Structural Studies on Diverse Betacyanin Classes in Matured Pigment-Rich Fruits of Basella alba L. and Basella alba L. var. ‘Rubra’ (Malabar Spinach)

Identification of betacyanins in Basella alba L. and Basella alba L. var. ‘Rubra’ fruits was performed by low- and high-resolution mass spectrometry (LRMS and HRMS) as well as 1H, 13C and two-dimensional NMR which revealed hitherto completely not known betacyanin classes in the plant kingdom. Especially, the presence of unique nitrogenous acyl moieties in the structures of the pigments was ascertained by the HRMS Orbitrap detection. Except for detected polar betacyanin glycosylated derivatives, presence of a series of previously not reported pigments such as malonylated betanidin 6-O-β-glusosides with their acyl migration isomers along with the evidence of the 3′′-hydroxy-butyrylated betacyanins is reported. The first complete NMR data were obtained for novel and principal acylated gomphrenins with hydroxycinnamic acids: 6′-O-E-caffeoyl-gomphrenin (malabarin), 6′-O-E-sinapoyl-gomphrenin (gandolin), 6′-O-E-4-coumaroyl-gomphrenin (globosin) and 6′-O-E-feruloyl-gomphrenin (basellin).


Introduction
The popularization of innovative nutraceuticals and functional foods has triggered research and exploration of alternative nutrients, including undoubtedly Basella alba L. and its variety Basella alba L. var. 'Rubra' (Figure 1), frequently known as Malabar spinach. These plants most widely cultivated in Asia belong to the Basellaceae family and are characterized by branched, climbing stems with alternating succulent and mucilaginous leaves. In summer, they abound with dark violet-blue, small stone fruits [1]. Traditional medicine, especially in India and China, uses different parts of said plants to treat many diseases [2]. In addition to the content of vitamins and minerals, the extracts of these plants can be attributed to antimicrobial, [3] anti-inflammatory and antidepressant properties [2]. Valuable sources of pro-health substances are both the stems and leaves of Malabar spinach [4], but also extracts from its fruits, which have been proven, among others, to show cytotoxic properties against human cervical cancer cells [5]. Malabar spinach fruits contain carbohydrates, proteins, lipids, niacin, ascorbic acid, tocopherols, as well as pigments-betalains, of which gomphrenin I (betanidin 6-O-β-D-glucoside) and its isoform dominate [6][7][8]. Betalains are a group of water-soluble colored compounds containing nitrogen in their structure ( Figure 2) [7,9]. They occur in most plants of the order Caryophyllales [10], and they were also found in some species of fungi of the genera Amanita and Hygrocybe [11,12]. These compounds consist of betalamic acid as the chromophore core that condenses with cyclo-DOPA or amino acids/amines to form red-violet betacyanins and yellow-orange betaxanthins, respectively. Gomphrenins, classified as betacyanins [7], are characterized by a hydroxyl group attached to the C-5 carbon and a glucose linked at the C-6 position [13]. Betacyanins found in plant extracts are usually accompanied by their respective isoforms (isobetacyanins). The reason for isomerization may be factors induced by the environment, e.g., postharvest change of reaction pH, as well as thermal treatment.
Different forms of betacyanins behave differently depending on the conditions, e.g., thermal treatment may increase the amount of one isoform, thereby reducing the amount of the other [14].
The pigment profile of fruit extracts is influenced by the extraction method and depends on the subsequent purification method of the betacyanin fractions. Effective in terms of efficiency and economy is, among others, the purification on ion exchange beds [15,16]. An interesting literature report is the purification of extracts by pre-fermentation [17]. Betalain pigments are strong antioxidants [12] which is valuable, e.g., due to the known participation in the reduction of reactive oxygen (ROS) and nitrogen (RNS) species in the development of cancer and other diseases, such as atherosclerosis. Compounds belonging to the group of antioxidants have also been found to inhibit or delay the development of certain neoplasms [18]. The literature provides reports on the ability of betalains to inhibit the proliferation of melanoma cancer cells as well as inhibit the development of prostate and breast cancer. Moreover, no significant negative impact of these compounds on the human body has been found [19]. Based on the above facts, betalains, including betacyanins, are natural chemopreventive compounds, although they are not yet fully studied [8].
In addition to the aforementioned pro-health potential, betacyanins can be used in many fields. Due to the high coefficient of molar extinction, the dyeing capacity of betalains is competitive with that of synthetic dyes [20]. Unlike the acid-stable anthocyanins popular in coloring food, betalains maintain their color fairly well over a broader pH range (3)(4)(5)(6)(7). However, the most optimal condition for them is an environment with a pH in the range of 4-6 and the stability increases under anaerobic conditions [21]. Changing to a lower pH, the absorption maximum is shifted towards shorter wavelengths [7]. The stability of betalains is closely related to their chemical structure, and the improvement in stability as a result of glycosylation and acylation with hydroxycinnamic acids was indicated [22].
Raw materials rich in betalains can be used in the production of functional food where betalains not only have a color function but also increase the nutritional value. Preliminary results show the possibility of their use while maintaining stability and health-promoting properties in the dyeing of banana and lemon juices [23] or ice creams [24]. Also of interest is the confirmed encapsulation of betalain extracts from B. var. 'Rubra' juices in the form of gummy candy [25].
The not fully explored properties and unexplored possibilities of using plants rich in betalains [26][27][28], as well as the presence of unknown compounds in the profiles of extracts from individual plants, make this topic very demanding for the development of science and trends in functional food and nutraceuticals.
This contribution reports on detailed profiles of betacyanins in fruits of B. alba and B. var. 'Rubra' which were completely overlooked in an increasing number of reports but represent a significant fraction of the novel pigments in the fruits.

Results and Discussion
Recent reports on B. alba betacyanins [2][3][4][5][6]17,[23][24][25] focused on the main pigment, gomphrenin, only scarcely mentioning some other acylated gomphrenins which were structurally identified two decades ago as the principal acylated pigments in purple Gomphrena globosa L. [7,29]. In this study, our detailed studies evidently detected new betacyanin pigments (Table 1, Figure 2) and also revealed completely novel structures with unique acylation patterns (Tables 2 and 3) as well as compared quantitatively the two plant varieties (Section 2.7) [30]. Determination of unequivocal elemental compositions of unknown genuine pigments was possible using the HRMS coupled to the HPLC separation system.  The 'soft' fragmentation experiments at an applied energy of 20 eV in the quadrupole collisional stage before the Orbitrap HRMS detection gave especially valuable information concerning the main part structures of the analyzed compounds.
The resulting betacyanin fingerprints in B. alba and var. 'Rubra' fruits in the form of chromatograms for selected ion monitoring obtained in the LC-DAD-MS system are presented in Figure 3A,B, respectively. The presence of unique betacyanins with acylating moieties containing nitrogen in their structures is confirmed in the samples based on the following analytical HRMS results. The complete 1 H, 13 C and 2-D NMR data were obtained for the principal acylated betacyanins isolated from B. alba fruits (15, 19, 20 and 21) for the first time (Section 2.6).  In the group of polar betacyanins identified in the fruits of B. alba and var. 'Rubra by LC-DAD-MS and co-elution experiments with the known references [7,26,[28][29][30][31][32], except for the known mono-and bi-glucosylated betacyanins 1, 4, 5 and 7 as well as their isoforms 1 , 4 , 5 and 7 (Table 1), novel pigments 2/2 , 3/3 and 6/6 were detected, which were not co-eluted with the reference betacyanins.
Betacyanins 2 and 6 showed protonated molecular ions at m/z 713 as well as their daughter ion fragments at m/z 551 and 389, respectively, in the positive ion mode LC-MS/MS ( Table 1). The molecular mass and the fragmentation pattern suggested a presence of a dihexosyl (713 − 389 = 2 × 162) of betanidin. The observed low absorption maxima λ max 536-537 nm suggested the 5-O-substitution pattern with a sugar system in betanidin, similarly to melocactin [7,28]. During the high-resolution mass spectrometric experiments on compounds 2 and 6 in the Orbitrap system, the molecular masses were obtained at m/z 713.2030 and 713.2033, respectively (C 30 H 37 N 2 O 18 , calculated m/z 713.2036), which together with the detected fragmentation ions ( Table 2) confirmed the elemental composition of 2 and 6.
Unexpectedly, compound 3 with [M+H] + ions at m/z 551 appeared as a hexosyl (551 − 389 = 162) of betanidin during the low-resolution mass spectrometric analysis, which together with the observed higher absorption maximum λ max 539 nm (Table 1) tentatively suggested a presence of a betanidin 6-O-substitution system. The 2 nm difference in λ max is always observed between betanins and gomphrenins acylated with aliphatic acids [7,28,31]. The obtained protonated molecular mass for 3 during the HRMS experiments corresponding to the ion at m/z 551.1505 (C 24 H 27 N 2 O 13 , calculated m/z 551.1508) and for its fragment ion ( Table 2) at m/z 389.0973 (C 18 H 17 N 2 O 8 , calculated m/z 389.0979) confirmed a presence of a novel isomeric betacyanin to the well-known betanin 4 and gomphrenin 7.

Malonylated Betanidin 6-O-β-Glusosides and Their Acyl Migration Derivatives
Further inspection of the chromatograms of the fruit extracts revealed two main peak groups with protonated molecular ions at m/z 637, 8a-8c/8a -8c and 10a-10d, apparently isomeric to phyllocactins [7,26,33]. The HPLC co-elution experiments with the authentic standards of phyllocactin/isophyllocactin from Hylocereus ocamponis fruits [26] excluded the presence of phyllocactin/isophyllocactin in the samples. Higher retention times of 8a-8c/8a -8c and 10a-10d than those of phyllocactins [26] suggested possible malonylation of gomphrenin/isogomphrenin at least in one of the detected groups, which is the first example of malonylation of gomphrenins.
The decisive data ( Table 2) were obtained by the high-resolution experiments in the Orbitrap system and confirmed that the structures of 8a-8c/8a -8c fit to the malonylated betacyanins (obtained mass for the most abounding isomer 8c at m/z 637.1513 (C 27 H 29 N 2 O 16 , calculated mass: 637.1512)), at the same time excluding the presence of malonylated derivatives in 10a-10d (discussed in the next section).
The position of 6-O-glucosylation in 8a-8c/8a -8c might be suggested by a bathochromic shift of their obtained absorption maxima λ max (537 nm), similar to the shift from 535 nm for betanin to 537 nm for gomphrenin in the applied chromatographic eluent [34]. This is the always-observed difference between betanins and gomphrenins acylated with aliphatic acids [7,28,31,34].

3-Hydroxy-Butyrylated Betanidin 6-O-β-Glusosides
Pigments 10a-10d, which were apparently isomeric to phyllocactins, were characterized with protonated molecular ions at m/z 637 and absorption maxima λ max 537-538 nm in the applied chromatographic eluent. Submitted to the LRMS analyses, formed fragmentation ions at m/z 593, 551 and 389 were similar to those obtained for the betacyanins 8a-8c/8a -8c . Subsequent HRMS experiments on compounds 10a-10d in the Orbitrap system excluded the presence of typical malonylated structures, instead, the acyl identity was readily proposed as 3 -hydroxy-butyryl for all the isomers 10a-10d yielding, e.g., m/z 637.1875 for the most prominent pigment 10c (C 28 H 33 N 2 O 15 , calculated m/z 637.1876), which suggested a presence of a 3 -hydroxy-butyrylated hexosyl of betanidin/isobetanidin ( Table 3). Taking the above data into consideration, some of the pigments 10a-10d can be tentatively identified as 3 -hydroxy-butyrylated gomphrenin/isogomphrenin (presumably the more abundant pair 10c/10d (Section 2.7). The lack of a carboxylic moiety in the 3 -hydroxy-butyryl substituent prevents the occurrence of the phenomenon of acyl migration in 10a-10d, therefore, one of the pairs might be betanin derivatives.
The 3 -hydroxy-butyryl substitution had been already tentatively suggested for the structures of the phyllocactin/isophyllocactin additional isomers detected in H. polyrhizus fruits [36], but further extensive studies proved that these isomers were the acyl migration products and no 3 -hydroxy-butyryl substitution could be considered at all [26,33]. Unfortunately, other recent reports (data not shown) cited this tentative assumption for other genera of H. polyrhizus without any proof and without taking the acyl migration into consideration. Now, according to our best knowledge, this study reports the first cases of tentatively identified 3 -hydroxy-butyrylated betacyanins 10a-10d.
The observed fragmentation pathway for the protonated molecular ion of 15 afforded fragments at m/z 551 and 389 (Table 2) The high-resolution mass spectrometric determination of the molecular mass for 15 by obtaining m/z 713.1820 (C 33 H 33 N 2 O 16 , calculated m/z 713.1825) as well as for its decarboxylated derivatives (Table 2) readily confirmed the substitution with the acyl moiety instead of another hexosyl in 15, thus, proposing its tentative identity as 6 O-E-caffeoylgomphrenin and a trivial name of "malabarin" of this endogenously present pigment in B. alba (Malabar spinach). Our NMR analysis of 15 finally confirmed this structure (Section 2.6).
Initial analyses of the structures of 12 and 18 brought the protonated molecular ions at m/z 859 and their fragmentation resulting in fragments at m/z 697, 551 and 389 (Table 2) (Table 2). Thus, the tentative structures of 12 and 18 are proposed as isomers of (hexosyl)-(coumaroyl-hexosyl)-betanidin.
Similarly, the cis-isomers of hydroxycinnamic acid conjugates of gomphrenin 16/17 as well as their isoforms 16 /17 were detected in the studied fruits, which was corroborated by the co-elution experiments with references obtained from G. globosa inflorescences and HRMS measurements (

Novel Natural Betacyanins Acylated with Nitrogenous Substituents
The most engaging was the detection of unusual betacyanins 9, 11, 13 and 14 as well as their isoforms 9 , 11 , 13 and 14 acylated with acids containing nitrogen in their structures. From this group of pigments contributing to a level of 4.4% of the total pigment content in fruits of B. alba, the most abundant were 9 and 14 (Section 2.7).
Subsequent HRMS experiments on compound 9 in the Orbitrap system confirmed these assumptions, yielding protonated molecular ions at m/z 744.1878 (C 33 H 34 N 3 O 17 , calculated m/z 744.1883), which readily disclosed the molecular formula of the acyl moiety (C 9 H 8 NO 4 ). This was also confirmed by the detection of the other fragments in the HRMS mode (Table 3). Interestingly, this result would fit with the presence of betalamic acid as acylating agent; however, the lack of an additional absorption maximum around λ max 422 nm, typical for this pigment, as well as the not increased reactivity of 9, which would be ascribed to the aldehyde functional group, suggests that this cannot be the case. Instead, rather another form of acyl related to betalamic acid should be considered.
Similar results were observed for the pigment 14 with lower absorption maximum λ max 538 nm and protonated molecular ions detected at m/z 688. The fragmentation pattern obtained for 14 was less abundant and accounted for ions at m/z 644 (-CO 2 ); 600 (-2CO 2 ); 551 (-acyl); and 389 (-acyl-glc), thus, indicating a presence of a betacyanin with a nitrogenous acyl group but not containing any carboxyl. In the HRMS experiments, a protonated molecular ion at m/z 688.1983 was reported (C 31 H 34 N 3 O 15 , calculated m/z 688.1984), which revealed the chemical formula of the acyl moiety (C 7 H 8 NO 2 ). The other fragments in the HRMS mode were also detected and unequivocally confirmed the molecular formula of 14 (Table 3).
Furthermore, the presence of pigments 11 and 13 was revealed in both the B. alba and var. 'Rubra' fruits, which appeared as differing from the structure of pigment 9 by a presence or absence, repectively, of a methoxy group resulting from the LC-DAD-MS/MS detection of protonated molecular ions at m/z 774 and 714, respectively, as well as the absorption maximum λ max 542 nm. In spite of the small quantities of pigment 11 (Table 3), it was possible to obtain, albeit scarce, fragmentation patterns with ions observed at m/z 742 (-CH 3 OH) and 389 (-acyl-glc), confirming a presence of methoxyl in the nitrogenous acyl moiety. Unexpectedly, no typical ion at m/z 551 was detected during the fragmentation, which would confirm the glucosylated betanidin fragment. In contrast, a more abundant fragmentation profile was monitored for 13 with ions at m/z 670 (-CO 2 ); 636 (-2CO 2 ); 582 (-3CO 2 ); 551 (-acyl); 538 (-4CO 2 ); and 389 (-acyl-glc), which was similar to the profile obtained for 9, also confirming four carboxylic groups present in a betacyanin 13.
The experiments performed in the HRMS mode in the Orbitrap system revealed much more prolific fragmentation patterns obtained for compound 11 after fragmentation of the protonated molecular ion at m/z 774.1975 (C 34 (Table 3) as well as deacylation and deglucosylation as in the case of 9. The chemical formula of the acyl moiety in 11 was established as C 10 H 10 NO 5 .
Determination of the molecular formula of [M+H] + ions of compound 13 in the HRMS mode (m/z 714.1776 (C 32 H 32 N 3 O 16 , calculated m/z 714.1777)) afforded to establish the chemical formula of the acyl moiety in 13 as C 8 H 6 NO 3 . This was unequivocally confirmed by the determination of the elemental composition of the fragmentation ions (Table 3). H-14ab, H-15) were assigned in 1 H NMR as well as COSY and TOCSY spectra [7,26,31,35,[37][38][39][40]. In each case, the betanidin system was readily distinguishable by the characteristic low-and high-field doublet signals for the H-11 and H-12 protons.

NMR Structural Elucidation of Acylated Gomphrenins
The spin system for H-2/H-3ab was observable in each pigment, indicating the presence of the carboxyl moiety at C-2. A significant upfield shift for the H-2 proton to 3.79-3.88 ppm (in 15, 20 and 21) or 4.72 (in 19) in comparison to non-acylated gomphrenin 7 was observed, suggesting intramolecular interactions [31,40]. This phenomenon is still not explored but is additionally suggested by the observed bathochromic shift of the visible absorption band in gomphrenins in relation to betanins [7,31]. The other spin system, for H-15/H-14ab, showed easily identifiable cross-peaks in the COSY and TOCSY spectra, also resulting from the presence of the carboxyl moiety at C-15.
The three singlets corresponding to H-4, H-7 and H-18 were detected in the spectra. A broad signal for H-18 in 15, 20 and 21 was observable by 1 H NMR and the correlation techniques for freshly prepared D 2 O solutions of the pigments avoiding the fast deuterium exchange [40]. In the case of 19, the acidic CD 3 OD solutions enabled observation of a stable narrow signal.
In contrast to previous reports [31,40], the presence of the characteristic interconnection system for gomphrenin could not be indicated (except of 19) based on the shift differences between H-4 and H-7 of 0.6-0.8 ppm, presumably because the measurements were performed in D 2 O instead of acidified CD 3 OD. Therefore, the well-developed confirmation of the C-6 phenolic moiety substitution in the betanidin system was obtained by the other techniques (NOESY and HMBC). Furthermore, this shift difference was not only lower than 0.2 ppm but also varied within the group of the studied acylated betacyanins 15, 20 and 21 (Table 4). In the case of the C-5 substitution, the expected differences of ca. 0.1 ppm or lower were observed in acidified CD 3 OD [40] but also in acidified D 2 O [37] and non-acidified D 2 O [35,37]. Table 4. The NMR data obtained in D 2 O (15, 20 and 21) and CD 3 OD/d-TFA (19) for the principal acylated betacyanins isolated from Basella alba L. fruits. The 1 H and 13 C spectra of the pigments are presented in Figures S1-S8.
The presence of the Z-isomers (data not shown) was also acknowledged for the acylated gomphrenins 15, 19, 20 and 21 [31,40]. The correlations between the Eand Z-protons being in equilibrium (at the signal ratio ca. 90:10) were noticeable in the TOCSY and NOESY spectra. The signals of the Z-protons were detected for the betanidin system (H-2, H-3ab, H-4, H-7, H-11, H-12, H-14ab and H-15) as well as for the caffeoyl moiety (H-2 , H-5 , H-6 , H-7 and H-8 ). Due to low signal intensities, the expected cross-peaks for the Z-protons of H-12 and H-14ab confirming the Z-configuration for C(12)=C (13) were not observable in the NOESY spectra. Such correlations were observed for more abundant (35%) Z-isomers in the other betalainic group, betaxanthins [41].
The other 13 C chemical shifts for carbons directly bound to protons were assigned by HSQC correlations. The presence of the anomeric proton H-1 indicating the sugar unit by its characteristic downfield shift was readily observed. The HMBC, COSY and TOCSY correlations clearly ascertained the glucosyl ring systems (Figure 4, Table 4) [7,26,31,35,[37][38][39][40]. The position of the glycosidic bond at the phenolic carbon C-6 was readily confirmed by the HMBC correlation with the anomeric proton H-1 . The β-linkage between the aglycone and glucopyranosyl moiety was denoted by the three-bond vicinal proton coupling constant 3 J 1 -2 ~6-7 Hz after re-registration of the 1 H spectra in other CD 3 OD/d-TFA solutions [37,40].
Definitive evidence of the acyl moiety position was provided by the downfield chemical shift of H-6 a/b protons in the glucosylic ring. Further confirmation of this linkage position was obtained by the HMBC correlations ( Figure 4) of C-9 to H-6 a and H-6 b.
The hydroxycinnamic acyl moieties were readily detected by their aromatic and olefinic protons (J = 16 Hz) and were differentiated by the presence or absence of the hydroxyl and methoxyl moieties at carbons C-3 and C-5 ( Figure 4, Table 4).
Above analyses completed the structural identification of the novel betacyanins:

Quantification of Betacyanins in the Fruits of B. alba and B. alba var. 'Rubra
For B. alba var. 'Rubra , a much higher total concentration of betacyanins expressed in betanin equivalents (Table 5) was obtained in the mature fruits (86.6 mg/100 g) than for B. alba (42.0 mg/100 g). This is roughly in accordance with previous reports on single varieties of the species [5,42]. The distribution of the pigments is also much different in both the fruit types. In B. alba, the fraction of acylated betacyanins is much higher (38.6%) than in var. 'Rubra (19.4%). Similarly, the percentage of the novel nitrogenous betacyanins in B. alba (4.4%) is twice as much as the fraction in var. 'Rubra fruits (2.2%). a Relative concentrations were expressed as percentage of the total peak area. Average of three measurements. b In betanin equivalents. c Abbreviations: hex-hexosyl; mal-malonyl; but-butyryl; caff-caffeoyl; coumcoumaroyl; fer-feruloyl; sin-sinapoyl; Bd-betanidin; Gp-gomphrenin.
From the polar pigments, gomphrenin 7 contributed to the total betacyanin content at 43.9% and 39.7% in var. 'Rubra' and B. alba, respectively. The portion of isogomphrenin 7' was reported at 13.2% and 13.7% in var. 'Rubra' and B. alba, respectively. Unexpectedly, the contribution of betanin 4 to the betacyanin total content (0.44% and 0.37% in var. 'Rubra' and B. alba, respectively) was much smaller than the fraction of the novel isomeric pigment, (hexosyl)-betanidin 3, which accounted for 17.5% and 3.7% in var. 'Rubra' and B. alba, respectively.

Reagents
All reagents were used as received. Formic acid, LC-MS grade methanol, and water were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The deionized water used throughout the experiments was purified through a Purix water purification system with a resistivity of 18.0 mΩ cm −1 at 295 K.

Plant Material
The seeds of Basella alba L. and Basella alba L. var. 'Rubra' obtained from the Botanical Garden of Jagiellonian University Institute of Botany (Cracow, Poland) were grown in a greenhouse of the University of Agriculture in Cracow (Faculty of Biotechnology and Horticulture). Sowing of the seeds was performed in a 3:1 ratio of soil and coconut pith mass and watered daily. The seedlings were transplanted to fertile soil with plenty of organic matter and a pH of 6.5-6.8. The plants were designed to support the climbing of the vines and were fast growing; therefore, they were trellised so that they reached up to 3 m in height. The plants were kept at consistent moisture and temperature to keep flowering and fruiting.

Preparation of Juice from B. alba and B. var. 'Rubra' Fruits
The fruits collected in the greenhouse (30 g for each variety) were squeezed and obtained liquid was centrifuged followed by filtering through a 0.2 mm i.d. pore size filter and then underwent threefold dilution with water before immediate chromatographic analysis of the pigment profiles or storage at −20 • C before the subsequent experiments.

Fast Betacyanin Screening in the Fruit Juice Samples
Betacyanin samples from the prepared fresh fruit juice of B. alba and var. 'Rubra' were immediately submitted to spectrophotometric as well as LC-MS analysis without any purification. For the pigment profile representation, a method of internal normalization of the chromatographic peaks derived from the MS signals was applied. For the measurement of the total concentration of the pigments, the extracts were analyzed by an Infinite 200 microplate reader (Tecan Austria GmbH, Grödig/Salzburg, Austria). The total concentration was expressed as mg betanin equivalents/100 g of fresh fruits. Quantification of betacyanins was evaluated taking a molar extinction coefficient of ε = 65,000 M −1 cm −1 at 536 nm for betanin in spectrophotometric calculations [30]. Three samples per species were analyzed according to this procedure.

Pigment Purification for LC-MS Experiments
For the further LC-MS analyses with detection by low-and high-resolution mass spectrometry, purification of B. alba extracts was performed to obtain preconcentrated samples. The pigment extracts were chromatographically purified by flash chromatography using a Shimadzu LC-20AD preparative chromatographic system (Kyoto, Japan) equipped with LC-20AP pumps, SPD-20AV UV−Vis detector, and LabSolutions 5.51 operating software. The separation was performed on Bionacom cartridges (Agela Technologies, Newark, DE) filled with non-endcapped silica C18 sorbent (porosity 60 Å and particle size 40-60 µm) [28]. After rinsing with water, the betacyanin fraction was eluted with 50% aqueous methanol acidified with 5% formic acid (v/v). The eluates were pooled and concentrated using a rotary evaporator under reduced pressure at 25 • C and freeze-dried. A similar purification procedure was performed for betacyanins from the reference material samples.

Preparation of Isolated Betacyanins from the Purified B. alba Extract
For the NMR study, betacyanins 15, 19, 20 and 21 were isolated from B. alba extract by chromatographic steps. The extract was initially purified by open column chromatography on a column (40 mm i.d. × 50 mm height) filled with Sepra™ ZT-SAX 30 µm Polymer, 85-Å (Phenomenex, Torrance, CA, USA). After application of the extract to the top of the column and rinsing the column with water, the betacyanin fraction was eluted with 50% aqueous methanol acidified with 5% formic acid (v/v). The eluates were pooled and concentrated using a rotary evaporator under reduced pressure at 25 • C before purification by flash chromatography on non-endcapped silica C18 sorbent (as described in Section 3.6) in a column of 40 mm i.d. × 140 mm height.
The concentrated eluates from the silica C18 sorbent were pooled and the pigments were separated using the Shimadzu LC-20AD system on an HPLC semipreparative column Synergi Hydro-RP 250 mm × 30 mm i.d., 10 µm (Phenomenex) with a 20 mm × 25 mm i.d. guard column of the same material (Phenomenex). A typical gradient system consisting of 1% aqueous formic acid (solvent A) and acetone (solvent B) was used as follows: 0 min, 15% B; increasing to 10 min, 17% B; increasing to 20 min, 20% B; increasing to 30 min, 22% B; increasing to 40 min; 80% B. The injection volume was 20 mL with a flow rate of 30 mL/min. Detection was performed using a UV/Vis detector at 538 and 480 nm, at a column temperature of 22 • C. The eluates were pooled and concentrated under reduced pressure at 25 • C and finally freeze-dried. All the solutions were concentrated in rotary evaporators at 25 • C under reduced pressure to remove the organic solvent and stored at −20 • C for further studies.

Chromatographic Analysis with Detection by a Low-Resolution Mass Spectrometric System (LC-DAD-ESI-MS/MS)
For the chromatographic and mass spectrometric analyses, an LCMS-8030 mass spectrometric system (Shimadzu, 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 (Shimadzu, 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-methanol. The gradient profile was: (t (min), % B), (0, 10), (12,40), (15,80), (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 N 2 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 in an arbitrary scale.

Chromatographic Analysis with Detection by a High-Resolution Mass Spectrometric System (LC-Q-Orbitrap-MS)
All high-resolution mass spectra were analyzed using Q Exactive Plus hybrid OrbiTrap quadrupole mass spectrometer (Thermo Fisher Scientific, Brema, Germany) coupled to an HPLC Dionex UltiMate 3000 chromatographic separation system. The chromatographic conditions were the same as for the LRMS system.
The detection of target betacyanins selected in the LRMS system was conducted in the full scan positive polarity mode. The MS data were acquired in the m/z 400−1000 range with a resolution (full width at half-maximum, FWHM, at m/z 200) of 70,000. The automatic gain control (AGC) target value was 200 000 in the full-scan mode. The maximum isolation time was set to auto mode.
Selected precursor ions were fragmented in the higher-energy collisional activated dissociation cell and the fragment (MS2) ions were analyzed in the Orbitrap analyzer. For the MS2 experiments, the fragment ions of selected target betacyanins were collected in the high-energy collision dissociation (HCD) mode at collision energies of 20 and 40 eV. The automatic gain control (AGC) target value and the resolution were 50,000 and 35,000, respectively. The m/z range was 70−900 and the maximum isolation time was set to auto mode. The number of microscans per MS/MS scan was set to 1. The LC-HRMS data acquisition and analysis were performed by using the software Chromeleon 7.2.10 and Xcalibur 4.3 (Thermo Fisher Scientific).

NMR Experiments
The NMR data were acquired on a Bruker Avance III 700 spectrometer (Bruker Corp., Billerica, MA, USA) using a QCI CryoProbe at 295 K in non-acidified D 2 O (15, 20 and 21) and CD 3 OD acidified by d-trifluoroacetic acid (19).

Conclusions
This is the first report on a series of novel betacyanins not reported in any plant and especially not in the B. alba varieties. The acylation with the discovered nitrogenous acyl substituents was established by high-resolution mass spectrometry and is a new phenomenon not only observed in the betacyanin group of pigments so far but also, to the best of our knowledge, in other polyphenolic compounds. The NMR structural experiments resulted in identification of the novel betacyanins, 6 O-E-caffeoyl-gomphrenin (malabarin) and 6 O-E-sinapoyl-gomphrenin (gandolin), as well as further confirming the presence of 6 -O-E-4-coumaroyl-gomphrenin (globosin) and 6 -O-E-feruloyl-gomphrenin (basellin) in B. alba matured fruit extracts by two-dimensional NMR techniques.
In this respect, further investigations of B. alba fruits as well as their processed products should significantly enhance our knowledge about the bioactivity of betacyanins and especially gomphrenins. Considering that the acylated gomphrenins are found together at relatively high concentrations in B. alba L. fruits, this makes this plant material an extremely valuable bioactive source of betacyanins for future food applications.