Thermal Degradation of Major Gomphrenin Pigments in the Fruit Juice

Article pubs.acs.org/JAFC

Thermal Degradation of Major Gomphrenin Pigments in the Fruit Juice of Basella alba L. (Malabar Spinach) Agnieszka Kumorkiewicz and Sławomir Wybraniec* Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24, Cracow 31-155, Poland ABSTRACT: Generation of decarboxylated and dehydrogenated gomphrenins during heating of Basella alba L. fruit juice containing high levels of betacyanin pigments was monitored by LC-DAD-ESI-MS/MS. The presence of principal decarboxylation products, 2-, 17-, and 2,17-decarboxy-gomphrenins, their diastereomers, as well as minor levels of their dehydrogenated derivatives are reported. In addition, determination of molecular masses of decarboxylated gomphrenins by highresolution mass spectrometry (LCMS-IT-TOF) was performed. Enzymatic deglucosylation of decarboxylated and dehydrogenated gomphrenins resulted in the generation of betanidin diagnostic derivatives for further identification processes. In addition, experiments were conducted to prove that the position of glucosylation of the chromophoric part of betacyanins (betanidin part) has decisive influence on different chromatographic properties of their decarboxylated derivatives. KEYWORDS: decarboxylated and dehydrogenated gomphrenins, betacyanins, betalains, betanidin, Basella alba, Malabar spinach, plant pigments

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INTRODUCTION Basella alba L. (Basellaceae), frequently known as Malabar spinach (as well as Indian spinach, Ceylon spinach, vine spinach, or climbing spinach), is a succulent, branched, smooth, perennial twining herbaceous vine that can reach several meters in length.1−4 The stem of Basella alba is green, but the stem, leaves, and petioles of the cultivar Basella alba ‘Rubra’ are redviolet. The fruits are fleshy, stalkless, ovoid or spherical, 5 to 6 mm long, and purple when mature; they contain betacyanins as the major pigments.1 The leaves of Basella alba are similar to spinach in that they can be prepared and consumed for potential health benefit, which makes them attractive for common diets.2 Promising applications of various parts of the plant for disease treatment and healing effects in humans have been reported.1 The plant, especially the leaves and stem, has been explored for its medicinal properties in ancient Indian and Chinese traditional medicine practices to treat constipation, as a diuretic, and as an anti-inflammatory agent.2−4 However, the purple fruits of Basella alba and Basella alba ‘Rubra’, rich in betacyanins and other bioactive phytochemicals, have not been investigated as food preparations and deserve exploration for possible medicinal and functional food applications. Recently, some anticarcinogenic activities of the fruit juice have been noted.3 The pigment-rich fruit extract was also tested as a natural colorant for ice cream.5 Betacyanins are water-soluble plant pigments that are a subgroup of betalains, which are found in most families of the Caryophyllales;6 they are extensively used in the food industry as food colorants.7 In addition, betacyanins have chemopreventive characteristics and strong antioxidant properties.8−18 Recent research has focused on new structures and derivatives of betacyanins, as well as their influence on health. These pigments are present mostly in plant fruits, flowers, and roots, as well as in tissues exposed to stress.6,7,16 © 2017 American Chemical Society

Despite the potential benefits and uses of betacyanins, systematic research of their activities is lacking. The 6-Oglycoside of betanidin is an extremely important betacyanin (Figure 1) and is present in high concentration in fruits of Basella alba L. and in leaves of its variety Basella alba ‘Rubra’. According to our recent studies, because of the presence of the phenolic group at carbon C-6 in gomphrenin I, the only possible quinonoid intermediate during oxidation of gomphrenin I is a dopachromic derivative.19−21 Therefore, gomphrenin I enables a unique possibility to observe reaction pathways complementary to betanin reaction routes, which is important for understanding the mechanism of betacyanin oxidation. This technique may also reveal other pro-health activities and chemical properties. To date, the highest antioxidant activity among betacyanins has been attributed to gomphrenin I.14 A series of reports have been published that detail several new groups of betacyanin degradation products, especially decarboxylated derivatives, in preparations subjected to thermal processing.22−26 Presumably, new gomphrenin derivatives should also have promising pro-health activities and important potentials for studies of betacyanin oxidation mechanism. For this reason, our research focused on the identification of colored degradation products in heated fruit juice of Basella alba L. We established the first tentative structures formed by decarboxylation and dehydrogenation of the main pigment present in the juice (gomphrenin I) and its diastereomer by means of liquid chromatography coupled to diode array detection and electrospray ionization tandem mass spectrometry (LC-DAD-ESI-MS/MS). To aid the identification process, Received: Revised: Accepted: Published: 7500

May 20, 2017 July 22, 2017 July 27, 2017 July 27, 2017 DOI: 10.1021/acs.jafc.7b02357 J. Agric. Food Chem. 2017, 65, 7500−7508

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of the detected gomphrenin and gomphrenin-based derivatives in Basella alba fruit juice submitted to heating at 85 °C. Cracow. In order to obtain the juice, 100 g of the fruits was manually squeezed, and 20 mL of obtained liquid was centrifuged and filtered through a 0.2 mm i.d. pore size filter and then 3-fold diluted with water for storage at −20 °C (typically for a few weeks) before the subsequent experiments. For semipreparative isolation of gomphrenin/isogomphrenin 1/1′, the juice was first filtered through a bed (10 cm height × 2 cm i.d.) of 0.063/0.200 mm silica (J.T. Baker, Deventer, Holland) to remove hydrocolloids and proteins to obtain a clear solution and subsequently through a 0.2 mm i.d. pore size filter. This solution was purified by semipreparative liquid chromatography. Heating Experiments on Basella alba Fruit Juice and Isolated Gomphrenins. 3 mL of Basella alba fruit juice was diluted

we performed enzymatic deglucosylation of the degradation products, which resulted in the formation of diagnostic betanidin derivatives. For the identification, we deglucosylated a series of already known betanin-based standards.

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MATERIALS AND METHODS

Reagents. Formic acid, LC-MS grade methanol, water, as well as almond β-glucosidase were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Preparation of Juice from Basella alba Fruits. Basella alba L. fruits were collected in a greenhouse at University of Agriculture in 7501

DOI: 10.1021/acs.jafc.7b02357 J. Agric. Food Chem. 2017, 65, 7500−7508

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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 85 °C

a

no.

compound

abbreviation

Rt [min]

λmax [nm]

m/z

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

1 1′ 2 2′ 3 4 4′ 5 5′ 6/6′ 7 8 9 10 11 12

gomphrenin isogomphrenin 17-decarboxy-gomphrenina 17-decarboxy-isogomphrenina 15-decarboxy-gomphrenina 2-decarboxy-gomphrenina 2-decarboxy-isogomphrenina 2,17-bidecarboxy-gomphrenina 2,17-bidecarboxy-isogomphrenina 2,17-bidecarboxy-2,3-dehydro-gomphrenina neogomphrenina 2,15,17-tridecarboxy-gomphrenina 2-decarboxy-neogomphrenina 2,17-bidecarboxy-2,3-dehydro-neogomphrenina 2,17-bidecarboxy-neogomphrenina 2-decarboxy-2,3-dehydro-neogomphrenina

Gp IGp 17-dGp 17-dIGp 15-dGp 2-dGp 2-dIGp 2,17-dGp 2,17-dIGp 2,17-dec-2,3-dHGp NGp 2,15,17-dGp 2-dNGp 2,17-dec-2,3-dHNGp 2,17-dec-NGp 2-dec-2,3-dHNGp

10.9 11.7 11.2 12.0 12.7 13.2 13.3 14.0 14.1 14.2 14.8 15.1 16.0 16.2 16.4 17.2

538 538 507 507 530 533 533 510 510 465 471 509 -b 418 467 424

551 551 507 507 507 507 507 463 463 461 549 419 505 459 461 503

389 389 345 345 345 345 345 301 301 299 387 257 343 297 299 341

Tentatively identified. bBecause of a coelution with impurities, the λmax could not be observed.

three times with water, acidified with 50 μL of glacial acetic acid, and heated at 85 °C in a water bath for 40 min according to previous studies.22,23 200 μL aliquots of the heated samples were collected for LC-DAD-ESI-MS/MS analysis every 5 min. For additional heating experiments of previously isolated single diastereomers of gomphrenin, 2- and 17-decarboxy-gomphrenin (for recognition of elution order of 4/4′ and 5/5′), their 1 mL solutions (100 μM) were acidified with 10 μL of glacial acetic acid and heated at 85 °C in a water bath for 10−20 min. 100 μL aliquots of the heated samples were collected every 5 min and analyzed by LC-DAD-ESI-MS/MS. Semisynthesis of Gomphrenin Derivatives. For the comparative hydrolysis experiments with single purified gomphrenin derivatives, thermal decarboxylation and dehydrogenation of gomphrenin and isogomphrenin in triple diluted 200 mL of Basella alba fruit juice were performed as described for heating experiments. Heating of gomphrenin and isogomphrenin present in the juice within 10−20 min resulted in the production of derivatives (Figure 1) differing in decarboxylation position (2/2′, 3, 4/4′, and 5/5′) as described in detail in the Results and Discussion section (Figure 1) (Table 1). Similarly, prolonged heating to 30−40 min generated increased levels of dehydrogenated derivatives: 2,17-bidecarboxy-2,3dehydro-neogomphrenin 10 and 2-decarboxy-2,3-dehydro-neogomphrenin 12. For the comparative studies, the analogous betaninderived compounds isolated previously by high-speed countercurrent chromatography and/or HPLC were prepared.19,27 Deglucosylation of Gomphrenin- and Betanin-Based Derivatives. β-Glucosidase hydrolysis for deglucosylation of previously isolated single diastereomers of selected gomphrenin derivatives as well as their corresponding betanin derivatives was performed in solutions containing 25 mM acetate buffer (pH 5), almond βglucosidase (15 units/mL), as well as 20−50 μM pigment at 30 °C for 30 min. For the chromatographic analyses, 20 μL samples of reaction mixtures were injected directly into the LC-DAD-MS/MS system without further purification. Semipreparative Chromatography. For the isolation of gomphrenin/isogomphrenin from the juice of Basella alba L. as well as gomphrenin-based derivatives obtained by heating of diluted Basella alba fruit juice, a flash chromatography system (preparative HPLC system with LC-20AP pumps, UV−vis SPD-20AV detector, and LabSolutions 5.51 operating software, Shimadzu Corp., Japan) equipped with a C18 (250 × 50 mm i.d., 30 μm) column (Interchim, France) was applied. Further separation and isolation of pigments was performed on an HPLC semipreparative column Luna C18(2) 250 × 10 mm i.d., 10 μm (Phenomenex, Torrance, CA, USA) with a 10 mm × 10 mm i.d. guard column of the same material (Phenomenex, Torrance, CA USA) under the following gradient system (System 1):

6% A in B at 0 min; gradient to 20% A in B at 30 min (A, acetonitrile; B, 1% (v/v) HCOOH in H2O). The injection volume was 2 mL, and the flow rate was 3 mL/min. Detection was performed at 538, 505, 480, and 440 nm with a PDA UV/vis detector; column temperature, 25 °C. The eluates were pooled and concentrated under reduced pressure at 25 °C and finally freeze-dried to obtain pure pigments. Chromatographic Analysis by LC-DAD-ESI-MS/MS System. For the chromatographic and mass spectrometric analyses, an LCMS8030 mass spectrometric system (Schimadzu, Kyoto, Japan) coupled to LC-20ADXR HPLC pumps, an injector model SIL-20ACXR, and a PDA detector (photo diode array) model SPD-M20A, all controlled with LabSolutions software, version 5.60 SP1 (Schimadzu, Japan), was used. The samples were eluted through a 150 mm × 4.6 mm i.d., 5.0 μm, Kinetex C18 chromatographic column preceded by a guard column of the same material (Phenomenex, Torrance, CA, USA). The injection volume was 20 μL, and the flow rate was 0.5 mL/min. The column was thermostated at 40 °C. The separation of the analytes was performed with a binary gradient elution. The mobile phases were: A, 2% formic acid in water, and B, pure methanol. The gradient profile was (t [min], % B), (0, 5), (12, 70), (15, 80), and (19, 80). The full range PDA signal was recorded, and chromatograms at 538, 505, 490, and 440 nm were individually displayed. Positive ion electrospray mass spectra were recorded on the LC-MS system which was controlled with LabSolutions software. The ionization electrospray source operated in positive mode (ESI+) at an electrospray voltage of 4.5 kV and capillary temperature at 250 °C, using N2 as a gas for the spray, recording total ion chromatograms, mass spectra, and ion chromatograms in selected ion monitoring mode (SIM) as well as the fragmentation spectra. Argon was used as the collision gas for the collision-induced dissociation (CID) experiments. The relative collision energies for MS/MS analyses were set at −35 V. Chromatographic Analysis with Detection by Ion-Trap Time-of-Flight System (LCMS-IT-TOF). All mass spectra were analyzed using a LCMS-IT-TOF mass spectrometer (Shimadzu) equipped with an electrospray (ESI) ion source and coupled to HPLC Prominence (Shimadzu). Separation of compounds was carried out on a 50 mm × 2.1 mm i.d., 1.9 μm Shim Pack GISS C18 column (Shimadzu). The injection volume was 2 μL, and the flow rate was 0.2 mL/min. The column was thermostated at 40 °C. The separation of the analytes was performed with a binary gradient elution. The mobile phases were A, 0.1% formic acid in water, and B, pure methanol. The gradient profile was (t [min], % B), (0, 5), (12, 30), (17, 80), and (19, 80). Parameters of the LCMS-IT-TOF spectrometer were set as follows: curved desolvation line (CDL) and heat block temperature, 230 °C; nebulizing gas flow rate, 1.5 L/min; and capillary voltage, 4.5 kV. All mass spectra, including fragmentation mass spectra, were 7502


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known.28 For simplicity, we will refer to the main pigment as “gomphrenin” instead of “gomphrenin I” and gomphrenins II and III will be named “acylated gomphrenins.” This is justified by the unique importance of gomphrenin, which we believe is comparable to another basic betacyanin: betanin; both pigments are basic O-glucosylated positional isomers. Additionally, the series of gomphrenin derivatives presumably have important pro-health properties and will be frequently studied. Therefore, deriving names from the “gomphrenin” root is also justified for simplification of the nomenclature of the multitude of the derivatives. The resulting high-performance liquid chromatographic (HPLC) profiles of decarboxylation/dehydrogenation products formed during the heating experiments are depicted in Figure 2B,C and their LC-DAD-MS fingerprints are presented in Table 1. For the most prominent decarboxylated gomphrenins, additional results from high-resolution liquid chromatography coupled with ion-trap and time-of-flight mass spectrometry (LCMS-IT-TOF) analyses are listed in Table 2. Generation of Monodecarboxy-Gomphrenins during the Heating of Basella alba L. Fruit Juice. The aqueous solutions acidified by acetic acid were heated for 10 to 15 min, and pigment profiles were obtained; the profiles were similar to the profiles of early products of Beta vulgaris L. root heating, but the retention times were shifted.22,24,26 This reflects the similar conditions of the experiments (aqueous solutions acidified by acetic acid at similar temperatures) to those applied previously for heating of Beta vulgaris L. juice.22 The temperature (85 °C) of the heating process was high enough for monitoring changes in the compositions of the resulting mixtures. The main resulting chromatographic peaks corresponded to well-separated compounds 2 and 2′, as well as a slightly resolved pair 4/4′ (Figure 2B). Additionally, lower signals for compound 3 and another slightly resolved pair 5/5′ were detected. All of the detected compounds (Figure 1) were less polar than their corresponding precursors (gomphrenin/ isogomphrenin 1/1′). Further interpretation of the LC-DAD and LC-MS/MS spectra revealed that the main products appeared to be monodecarboxylated derivatives with absorption maxima at λmax 507 nm for 2/2′ and λmax 533 nm for 4/4′ (Table 1) with characteristic pseudomolecular ions with m/z 507 due to the loss of CO2 from the corresponding precursors (gomphrenin/isogomphrenin 1/1′). In the collision-induced

recorded in the positive ion mode with a mass range of 100−2000 Da and collision energy between 12−50% depending on the compound’s structure. The results of high resolution mass spectrometry experiments (HRMS) were studied using the Formula Predictor within the LCMS Solution software. Only empirical formulas with a mass error below 5 ppm were taken into account.

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RESULTS AND DISCUSSION The chromatogram in Figure 2A depicts a typical betacyanin profile in Basella alba fruit juice. The dominant presence of the

Figure 2. Chromatographic DAD traces of Basella alba fruit juice (A) and the juice heating products obtained after 10 min (B) and 30 min (C) monitored at 440 nm.

known gomphrenin 1 and its isoform 1′ with minor quantities of gomphrenin II and III (acylated gomphrenins) is well-

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 85°C as Well as for Their Fragmentation Ions (m/z 301) no.

compound

2

17-decarboxy-gomphrenin bidecarboxy-betanidin 17-decarboxy-isogomphrenin bidecarboxy-betanidin 15-decarboxy-gomphrenin bidecarboxy-betanidin 2-decarboxy-gomphrenin bidecarboxy-betanidin 2-decarboxy-isogomphrenin bidecarboxy-betanidin 2,17-bidecarboxy-gomphrenin 2,17-bidecarboxy-betanidin 2,17-bidecarboxy-isogomphrenin 2,17-bidecarboxy-betanidin

2′ 3 4 4′ 5 5′

molecular formula C23 C16 C23 C16 C23 C16 C23 C16 C23 C16 C22 C16 C22 C16

H26 H16 H26 H16 H26 H16 H26 H16 H26 H16 H26 H16 H26 H16

N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2

O11 O4 O11 O4 O11 O4 O11 O4 O11 O4 O9 O4 O9 O4

[M + H]+ observed

[M + H]+ predicted

error [mDa]

error [ppm]

MS2 ions

507.1603 301.1169 507.1613 301.1179 507.1614 301.1168 507.1619 301.1172 507.1601 301.1188 463.1709 301.1174 463.1721 301.1180

507.1609 301.1183 507.1609 301.1183 507.1609 301.1183 507.1609 301.1183 507.1609 301.1183 463.1711 301.1183 463.1711 301.1183

−0.6 −1.4 0.4 −0.4 0.5 −1.5 1.0 −1.1 −0.8 0.5 −0.2 −0.9 1.0 −0.3

−1.18 −4.65 0.79 −1.33 0.99 −4.98 1.97 −3.65 −1.58 1.66 −0.43 −2.99 2.16 −1.00

463; 345; 301

7503

463; 345; 301 463; 345; 301 463; 345; 301 463; 345; 301 301 301


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Journal of Agricultural and Food Chemistry fragmentation experiments, the daughter ion spectra displayed fragments of [M + H]+ at m/z 345 in each case, which represented the decarboxylated aglycone (betanidin) part of the molecules. Additionally, LC-IT-TOF analyses yielding m/z 507.1603, 507.1613, 507.1619, and 507.1601 (C23H26N2O11, calculated mass: 507.1609) supported the identification of decarboxylated gomphrenins 2/2′ and 4/4′ (Table 2). Previous absorption data indicated that 17-decarboxy-betanin has a characteristic absorption of approximately λmax 506 nm.22,24 Therefore, it was possible to conclude that the pair 2 and 2′ were 17-decarboxy-gomphrenin and 17-decarboxyisogomphrenin, respectively. The slightly higher retention times of 2 and 2′ compared to their corresponding precursors 1 and 1′ support this assumption. For 4/4′, the only possible pair of diastereomers were 2decarboxylated derivatives. This conclusion was supported by an absorption maximum (λmax 533 nm) and chromatographic retention (not completely resolved pair of peaks) that were similar to those of betanin thermal degradation products (2decarboxy-betanin/-isobetanin) in heated red beet juice,22,24,26 as well as in endogenously present dopamine-derived 2decarboxy-betacyanins in hairy roots of Beta vulgaris and Carpobrotus acinaciformis.29,30 Therefore, the pair 4 and 4′ was assigned to 2-decarboxy-gomphrenin and 2-decarboxy-isogomphrenin, respectively. Interestingly, these pigments were much better separated in reversed-phase HPLC, especially at low concentrations of formic acid in the eluent, than their corresponding betanin derivatives.22,23 The elution order of 4 and 4′ on the C18 HPLC column was established by analysis of decarboxylation products of previously isolated gomphrenin/isogomphrenin (1/1′), assuming that isomerization is less strong than decarboxylation. This procedure was previously successfully applied in experiments of 2-decarboxy-betanin/-isobetanin elution order recognition in which the reversed order was revealed compared to the precursor pair (betanin/isobetanin) and the pair of 17decarboxy-betanin/-isobetanin.22 Figure 3 represents the results of the experiment in which, rather unexpectedly, the elution order for 4 and 4′ appeared the same as for 1 and 1′ (the form 4 is eluted earlier than the isoform 4′). This finding is in contrast to betanin-based 2decarboxylated derivatives.22 In addition, during the experiment, equal quantities of both forms of 17-decarboxygomphrenin/-isogomphrenin (2/2′) were generated, irrespective of the starting epimer of gomphrenin (1/1′) resembling the generation profile of the corresponding 17-decarboxybetanin/-isobetanin.22 The mechanism of epimerization at carbon C-15 in betanidin had already been explained by Dunkelblum et al.31 Further experiments based on a method of hydrolysis and cross-recondensation of selected mixtures of betacyanins are needed to confirm the elution order of 4 and 4′.32 Further analysis of the data indicated the presence of a small peak (Figure 2) likely corresponding to 15-decarboxygomphrenin (3), which is formed by decarboxylation (detection of protonated molecular ions at m/z 507 with their fragmentation to ions of m/z 345) with a loss of the chiral center at carbon C-15 in gomphrenin.10 Therefore, the presence of only one form of 3 suggested that this compound was similar to 15-decarboxy-betanin, which was previously detected in red beet extract.33 Indeed, a similar absorption maximum of 3 at λmax 530 nm and elution between 17decarboxy-isogomphrenin (2′) and 2-decarboxy-gomphrenin

Figure 3. Results of the elution order recognition for 2-decarboxygomphrenin/-isogomphrenin 4/4′ on the C18 HPLC column established by decarboxylation of isolated gomphrenin/isogomphrenin 1/1′. The experiment results indicate that the elution order for 4 and 4′ (chromatograms B and D, respectively) is the same as that for 1 and 1′ (chromatograms A and C, respectively).

(4) supported the assignment of 3 (C23H26N2O11, m/z 507.1614; calculated mass, 507.1609). Bidecarboxy- and Tridecarboxy-Gomphrenins. After prolonged heating (30 min) of Basella alba L. fruit juice, higher quantities of compounds corresponding to chromatographic peaks 5/5′ were detected (Figure 2). These pigments displayed absorption maxima at λmax 510 nm and pseudomolecular ions at m/z 463, clearly indicating a loss of two CO2 molecules from the starting gomphrenin/isogomphrenin, 1/1′ (Figure 1). Subsequent fragmentation to ions of m/z 301 confirmed the existence of a bidecarboxylated fragment of betanidin and suggested the formation of bidecarboxylated gomphrenin/ isogomphrenin. The detection of the two key epimers clearly indicated the position of double decarboxylation and, therefore, the presence of 2,17-bidecarboxy-gomphrenin and its isoform 5/5′ (C22H26N2O9, m/z 463.1709, calculated mass: 463.1711). Interestingly, these epimers were well separated in the applied HPLC system, especially in eluents containing diluted formic acid. This is in contrast to the lack of separation between the epimers of 2,17-bidecarboxy-betanin and -isobetanin, which had been separated only in ion-pair chromatography.22 Accordingly, taking into account the properties of 2decarboxy-gomphrenin/-isogomphrenin (4/4′) and 2,17-bidecarboxy-gomphrenin/-isogomphrenin (5/5′), the position of 7504


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gomphrenin-based diastereomeric pairs (1/1′, 2/2′, 4/4′, and 5/5′) were the same. Previously, similar experiments were performed on betaninbased (phyllocactin and hylocerenin) decarboxylated derivatives in aqueous or ethanolic solutions: the experiments resulted in the generation of equal quantities of both of the respective bidecarboxy-betacyanin isomers.23 Consequently, the generation of equal quantities of the respective bidecarboxybetacyanin isomers prevented deduction of their elution order. Additionally, for 2,17-bidecarboxy-betanin/-isobetanin, the detection of both forms was possible only in an ion-pairing HPLC system, which yielded the same negative results.22 Prolonged heating in acetic acid solutions released small quantities of compound 8 (Figures 1 and 2). It was slightly less polar than 2,17-bidecarboxy-gomphrenin and, in LC-MS analysis, displayed a pseudomolecular ion at m/z 419 and an absorption maximum of λmax 509 nm. This suggested the presence of a tridecarboxy-gomphrenin for which the only possible structure was 2,15,17-tridecarboxy-gomphrenin. This conclusion was supported by the detection of only one chromatographic peak 8 in the HPLC system, which resulted from the loss of the chiral center at carbon C-15 in 8. Subsequent fragmentation experiments on the pseudomolecular ion at m/z 419 revealed fragmentation ions at m/z 257, which proved the existence of the tridecarboxylated fragment of betanidin. Comparative Enzymatic Deglucosylation Studies on Gomphrenin- and Betanin-Based Decarboxylated Derivatives. The presence of the same aglycones (decarboxylated betanidins) in the structures of betanin- and gomphrenin-based decarboxylated derivatives enabled further chromatographic confirmation of the identities of the decarboxylated gomphrenins. For this aim, we performed experiments based on a βglucosidase assay, a sensitive tool for β-deglucosylation of such pigments as betanin or gomphrenin (Figure 1) and other nonacylated betacyanins at the first β-glucosidic ring.34 The assays were conducted with almond β-glucosidase and yielded 2-, 15-, 17-, and 2,17-decarboxy-betanidins (Table 3) as a result of β-deglucosylation34 of the starting glucosylated substrates (the corresponding 2-, 15-, 17-, and 2,17-decarboxygomphrenins (Figure 1) and the analogous decarboxylated betanins). Their identities were confirmed by the same retention times and spectrophotometric and mass spectrometric data obtained for the products (decarboxylated betanidins). Additionally, the corresponding diastereomers (where applicable) were also positively tested. Because of the scarce quantities of 2,15,17-tridecarboxy-betanin that were generated, tests for this pigment were not performed. The generated betanidin derivatives were sufficiently stable for performing the chromatographic analyses, even after several hours of the reaction and despite the elevated temperature of the enzymatic process. Generation of Dehydrogenated Gomphrenins during the Heating of Basella alba Fruit Juice. Dehydrogenated betacyanins are formed as a result of oxidation of the corresponding betacyanins and their decarboxylated derivatives. In the case of red beet juice,22,24 a series of dehydrogenated betanin-like derivatives were identified after prolonged heating experiments. Similarly, a complex mixture of dehydrogenated betacyanins was derived from the juice of Hylocereus polyrhizus fruits containing three main betacyanins: betanin, phyllocactin, and hylocerenin, as well as their isoforms.23

glucosylation of the betanidin at carbon C-5 or C-6 has decisive influence on the chromatographic differences between betanin and gomphrenin derivatives. Subsequent experiments designed to recognize the elution order of 2,17-bidecarboxy-gomphrenin/-isogomphrenin (5/5′) were performed by heating of each single form of previously isolated 17-decarboxy-gomphrenin/-isogomphrenin (2/2′) and 2-decarboxy-gomphrenin/-isogomphrenin (4/4′). The results of the experiments are presented in Figure 4. Interestingly, in

Figure 4. Results of the elution order recognition for 2,17bidecarboxy-gomphrenin/-isogomphrenin 5/5′ on the C18 HPLC column established by decarboxylation of isolated 17-decarboxygomphrenin/-isogomphrenin 2/2′ (chromatograms A and B, respectively) as well as 2-decarboxy-gomphrenin/-isogomphrenin 4/ 4′ (chromatograms C and D, respectively). The experiment results indicate that the elution order for 5 and 5′ is the same as that for 1/1′, 2/2′, and 4/4′.

each case, a positive result of the experiment was obtained (in contrast to experiments with betanin-based derivatives). Specifically, for each single 15S form of the substrate (2- and 17-decarboxy-gomphrenin), an evident peak of 2,17-bidecarboxy-gomphrenin 5 was obtained; the peak was accompanied by a much smaller peak of the 15R form (the isoform) that likely resulted from the epimerization effect. Analogous results were obtained for heated single 15R forms of the substrates (2and 17-decarboxy-isogomphrenin). Generated single products indicated the elution order of 5/5′ and confirmed that the isoform is eluted after the 15S form. Therefore, in contrast to betanin-based derivatives, the elution orders for all the tested 7505


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Table 3. Results of Identity Confirmation of Gomphrenin-Based Derivatives Enzymatically Deglucosylated18 along with the Corresponding Betanin-Based Standardsa no. 1 2 3 4 5 6 7 8

9

substrate 17-decarboxy-gomphrenin 2 17-decarboxy-betanin 17-decarboxy-isogomphrenin 2′ 17-decarboxy-isobetanin 15-decarboxy-gomphrenin 3 15-decarboxy-betanin 2-decarboxy-gomphrenin 4 2-decarboxy-betanin 2-decarboxy-isogomphrenin 4′ 2-decarboxy-isobetanin 2,17-bidecarboxy-gomphrenin 5 2,17-bidecarboxy-betanin 2,17-bidecarboxy-isogomphrenin 5′ 2,17-bidecarboxy-isobetanin 2,17-bidecarboxy-2,3-dehydro-neogomphrenin 10 2,17-bidecarboxy-2,3-dehydro-neobetanin 2-decarboxy-2,3-dehydro-neogomphrenin 12 2-decarboxy-2,3-dehydro-neobetanin

Rt [min]

λmax [nm]

m/z

11.2 9.6 12.0 10.3 12.7 11.0 13.2 11.7 13.3 11.7 14.0 12.5 14.1 12.5 16.2

507 505 507 505 530 527 533 533 533 533 510 507 510 507 418

507 507 507 507 507 507 507 507 507 507 463 463 463 463 459

14.9 17.2 16.8

414 424 422

459 503 503

Rt [min]

λmax [nm]

m/z

17-decarboxy-betanidin

11.7

510

345

17-decarboxy-isobetanidin

12.5

510

345


13.6

531

345


14.4

536

345

2-decarboxy-isobetanidin

14.4

536

345

2,17-bidecarboxy-betanidin

15.0

508

301

2,17-bidecarboxy-isobetanidin

15.0

508

301

2,17-bidecarboxy-2,3-dehydroneobetanidin

17.1

415

297

2-decarboxy-2,3-dehydro-neobetanidin

17.5

433

341

deglucosylation diagnostic product

a Obtained deglucosylation diagnostic products are identical for each tested pair of the corresponding substrates according to their chromatographic, spectrophotometric, and mass spectrometric data.

only one chromatographic peak, resulting from the loss of the chiral center at carbon C-15, was expected. Unfortunately, no ultraviolet−visible spectrum was able to be detected as a result of peak overlap with impurities. Further inspection of the LC-DAD-MS/MS data of longerheated samples revealed two peaks, 6 and 11 (Figure 2), that both corresponded to pseudomolecular ions at m/z 461, suggesting a loss of 2H from the more polar compound (with a lower retention time) 2,17-bidecarboxy-gomphrenin 5 (m/z 463). A subsequent fragmentation ion at m/z 299 from the loss of a glucose moiety in both cases supported the suggestion of the presence of a bidecarboxylated dehydrogenated fragment of betanidin. In the case of 11, the absorption maximum found at λmax 467 nm and its relatively high hydrophobic nature were similar to its analogue from the betanin group (2,17bidecarboxy-neobetanin).20,22 For this reason, the presence of 2,17-bidecarboxy-neogomphrenin 11 was inferred (Figure 1). In contrast, the more polar character of 6 and its close retention to 2,17-bidecarboxy-gomphrenin 5 indicated that this compound may be 2,17-bidecarboxy-2,3-dehydrogomphrenin (analogous to 2,17-bidecarboxy-2,3-dehydrobetanin formed after direct oxidation of 2,17-bidecarboxy-betanin).20 Because of the fact that the chiral center is present at carbon C-15 (Figure 1), the peaks must correspond to two unresolved epimers 6/6′. No additional identification for 6/6′ could be performed because no absorption maximum could be registered for 6/6′ due to overlap with 5/5′. The identity of 6/6′ will be verified by direct oxidation (e.g., by ABTS cation radicals20) of 2,17-bidecarboxygomphrenin 5/5′, which will possibly generate compounds identical to 6/6′. The presence of two dehydrogenated neo-derivatives of gomphrenin corresponding to peaks 10 and 12 were also detected in the LC-DAD-MS/MS data of the juice samples (Figure 2). Compound 10 was likely a result of the oxidation of 11 (analogous to a betanin derivative20); its mass spectrometric data (pseudomolecular ion at m/z 459 and fragmentation ion at m/z 297), its absorption maximum (λmax 418 nm), and its

Direct oxidation of betanin results in the generation of neobetanin (14,15-dehydrobetanin) and 2-decarboxy-2,3-dehydrobetanin (result of oxidative decarboxylation).19,20 The final result depends on the matrix composition, as well as additional factors such as buffers, pH, or oxidizing agents. The autooxidation of the pigments assisted by air-derived oxygen is also possible because of the relatively high antioxidant activity of betacyanins.8,11−15,17 Consequently, the formation of a group of gomphreninbased dehydrogenated derivatives was also expected in the heated Basella alba juice. Indeed, the presence of neogomphrenin 7 was detected in the heated juice. However, it was present at low concentration levels (Figure 2). Formation of neogomphrenin results in the loss of the chiral center at carbon C-15, yielding only one chromatographic peak. Neogomphrenin has lower polarity than gomphrenin/isogomphrenin, which results in a longer retention time. This is a general trend for neobetacyanins that is frequently observed during HPLC analyses.22−25 Subsequent confirmation was indicated by an absorption maximum at λmax 471 nm (similar for neobetacyanins) and m/z 549 obtained for pseudomolecular ions in the LC-MS/MS system, indicating the loss of 2H from gomphrenin/isogomphrenin during the heating experiment. The subsequent fragmentation ion at m/z 387 from the loss of a glucose moiety supported the formation of a dehydrogenated betanidin structure. Generated neogomphrenin 7 can undergo a further decarboxylation (most probably at carbon C-2 or C-1720,22) during heating. Therefore, in this study, we investigated the presence of the 2- or 17-decarboxy-neogomphrenin. However, based on its high retention time, as well as LC-MS/MS data, only 2-decarboxy-neogomphrenin 9 was detected. Detection of a pseudomolecular ion at m/z 505 and its subsequent fragment at m/z 343 from the loss of a glucose moiety confirmed the generation of decarboxylated and dehydrogenated betanidin and suggested a possibility that peak 9 may correspond to 2decarboxy-neogomphrenin. As in the case of neogomphrenin, 7506


Article

Journal of Agricultural and Food Chemistry Funding

relatively high hydrophobic nature indicated the presence of 2,17-bidecarboxy-2,3-dehydro-neogomphrenin 10. Similarly, high hydrophobicity, absorption data (λmax 424 nm), and mass spectrometric data (pseudomolecular ion at m/z 503 and fragmentation ion at m/z 341) strongly indicated that the identity of 12 was 2-decarboxy-2,3-dehydro-neogomphrenin (analogous to a betanin derivative20). As in the case of the oxidized structures mentioned above, the identities of 10 and 12 were supported by further oxidation experiments of a series of isolated and purified gomphrenin derivatives. Comparative Enzymatic Deglucosylation Studies on Betanin- and Gomphrenin-Based Dehydrogenated Derivatives. Further confirmation of the identities of the dehydrogenated neogomphrenins 10 and 12 were performed by the β-glucosidase assay with the use of additional betaninderived standards obtained in the previous oxidation study.20 The assay yielded 2-decarboxy-2,3-dehydro-betanidin and 2,17bidecarboxy-2,3-dehydro-betanidin (Table 3), which resulted from β-deglucosylation of the starting glucosylated substrates (the corresponding 2,17-bidecarboxy-2,3-dehydro-neogomphrenin 10 and 2-decarboxy-2,3-dehydro-neogomphrenin 12, as well as the analogous betanin derivatives). As mentioned, the identities were confirmed by the same retention times and the spectrophotometric and mass spectrometric data obtained for the products. The assay was not performed for the other dehydrogenated pigments due to the scarce quantities of the generated compounds. This is the first report on the generation of mono-, bi-, and tridecarboxylated gomphrenins and their dehydrogenated derivatives in general, but also specifically, in degradation products of heated Basella alba fruit juice, which can be used for various food applications. The health-promoting actions and colorant properties of these compounds should be different not only because of the matrix effect but also because of different activities of gomphrenin compared to those of gomphreninbased derivatives. Interestingly, from an analytical point of view, our results indicate that the position of glucosylation of betanidin at carbon C-5 or C-6 has significant influence on the chromatographic differences between betanin and gomphrenin derivatives. Further, the higher antioxidant activity of gomphrenin compared to that of betanin14 likely results from the favorable position of the glycosidic bond, which enables the formation of the aminochromic intermediate during oxidation.19,20 This also opens a question for other promising properties that may differ from the known activities of the commonly known betanins. In this respect, further investigation of Basella alba fruit juice, as well as its processed products, should significantly enhance our knowledge about the action of gomphrenins. Considering that gomphrenin is found in few other plant species (mostly at low concentration levels in Gomphrena globosa L.35 and Bougainvillea glabra Choisy36), its presence at high concentrations in Basella alba L. fruits3 renders this plant an extremely valuable source of gomphrenin for future applications.

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This research was financed by Polish National Science Centre for years 2015−2018 (Project No. UMO-2014/13/B/ST4/ 04854). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Beata Wileńska Ph.D., eng. and Bartłomiej Fedorczyk M.Sc. from Laboratory of Biologically Active Compounds (Warsaw University) for the excellent technical assistance with LCMS-IT-TOF experiments.

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REFERENCES

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Sławomir Wybraniec: 0000-0002-1263-4188 7507


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Thermal Degradation of Major Gomphrenin Pigments in the Fruit Juice

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