small peak (Figure 2) likely

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corresponding to 15-decarboxy-gomphrenin (3), which is formed by decarboxylation

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(detection of protonated molecular ions at m/z 507 with their fragmentation to ions of m/z

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345) with a loss of the chiral center at carbon C-15 in gomphrenin.10 Therefore, the presence

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of only one form of 3 suggested that this compound was similar to 15-decarboxy-betanin,

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which was previously detected in red beet extract.33 Indeed, a similar absorption maximum of

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3 at λmax 528 nm and elution between 17-decarboxy-isogomphrenin (2') and 2-decarboxy-

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gomphrenin (4) supported the assignment of 3 (C23H26N2O11, m/z 507.1614, calculated mass:

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507.1609).

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Bi-decarboxy- and tri-decarboxy-gomphrenins. After prolonged heating (30 minutes) of

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Basella alba L. fruit juice, higher quantities of compounds corresponding to chromatographic

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peaks 5/5' were detected (Figure 2). These pigments displayed absorption maxima at λmax 507

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nm and pseudomolecular ions at m/z 463, clearly indicating a loss of two CO2 moieties from

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the starting gomphrenin/isogomphrenin, 1/1' (Figure 1). Subsequent fragmentation to ions of

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m/z 301 confirmed the existence of a bidecarboxylated fragment of betanidin and suggested

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the formation of bidecarboxylated gomphrenin/isogomphrenin. The detection of the two key

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epimers clearly indicated the position of double decarboxylation and, therefore, the presence

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of 2,17-bidecarboxy-gomphrenin and its isoform 5/5' (C22H26N2O9, m/z 463.1709, calculated

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mass: 463.1711).

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Interestingly, these epimers were well separated in the applied HPLC system, especially in

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eluents containing diluted formic acid. This is in contrast to the lack of separation between the

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epimers of 2,17-bidecarboxy-betanin and -isobetanin, which had been separated only in ion-

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pair chromatography.22 Accordingly, taking into account the properties of 2-decarboxy-

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gomphrenin/-isogomphrenin (4/4') and 2,17-bidecarboxy-gomphrenin/-isogomphrenin (5/5'),

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the position of glucosylation of the betanidin at carbon C-5 or C-6 has decisive influence on

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the chromatographic differences between betanin and gomphrenin derivatives.

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Subsequent experiments designed to recognize the elution order of 2,17-bidecarboxy-

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gomphrenin/-isogomphrenin (5/5') were performed by heating of each single form of

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previously isolated 17-decarboxy-gomphrenin/-isogomphrenin (2/2') and 2-decarboxy-

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gomphrenin/-isogomphrenin (4/4'). The results of the experiments are presented in Figure 4.

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Interestingly, in each case, a positive result of the experiment was obtained (in contrast to

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experiments with betanin-based derivatives)., Specifically, for each single 15S form of

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substrate (2- and 17-decarboxy-gomphrenin), an evident peak of 2,17-bidecarboxy-

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gomphrenin 5 was obtained; the peak was accompanied by a much smaller peak of the 15R

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form (the isoform) that likely resulted from the epimerization effect. Analogous results were

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obtained for heated single 15R forms of the substrates (2- and 17-decarboxy-isogomphrenin).

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Generated single products indicated the elution order of 5/5' and confirmed that the isoform is

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eluted after the 15S form. Therefore, in contrast to betanin-based derivatives, the elution

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orders for all the tested gomphrenin-based diastereomeric pairs (1/1', 2/2', 4/4' and 5/5') were

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the same.

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Previously, similar experiments were performed on betanin-based (phyllocactin and

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hylocerenin) decarboxylated derivatives in aqueous or ethanolic solutions: the experiments

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resulted in the generation of equal quantities of both of the respective bidecarboxy-betacyanin

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isomers.23 Consequently, the generation of equal quantities of the respective bidecarboxy-

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betacyanin isomers prevented deduction of their elution order. Additionally, for 2,17-

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bidecarboxy-betanin/-isobetanin, the detection of both forms was possible only in an ion-

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pairing HPLC system, which yielded the same negative results.22

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Prolonged heating in acetic acid solutions released small quantities of compound 8 (Figures 1

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and 2). It was slightly less polar than 2,17-bidecarboxy-gomphrenin and, in LC-MS analysis,

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displayed a pseudomolecular ion at m/z 419 and an absorption maximum of λmax 506 nm. This

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suggested the presence of a tridecarboxy-gomphrenin for which the only possible structure

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was 2,15,17-tridecarboxy-gomphrenin. This conclusion was supported by the detection of

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only one chromatographic peak 8 in the HPLC system, which resulted from the loss of the

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chiral center at carbon C-15 in 8. Subsequent fragmentation experiments on the

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pseudomolecular ion at m/z 419 revealed fragmentation ions at m/z 257, which proved the

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existence of the tridecarboxylated fragment of betanidin.

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Comparative enzymatic deglucosylation studies on gomphrenin- and betanin-based

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decarboxylated derivatives. The presence of the same aglycones (decarboxylated betanidins)

326

in the structures of betanin- and gomphrenin-based decarboxylated derivatives enabled further

327

chromatographic confirmation of the identities of the decarboxylated gomphrenins. For this

328

aim, we performed experiments based on a β-glucosidase assay, a sensitive tool for β-

329

deglucosylation of such pigments as betanin or gomphrenin (Figure 1) and other non-acylated

330

betacyanins at the first β-glucosidic ring.34

331

The assays were conducted with almond β-glucosidase and yielded 2-, 15-, 17-, and 2,17-

332

decarboxy-betanidins (Table 3) as a result of β-deglucosylation34 of the starting glucosylated

333

substrates (the corresponding 2-, 15-, 17-, and 2,17-decarboxy-gomphrenins (Figure 1) and

334

the analogous decarboxylated betanins). Their identities were confirmed by the same retention

335

times and spectrophotometric and mass spectrometric data obtained for the products

336

(decarboxylated betanidins). Additionally, the corresponding diastereomers (where

337

applicable) were also positively tested. Because of the scarce quantities of 2,15,17-

338

tridecarboxy-betanin that were generated, tests for this pigment were not performed. The

339

generated betanidin derivatives were sufficiently stable for performing the chromatographic

340

analyses, even after several hours of the reaction and despite the elevated temperature of the

341

enzymatic process.

342 343

Generation of dehydrogenated gomphrenins during heating of Basella alba fruit juice.

344

Dehydrogenated betacyanins are formed as a result of oxidation of the corresponding

345

betacyanins and their decarboxylated derivatives. In the case of red beet juice,22,24 a series of

346

dehydrogenated betanin-like derivatives were identified after prolonged heating experiments.

347

Similarly, a complex mixture of dehydrogenated betacyanins was derived from juice of

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348

Hylocereus polyrhizus fruits containing three main betacyanins: betanin, phyllocactin, and

349

hylocerenin, as well as their isoforms.23

350

Direct oxidation of betanin results in generation of neobetanin (14,15-dehydrobetanin) and 2-

351

decarboxy-2,3-dehydrobetanin (result of oxidative decarboxylation).19,20 The final result

352

depends on the matrix composition, as well as additional factors such as buffers, pH, or

353

oxidizing agents. The auto-oxidation of the pigments assisted by air-derived oxygen is also

354

possible because of the relatively high antioxidant activity of betacyanins.8,11-15,17

355

Consequently, the formation of a group of gomphrenin-based dehydrogenated derivatives was

356

also expected in the heated Basella alba juice. Indeed, the presence of neogomphrenin 7 was

357

detected in the heated juice. However, it was present at low concentration levels (Figure 2).

358

Formation of neogomphrenin results in the loss of the chiral center at carbon C-15, yielding

359

only one chromatographic peak. Neogomphrenin has lower polarity than

360

gomphrenin/isogomphrenin, which resulted in a longer retention time. This is a general trend

361

for neobetacyanins that is frequently observed during HPLC analyses.22-25 Subsequent

362

confirmation was indicated by an absorption maximum at λmax 471 nm (similar for

363

neobetacyanins) and m/z 549 obtained for pseudomolecular ions in the LC-MS/MS system,

364

indicating the loss of 2H from gomphrenin/isogomphrenin during the heating experiment. The

365

subsequent fragmentation ion at m/z 387 from the loss of a glucose moiety supported the

366

formation of a dehydrogenated betanidin structure.

367

Generated neogomphrenin 7 can undergo a further decarboxylation (most probably at carbon

368

C-2 or C-1720,22) during heating. Therefore, in this study, we investigated the presence of the

369

2- or 17-decarboxy-neogomphrenin. However, based on its high retention time, as well as LC-

370

MS/MS data, only 2-decarboxy-neogomphrenin 9 was detected. Detection of a

371

pseudomolecular ion at m/z 505 and its subsequent fragment at m/z 343 from the loss of a

372

glucose moiety confirmed the generation of decarboxylated and dehydrogenated betanidin

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Journal of Agricultural and Food Chemistry

373

and suggested a possibility that peak 9 may correspond to 2-decarboxy-neogomphrenin. As in

374

the case of neogomphrenin, only one chromatographic peak, resulting from the loss of the

375

chiral center at carbon C-15, was expected. Unfortunately, no ultraviolet-visible spectrum was

376

able to be detected as a result of peak overlap with impurities.

377

Further inspection of the LC-DAD-MS/MS data of longer-heated samples revealed two

378

peaks— 6 and 11 (Figure 2)—that both corresponded to pseudomolecular ions at m/z 461,

379

suggesting a loss of 2H from the more polar compound (with a lower retention time) 2,17-

380

bidecarboxy-gomphrenin 5 (m/z 463). A subsequent fragmentation ion at m/z 299 from the

381

loss of a glucose moiety in both cases supported the suggestion of the presence of a

382

bidecarboxylated dehydrogenated fragment of betanidin. In the case of 11, the absorption

383

maximum found at λmax 467 nm and its relatively high hydrophobic nature were similar to its

384

analogue from the betanin group (2,17-bidecarboxy-neobetanin).20,22 For this reason, the

385

presence of 2,17-bidecarboxy-neogomphrenin 11 was inferred (Figure 1). In contrast, the

386

more polar character of 6 and its close retention to 2,17-bidecarboxy-gomphrenin 5 indicated

387

that this compound may be 2,17-bidecarboxy-2,3-dehydrogomphrenin (analogous to 2,17-

388

bidecarboxy-2,3-dehydrobetanin formed after direct oxidation of 2,17-bidecarboxy-betanin).20

389

Due to the fact that the chiral center is present at carbon C-15 (Figure 1), the peaks must

390

correspond to two unresolved epimers 6/6'. No additional identification for 6/6' could be

391

performed because no absorption maximum could be registered for 6/6' due to overlap with

392

5/5'. The identity of 6/6' will be verified by direct oxidation (e.g., by ABTS cation radicals20)

393

of 2,17-bidecarboxy-gomphrenin 5/5', which will possibly generate compounds identical to

394

6/6'.

395

The presence of two dehydrogenated neo-derivatives of gomphrenin corresponding to peaks

396

10 and 12 were also detected in the LC-DAD-MS/MS data of the juice samples (Figure 2).

397

Compound 10 was likely a result of oxidation of 11 (analogous to a betanin derivative20); its

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398

mass spectrometric data (pseudomolecular ion at m/z 459 and fragmentation ion at m/z 297),

399

its absorption maximum (λmax 414 nm), and its relatively high hydrophobic nature indicated

400

the presence of 2,17-bidecarboxy-2,3-dehydro-neogomphrenin 10. Similarly, high

401

hydrophobicity, absorption data (λmax 420 nm), and mass spectrometric data (pseudomolecular

402

ion at m/z 503 and fragmentation ion at m/z 341) strongly indicated that the identity of 12 was

403

2-decarboxy-2,3-dehydro-neogomphrenin (analogous to a betanin derivative20). As in the case

404

of the oxidized structures mentioned above, the identities of 10 and 12 were supported by

405

further oxidation experiments of a series of isolated and purified gomphrenin derivatives.

406 407

Comparative enzymatic deglucosylation studies on betanin- and gomphrenin-based

408

dehydrogenated derivatives. Further confirmation of the identities of the dehydrogenated

409

neogomphrenins 10 and 12 were performed by the β-glucosidase assay with the use of

410

additional betanin-derived standards obtained in the previous oxidation study.20 The assay

411

yielded 2-decarboxy-2,3-dehydro-betanidin and 2,17-bidecarboxy-2,3-dehydro-betanidin

412

(Table 3), which resulted from β-deglucosylation of the starting glucosylated substrates (the

413

corresponding 2,17-bidecarboxy-2,3-dehydro-neogomphrenin 10 and 2-decarboxy-2,3-

414

dehydro-neogomphrenin 12, as well as the analogous betanin derivatives). As mentioned, the

415

identities were confirmed by the same retention times and the spectrophotometric and mass

416

spectrometric data obtained for the products. The assay was not performed for the other

417

dehydrogenated pigments due to the scarce quantities of the generated compounds.

418

This is the first report on the generation of mono-, bi-, and tri-decarboxylated gomphrenins

419

and their dehydrogenated derivatives in general, but also specifically, in degradation products

420

of heated Basella alba fruit juice, which can be used for various food applications. The

421

health-promoting actions and colorant properties of these compounds are different not only

422

because of the matrix effect but also because of different activities of gomphrenin compared

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Journal of Agricultural and Food Chemistry

423

to gomphrenin-based derivatives. Interestingly, from an analytical point of view, our results

424

indicate that the position of glucosylation of betanidin at carbon C-5 or C-6 has significant

425

influence on the chromatographic differences between betanin and gomphrenin derivatives.

426

Further, the higher antioxidant activity of gomphrenin compared to betanin14 likely results

427

from the favorable position of the glycosidic bond, which enables the formation of the

428

aminochromic intermediate during oxidation.19,20 This also opens a question for other

429

promising properties that may differ from the known activities of the commonly known

430

betanins. In this respect, further investigation of Basella alba fruit juice, as well as its

431

processed products, should significantly enhance our knowledge about the action of

432

gomphrenins. Considering that gomphrenin is found in few other plant species (mostly at low

433

concentration levels in Gomphrena globosa L.35 and Bougainvillea glabra Choisy36), its

434

presence at high concentrations in Basella alba L. fruits3 renders this plant an extremely

435

valuable source of gomphrenin for future applications.

436 437

Acknowledgements

438 439

This research was financed by Polish National Science Centre for years 2015-2018 (Project

440

No. UMO-2014/13/B/ST4/04854). The authors thank Beata Wileńska Ph.D., eng. and

441

Bartłomiej Fedorczyk M.Sc. from Laboratory of Biologically Active Compounds (Warsaw

442

University) for the excellent technical assistance with LCMS-IT-TOF experiments.

443 444

References:

445 446

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447

alba L. Int. J. Pharm. Sci. Drug Res. 2012, 4, 110-114.

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(2) Kumar, S.S.; Manoj, P.; Giridhar, P. Nutrition facts and functional attributes of foliage of

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Basella spp. LWT-Food Sci. Technol. 2015, 64, 468-474.

450

(3) Kumar, S.S.; Manoj, P.; Giridhar, P.; Shrivastava, R.; Bharadwaj, M. Fruit extracts of

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Basella rubra that are rich in bioactives and betalains exhibit antioxidant activity and

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cytotoxicity against human cervical carcinoma cells. J. Funct. Foods 2015, 15, 509-515.

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(4) Kumar, S.S.; Manoj, P.; Giridhar, P. A method for red-violet pigments extraction from

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fruits of Malabar spinach (Basella rubra) with enhanced antioxidant potential under

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fermentation. J. Food Sci. Technol. 2015, 52, 3037-3043.

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(5) Kumar, S.S.; Manoj, P.; Shetty, N.P.; Prakash, M.; Giridhar, P. Characterization of major

457

betalain pigments-gomphrenin, betanin and isobetanin from Basella rubra L. fruit and

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evaluation of efficacy as a natural colourant in product (ice cream) development. J. Food Sci.

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Technol. 2015, 52, 4994-5002.

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(6) Khan, M.I.; Giridhar P. Plant betalains: Chemistry and biochemistry. Phytochemistry

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2015, 117, 267-295.

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(7) Azeredo, H.M.C. Betalains: properties, sources, applications, and stability – a review. Int.

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(8) Wybraniec, S.; Starzak, K.; Pietrzkowski, Z. Chlorination of betacyanins in several

465

hypochlorous acid systems. J. Agric. Food Chem. 2016, 64, 2865−2874.

466

(9) Tesoriere, L.; Butera, D.; Allegra, M.; Fazzari, M.; Livrea, M. A. Distribution of betalain

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pigments in red blood cells after consumption of cactus pear fruits and increased resistance of

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the cells to ex vivo induced oxidative hemolysis in humans. J. Agric. Food Chem. 2005, 53,

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1266-1270.

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(10) Kapadia, G. J.; Azuine, M. A.; Sridhar, R.; Okuda, Y.; Tsuruta, A.; Ichiishi, E.;

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Mukainake, T.; Takasaki, M.; Konoshima, T.; Nishino, H.; Tokuda, H. Chemoprevention of

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DMBA-induced UV-B promoted, NOR-1-induced TPA promoted skin carcinogenesis, and

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DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot. Pharmacol.

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Res. 2003, 47, 141-148.

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(11) Kanner, J.; Harel, S.; Granit, R. Betalains - A new class of dietary cationized

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antioxidants. J. Agric. Food Chem. 2001, 49, 5178-5185.

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(12) Escribano, J.; Pedreño, M. A.; García-Carmona, F.; Muñoz, R. Characterization of the

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antiradical activity of betalains from Beta vulgaris L. roots. Phytochem. Anal. 1998, 9, 124-

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127.

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(13) Butera, D.; Tesoriere, L.; Di Gaudio, F.; Bongiorno, A.; Allegra, M.; Pintaudi,

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A. M.; Kohen, R.; Livrea, M. A. Antioxidant activities of Sicilian prickly pear (Opuntia ficus-

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indica) fruit extracts and reducing properties of its betalains: betanin and indicaxanthin. J.

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Agric. Food Chem. 2002, 50, 6895–6901.

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(14) Cai, Y.; Sun, M.; Corke, H. Antioxidant activity of betalains from plants of the

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Amaranthaceae. J. Agric. Food Chem. 2003, 51, 2288-2294.

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(15) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. Structural implications on color,

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fluorescence, and antiradical activity in betalains. Planta 2010, 232, 449-460.

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(16) Wybraniec, S.; Stalica, P.; Spórna, A.; Mizrahi, Y. Profiles of betacyanins in epidermal

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layers of grafted and light-stressed cacti studied by LC-DAD-ESI-MS/MS. J. Agric. Food

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Chem. 2010, 58, 5347-5354.

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(17) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. The role of phenolic hydroxy

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groups in the free radical scavenging activity of betalains. J. Nat. Prod. 2009, 72, 1142-1146.

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(18) Tesoriere, L.; Butera, D.; D’Arpa, D.; Di Gaudio, F.; Allegra, M.; Gentile, C.; Livrea, M.

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A. Increased resistance to oxidation of betalain enriched human low density lipoproteins. Free

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Radic. Res. 2003, 37, 689–696.

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(19) Wybraniec, S.; Michałowski, T. New pathways of betanidin and betanin enzymatic

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oxidation studied by LC-DAD-ESI-MS/MS. J. Agric. Food Chem. 2011, 59, 9612–9622.

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(20) Wybraniec, S.; Starzak, K.; Skopinska, A.; Nemzer, B.; Pietrzkowski, Z.; Michalowski,

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T. Studies on nonenzymatic oxidation mechanisms in neobetanin, betanin, and

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decarboxylated betanins. J. Agric. Food Chem. 2013, 61, 6465–6476.

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(21) Wybraniec, S.; Stalica, P.; Spórna, A.; Nemzer, B.; Pietrzkowski, Z.; Michałowski, T.

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Antioxidant activity of betanidin: electrochemical study in aqueous media. J. Agric. Food

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Chem. 2011, 59, 12163-12170.

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(22) Wybraniec, S. Formation of decarboxylated betacyanins in heated purified betacyanin

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fractions from red beet root (Beta vulgaris L.) monitored by LC-MS/MS. J. Agric. Food

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Chem. 2005, 59, 3483-3487.

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(23) Wybraniec, S.; Mizrahi, Y. Generation of decarboxylated and dehydrogenated

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betacyanins in thermally treated purified fruit extract from purple pitaya (Hylocereus

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polyrhizus) monitored by LC-MS/MS. J. Agric. Food Chem. 2005, 53, 6704-6712.

510

(24) Herbach, K. M.; Stintzing, F. C.; Carle, R. Impact of thermal treatment on color and

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pigment pattern of red beet (Beta vulgaris L.) preparations. J. Food Sci. 2004, 69, 491-498.

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(25) Herbach, K. M.; Stintzing, F. C.; Carle, R. Betalain stability and degradation-structural

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and chromatic aspects. J. Food Sci. 2006, 71, R41-R50.

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(26) Wybraniec, S.; Nowak-Wydra, B.; Mizrahi, Y. 1H and 13C NMR spectroscopic structural

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elucidation of new decarboxylated betacyanins. Tetrahedron Lett. 2006, 47, 1725-1728.

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(27) Spórna-Kucab, A.; Ignatova, S.; Garrard, I.; Wybraniec, S. Versatile solvent systems for

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the separation of betalains from processed Beta vulgaris L. juice using counter-current

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chromatography. J. Chromatogr. B 2013, 941, 54-61.

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(28) Glassen, W.E.; Metzger, J.W.; Heuer, S.; Strack, D. Betacyanins from fruits of Basella

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rubra. Phytochemistry 1993, 33, 1525-1527.

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

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

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(30) Piattelli, M.; Impellizzeri, G. 2-Descarboxybetanidin, a minor betacyanin from

524

Carpobrotus acinaciformis. Phytochemistry 1970, 9, 2553-2556.

525

(31) Dunkelblum, E.; Miller, H. E.; Dreiding, A. S. On the mechanism of decarboxylation of

526

betanidine. A contribution to the interpretation of the biosynthesis of betalaines. Helv. Chim.

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Acta 1972, 55, 642-648.

528

(32) Wybraniec S. A method for identification of diastereomers of 2-decarboxy-betacyanins

529

and 2,17-bidecarboxy-betacyanins in reversed phase HPLC. Anal. Bioanal. Chem. 2007, 389,

530

1611-1621.

531

(33) Nemzer, B.; Pietrzkowski, Z.; Spórna, A.; Stalica, P.; Thresher, W.; Michałowski, T.;

532

Wybraniec, S. Betalainic and nutritional profiles of pigment-enriched red beet root (Beta

533

vulgaris L.) dried extracts. Food Chem. 2011, 127, 42-53.

534

(34) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. Characterization of the activity of

535

tyrosinase on betanidin. J. Agric. Food Chem. 2007, 55, 1546-1551.

536

(35) Heuer, S.; Wray, V.; Metzger, J.W.; Strack, D. Betacyanins from flowers of Gomphrena

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globosa. Phytochemistry 1992, 31, 1801-1807.

538

(36) Wybraniec, S.; Jerz, G.; Gebers, N.; Winterhalter, P. Ion-pair high speed countercurrent

539

chromatography fractionation of a high-molecular weight variation of acyl-oligosaccharide

540

linked betacyanins from purple bracts of Bougainvillea glabra. J. Chromatogr. B 2010, 878,

541

538-550.

542 543 544 545 546

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Journal of Agricultural and Food Chemistry

Table 1. Chromatographic, spectrophotometric and mass spectrometric data of the analyzed gomphrenin-based betacyanins present in Basella alba L. fruit juice submitted to heating at 85ºC.

No.

Compound

Abbreviation

Rt [min]

λmax

m/z from m/z

[nm]

MS/MS of [M+H]+

1

gomphrenin

Gp

10.9

538

551

389

1′

isogomphrenin

IGp

11.7

538

551

389

2

17-decarboxy-gomphrenina

17-dGp

11.2

507

507

345

17-dIGp

12.0

507

507

345

15-dGp

12.7

530

507

345

a

17-decarboxy-isogomphrenin

2′

a

15-decarboxy-gomphrenin

3

a

4

2-decarboxy-gomphrenin

2-dGp

13.2

533

507

345

4′

2-decarboxy-isogomphrenina

2-dIGp

13.3

533

507

345

2,17-dGp

14.0

510

463

301

2,17-dIGp

14.1

510

463

301

a

2,17-bidecarboxy-gomphrenin

5

a

2,17-bidecarboxy-isogomphrenin

5′

a

2,17-bidecarboxy-2,3-dehydro-gomphrenin

2,17-dec-2,3-dHGp

14.2

465

461

299

7

neogomphrenina

NGp

14.8

471

549

387

8

2,15,17-tridecarboxy-gomphrenina

2,15,17-dGp

15.1

509

419

257

2-dNGp

16.0

-

b

505

343

2,17-dec-2,3-dHNGp

16.2

418

459

297

6/6’

a

2-decarboxy-neogomphrenin

9

a

2,17-bidecarboxy-2,3-dehydro-neogomphrenin

10

a

11

2,17-bidecarboxy-neogomphrenin

12

2-decarboxy-2,3-dehydro-neogomphrenina

2,17-dec-NGp

16.4

467

461

299

2-dec-2,3-dHNGp

17.2

424

503

341

a

Tentatively identified.

b

Due to a coelution with impurities, the λmax could not be observed.

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Journal of Agricultural and Food Chemistry

Page 24 of 31

Table 2. High-resolution mass spectrometric data obtained by IT-TOF of decarboxylated gomphrenins present in Basella alba L. fruit juice submitted to heating at 85ºC as well as for their fragmentation ions (m/z 301). No. 2

2′

3

4

4′

5

5′

Molecular

[M+H]+

[M+H]+

Error

Error

formula

observed

predicted

[mDa]

[ppm]

17-decarboxy-gomphrenin

C23 H26 N2 O11

507.1603

507.1609

-0.6

-1.18

463; 345; 301

bidecarboxy-betanidin

C16 H16 N2 O4

301.1169

301.1183

-1.4

-4.65

-

17-decarboxy-isogomphrenin

C23 H26 N2 O11

507.1613

507.1609

0.4

0.79

463; 345; 301

bidecarboxy-betanidin

C16 H16 N2 O4

301.1179

301.1183

-0.4

-1.33

-

Compound

MS2 ions

15-decarboxy-gomphrenin

C23 H26 N2 O11

507.1614

507.1609

0.5

0.99

463; 345; 301

bidecarboxy-betanidin

C16 H16 N2 O4

301.1168

301.1183

-1.5

-4.98

-

2-decarboxy-gomphrenin

C23 H26 N2 O11

507.1619

507.1609

1.0

1.97

463; 345; 301

bidecarboxy-betanidin

C16 H16 N2 O4

301.1172

301.1183

-1.1

-3.65

-

2-decarboxy-isogomphrenin

C23 H26 N2 O11

507.1601

507.1609

-0.8

-1.58

463; 345; 301

bidecarboxy-betanidin

C16 H16 N2 O4

301.1188

301.1183

0.5

1.66

-

2,17-bidecarboxy-gomphrenin

C22 H26 N2 O9

463.1709

463.1711

-0.2

-0.43

301

2,17-bidecarboxy-betanidin

C16 H16 N2 O4

301.1174

301.1183

-0.9

-2.99

-

2,17-bidecarboxy-isogomphrenin

C22 H26 N2 O9

463.1721

463.1711

1.0

2.16

301

2,17-bidecarboxy-betanidin

C16 H16 N2 O4

301.1180

301.1183

-0.3

-1.00

-

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Journal of Agricultural and Food Chemistry

Table 3. Results of identity confirmation of gomphrenin-based derivatives enzymatically deglucosylated18 along with the corresponding betanin-based standards. Obtained deglucosylation diagnostic products are identical for each tested pair of the corresponding substrates according to their chromatographic, spectrophotometric and mass spectrometric data. No. 1

Rt

Substrate

[min] [nm]

17-decarboxy-gomphrenin 2

3

4

5

6

507

9.6 505

507

17-decarboxy-isogomphrenin 2′

12.0 507

507

17-decarboxy-isobetanin

10.3 505

507

15-decarboxy-gomphrenin 3

12.7 530

507

15-decarboxy-betanin

11.0 527

507

2-decarboxy-gomphrenin 4

13.2 533

507

2-decarboxy-betanin

11.7 533

507

2-decarboxy-isogomphrenin 4′

13.3 533

507

2-decarboxy-isobetanin

11.7 533

507

2,17-bidecarboxy-gomphrenin 5

14.0 510

463

2,17-bidecarboxy-betanin

12.5 507

463

14.1 510

463

2,17-bidecarboxy7

-isogomphrenin 5′ 2,17-bidecarboxy-isobetanin 2,17-bidecarboxy-2,3-dehydro-

8

neogomphrenin 10 2,17-bidecarboxy-2,3-dehydroneobetanin 2-decarboxy-2,3-dehydro-

9

m/z

11.2 507

17-decarboxy-betanin 2

λmax

neogomphrenin 12 2-decarboxy-2,3-dehydroneobetanin

12.5 507

463

16.2 418

459

14.9 414

459

17.2 424

503

16.8 422

503

Deglucosylation diagnostic product

Rt

λmax

[min] [nm]

m/z

17-decarboxy-betanidin

11.7

510

345

17-decarboxy-isobetanidin

12.5

510

345

15-decarboxy-betanidin

13.6

531

345

2-decarboxy-betanidin

14.4

536

345

2-decarboxy-isobetanidin

14.4

536

345

2,17-bidecarboxy-betanidin

15.0

508

301

15.0

508

301

17.1

415

297

17.5

433

341

2,17-bidecarboxyisobetanidin

2,17-bidecarboxy-2,3dehydro-neobetanidin

2-decarboxy-2,3-dehydroneobetanidin

25 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1 2

List of figures

3 4

Figure 1. Chemical structures of the detected gomphrenin and gomphrenin-based derivatives

5

in Basella alba fruit juice submitted to heating at 85ºC.

6 7

Figure 2. Chromatographic DAD traces of Basella alba fruit juice (A) and the juice heating

8

products obtained after 10 min (B) and 30 min (C) monitored at 440 nm.

9 10

Figure 3. Results of the elution order recognition for 2-decarboxy-gomphrenin/-

11

isogomphrenin 4/4' on the C18 HPLC column established by decarboxylation of isolated

12

gomphrenin/isogomphrenin 1/1'. The experiment results indicate that the elution order for 4

13

and 4' (chromatograms B and D, respectively) is the same as for 1 and 1' (chromatograms A

14

and C, respectively).

15 16

Figure 4. Results of the elution order recognition for 2,17-bidecarboxy-gomphrenin/-

17

isogomphrenin 5/5' on the C18 HPLC column established by decarboxylation of isolated 17-

18

decarboxy-gomphrenin/-isogomphrenin 2/2' (chromatograms A and B, respectively) as well

19

as 2-decarboxy-gomphrenin/-isogomphrenin 4/4' (chromatograms C and D, respectively). The

20

experiment results indicate that the elution order for 5 and 5' is the same as for 1/1', 2/2' and

21

4/4'.

22 23 24 25

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Figure 1

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30 31

Figure 2

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Figure 3

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Figure 4

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TOC Graphic

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31 ACS Paragon Plus Environment