Journal of Chromatography A, 1344 (2014) 42–50
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Separation of amaranthine-type betacyanins by ion-pair high-speed countercurrent chromatography Gerold Jerz a , Nadine Gebers a , Dominika Szot b , Maciej Szaleniec c , Peter Winterhalter a , Slawomir Wybraniec b,∗ a
Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany Faculty of Analytical Chemistry, Institute C-1, Department of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24, Cracow 31-155, Poland c Joint Laboratory of Biotechnology and Enzyme Catalysis at Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, 30-239 Cracow, Poland b
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 29 March 2014 Accepted 31 March 2014 Available online 12 April 2014 Keywords: Acylated betacyanins Betanin Betalains Ion-pair countercurrent chromatography IP-HSCCC Iresinin
a b s t r a c t Betacyanins, red-violet plant pigments, were fractionated by ion-pair high-speed countercurrent chromatography (IP-HSCCC) from leaves extract of Iresine lindenii Van Houtte, an ornamental plant of the family Amaranthaceae. An HSCCC solvent system consisting of TBME–1-BuOH–ACN–H2 O (1:3:1:5, v/v/v/v) was applied using ion-pair forming heptafluorobutyric acid (HFBA). Significantly different elution profiles of betacyanin diastereomeric pairs (derivatives based on betanidin and isobetanidin) observed in the HSCCC in comparison to HPLC systems indicate a complementarity of both techniques’ fractionation capabilities. The numerous diastereomeric pairs can be selectively separated from each other using the HSCCC system simplifying the pigment purification process. Apart from the three well known highly abundant pigments (amaranthine, betanin and iresinin I) together with their isoforms, three new acylated (feruloylated and sinapoylated) betacyanins as well as known pigment hylocerenin (previously isolated from cacti fruits) were characterized in the plant for the first time and they are new for the whole Amaranthaceae family. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Betacyanins, erroneously named as nitrogenous anthocyanins before their basic structure was disclosed in the 60s of the last century, are distinctively different from the anthocyanin plant pigments by the characteristic diazaheptamethine chromophoric system [1,2]. Betacyanins can be considered as condensation products of betalamic acid with cyclodopa or its O-glycosylated (in most cases 5-O-glucosylated) derivatives. Further esterification of the Oglycosides with acids such as ferulic, p-coumaric or malonic acid is very common [1,2]. Betalains exist in two diastereomeric forms differing by the configuration of the C-15 carbon (Fig. 1) [1–3]. Betanin, the simplest 5-O-glucosylated betacyanin and its C-15 isoform are frequently derived from red beet root (Beta vulgaris L.) [1–4]. Another edible rich source of betacyanins are recently investigated cacti species of Hylocereus containing betanin, phyllocactin
∗ Corresponding author. Tel.: +48 12 628 3074; fax: +48 12 628 2036. E-mail address: [email protected] (S. Wybraniec). http://dx.doi.org/10.1016/j.chroma.2014.03.085 0021-9673/© 2014 Elsevier B.V. All rights reserved.
and hylocerenin as the most abundant pigments [5,6] as well as Opuntia [7] or Mammillaria [8]. Betacyanin’s early investigations resulted in publication of a series of their chemical structures present in many plants, however, due to the lack of appropriate techniques, many of the these red-violet pigments remained unknown [1,2]. One of the most investigated species was Iresine herbstii, an ornamental plant of the family Amaranthaceae [9–11]. In spite of extensive studies performed, only two highly abundant betacyanins were fully characterized in the plant by chemical means [12] and mass spectrometry [9–11]. Minale et al. published in 1966 their comprehensive report on acylated betacyanins present in Iresine herbstii leaves [12] and Cai et al. surveyed the pigments in a broad range of Amaranthaceae species [9–11]. A series of betacyanins was assigned with the trivial names as ‘iresinin I–IV’ from which only ‘iresinin I’ as well as its C-15 diastereoisomer (‘isoiresinin I’) had been structurally identified completely [12]. According to comprehensive studies on betacyanins of the species of the Amaranthaceae family, their pigments have high potential for use as colorants in food products [9,10]. Some Amaranthus genotypes produce high biomass and contain more betacyanins than red beet [9,10].
G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
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Choosing a suitable solvent system for betalains purification is challenging due to their low solubility in organic solvents as a result of their ionization. The addition of ion-pair reagents results in a different chromatographic behaviour of betalains, e.g. longer retention times of some betalains in RP-HPLC [25]. This is due to a formation of much more lipophilic ion-pairs between the counterions and betalains and thereby a better solubility of the compounds in the organic stationary phase. The addition of ion-pair reagents shifts the partition coefficient of betalains and creates a new possibility for CCC separation of these ionic plant pigments, including betanin and its diastereomer [22,23,6,24]. Except of the ion-pairing action, the acidification suppresses the carboxylic group’s dissociation, further enhancing the demanded hydrophobicity of the pigments. The most challenging is the separation of the most polar betacyanins for which appropriate organic phases dissolving the pigments should be elaborated. This contribution reports on the first results of HSCCC fractionation of betacyanins from Iresine lindenii leaves including a group of amaranthine-type pigments for their structural characterization.
2. Experimental 2.1. Reagents Formic acid, heptafluorobutyric acid (HFBA), HPLC-grade acetonitrile, TMBE, 1-butanol, methanol and HPLC-grade water were obtained from Merck (Darmstadt, Germany).
2.2. Preparation of crude pigment extract
Fig. 1. Chemical structures of amaranthine-, betanin- and gomphrenin-type betacyanins identified in I. lindenii Van Houtte leaves (for the full list of the pigments; see Table 1).
Betalains can be easily oxidized enzymatically and nonenzymatically [13–18] and having strong antioxidant properties are arising as interesting chemopreventive natural compounds. Therefore, in connection to their good colorant properties, a growing interest in betalains has been noticed during the past decade [19] and some of these compounds have not been identified yet. High-speed countercurrent chromatography (HSCCC) has been proved to be a very useful technique for preparative isolation of natural compounds from plant extracts [20,21]. Preparative isolation of betalains is problematic, therefore, new separation methods such as countercurrent chromatography create an important possibility of obtaining pure pigments. In addition, new interesting betalain structures are usually present in plant materials at low concentration and their isolation requires tedious and time consuming procedures. Hitherto, successful isolation and purification of betalains for further analytical investigations (structure elucidation) was carried out only in the HSCCC technique in a solvent system consisting either of TBME–1-BuOH–ACN–water (acidified with TFA or HFBA) [22,23,6,24].
For semipreparative pigment isolation, the leaves of I. lindenii Van Houtte (100 g) were extracted with 1000 mL of water in a blender and subsequently filtered through a 0.2 m i.d. pore size filter (Millipore, Bedford, MA). The extract was concentrated using a freeze-drier. For the co-injection experiments, the extracts of Hylocereus polyrhizus fruits from a previous study [6], Iresine herbstii leaves [9,10,12], Celosia argentea var. cristata flowers [26] as well as Gomphrena globosa flowers [27–29] were processed by a similar procedure. The pigment extract was chromatographically concentrated by solid phase extraction on C18 cartridges (Merck, Darmstadt, Germany) according to the procedure of Stintzing et al. [7]. After rinsing with water and acetonitrile the betacyanin fraction was eluted with acidified methanol (methanol/3% TFA, 95:5, v/v). The eluates were pooled and concentrated using a rotary evaporator under reduced pressure at 25 ◦ C and freeze-dried. The freeze-dried residue was submitted to semipreparative HPLC for isolation of betacyanins.
2.3. Reference compounds For structure confirmation, completely elucidated reference betacyanins as well as C-15 diastereomers (mostly by ESI-MS/MS and 2D-NMR) were derived from extracts of fruits, flowers or leaves of the following plants:betanidin 5-O-ˇ-glucuronosyl-glucoside (amaranthine) from Iresine herbstii [9,10,12], betanidin 5-O-(6 (hylocerenin) O-3 -hydroxy-3 -methyl-glutaryl)-ˇ-glucoside from Hylocereus polyrhizus [6], (2 -O-E-feruloyl)-amaranthine (celosianin II) from Celosia argentea var. cristata [26], betanidin 5-O(6 -O-3 -hydroxy-3 -methyl-glutaryl)-ˇ-glucuronosyl-glucoside (iresinin I) from Iresine herbstii [9,10,12], and betanidin 5-O-(6 -OE-feruloyl)-ˇ-glucoside (gomphrenin III) from Gomphrena globosa [27–29].
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G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
2.4. Apparatus The HSCCC separation was carried out on a triple-coil highspeed countercurrent chromatograph (HSCCC) model CCC-1000 (Pharma-Tech. Res. Corp., USA) equipped with three preparative coils made of PTFE tubing (2.6 mm × 160 m i.d.), which were connected in a series (850 mL total volume). A manual sample injection valve with a 25 mL loop was used to introduce the sample into the coil system. All solvents were delivered by a Biotronik HPLC pump BT 3020 (Jasco, Gross-Umstadt, Germany). A Gynkotek HPLC system with UVD340U, Gynkotek HPLC Pump Series LPG-3400A and thermostat (Gynkotek Separations, H.I. Ambacht, The Netherlands) was used for the chromatographic analysis. For the data acquisition, the software package Chromeleon 6.0 (Gynkotek Separations) was used. For the separation of betalains, an ONYX monolithic column 100 mm × 4.6 mm i.d., protected by a guard column (Phenomenex, Torrance, CA, USA) was used. The positive ion electrospray mass spectra were recorded on ThermoFinnigan LCQ Advantage (electrospray voltage 4.5 kV; capillary 250 ◦ C; sheath gas: N2 ) coupled to ThermoFinnigan LC Surveyor pump utilizing the HPLC gradient. The MS was controlled and total ion chromatograms and mass spectra were recorded using ThermoFinnigan Xcalibur software (San Jose, CA, USA). The relative collision energies for the CID experiments were set at 30% (according to a relative energy scale). Helium was used to improve trapping efficiency and as the collision gas for the CID experiments. 2.5. Selection of the two-phase solvent system For the evaluation of suitable solvent systems with ion-pair forming capacity, the previously published solvent systems containing differently concentrated perfluorinated acids (aqueous TFA or HFBA solutions) were compared in a system TBME–1BuOH–ACN–H2 O (0.7% or 1% perfluorinated acid) 1:3:1:5 (v/v/v/v). Investigation of polar solvent systems suitable for preparative HSCCC separations of betalains clearly indicated that acetonitrile cannot be replaced by methanol because of a strong decomposition of the pigments in the latter solvent. The partition of pigment components was determined in the systems after shaking of 100 L TBME, 500 L 1-BuOH, 100 L ACN and 600 L acidified H2 O (0.4% or 0.7% perfluorinated acid) I. lindenii crude betacyanin extract in 2 mL vials. The estimation of the pigment distribution was performed by LC-DAD. For systems containing HFBA, a very good distribution of the polar as well as more lipophilic pigments between the organic (stationary) and aqueous (mobile) phase (head-to-tail mode) was observed, therefore, the systems containing 0.7% and 1% HFBA were chosen for further experiments in HSCCC apparatus. Because of the highest retention of its stationary phase [6], the system with 0.7% HFBA was elaborated further, enabling much better separation of the most polar betacyanins. 2.6. HSCCC separation of I. lindenii Van Houtte leaves extract The separation of the freeze-dried I. lindenii leaves crude betacyanin extract (C18 -SPE-cleaned, 1677 mg) was performed in the HSCCC system operated in the ‘head-to-tail’ mode applying the novel biphasic solvent system TBME–1-BuOH–ACN–H2 O (0.7% HFBA) 1:3:1:5 (v/v/v/v). The flow rate was 3.0 mL/min and the rotation velocity was set to 850 rpm. After separation, the solvent in the coil column was ejected with nitrogen gas. Analysis of CCC fractions was performed by HPLC-DAD and HPLC-ESI-MS/MS. Recovered fractions (No. 2–14) from the IP-HSCCC separation (injection amount 1677 mg of I. lindenii crude pigment extract): F2 (257 mg), F3 (349 mg), F4 (139 mg), F5 (195 mg), F6 (256 mg), F7 (138 mg), F8 (43.5 mg), F9 (7.4 mg), F10 (27.1 mg), F11 (17.7 mg),
F12 (3.2 mg), F13 (1.9 mg), F14-coil (with no betacyanins, 190 mg); sum of recovered betacyanin yields from HSCCC-fractions: 1435 mg (∼86%). 2.7. Analytical HPLC system For the separation of the analytes, the following gradient system was used: 95% A with 5% B at 0 min; gradient to 70% A with 30% B at 40 min (Solvent A: 1% HCOOH, Solvent B: acetonitrile). The injection volume was 10 L, and the flow rate was maintained at 0.5 mL/min. The detection of analytes was performed typically at 538, 505, 480, 450 and 310 nm. For the UV–vis spectra acquisition, the detection was performed in the DAD (diode-array detection) mode. The column was thermostated at 35 ◦ C. The same chromatographic conditions were applied for the HPLC-ESI-MS/MS analyses. 2.8. Enzymatic hydrolysis of I. lindenii pigments For the identification of the main structural units in the analyzed betacyanins, enzymatic hydrolysis for 90 min of the purified I. lindenii pigments by -glucuronidase from Helix pomatia (Sigma–Aldrich) at pH 5.0 and 37 ◦ C was performed [12]. The assays were performed for the newly identified pigments in comparison to the known betacyanins, amaranthine 1 and iresinin I 3 in which the glucuronosyl moiety is hydrolytically detached and the deglucuronosylated betacyanins (betanin and hylocerenin, respectively) can be detected by LC-MS. Acylation of the glucuronosyl ring prevents betacyanins from the action of the enzyme [12]. 2.9. Carbohydrate linkage analysis The linkage between the carbohydrate moieties was established by GC-MS analysis of partially methylated alditol acetates prepared by permethylation of betacyanins in basic conditions (dispersed NaOH in DMSO) using methyl iodide with subsequent hydrolysis, reduction and peracetylation as described by Anumula and Taylor [30]. Any existing acyl-linked organic residues are lost under these conditions. The GC-MS analyses were performed on a Finnigan gas chromatograph equipped with a 30-m Rtx-5 capillary column (Restek Co., Bellefonte, PA) connected to a Finnigan GCQ ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA) running in the 70 eV electron-impact mode. The gas chromatographic program used was 80 ◦ C (1 min)–10 ◦ C/min–300 ◦ C. The analytes were identified by their retention times and characteristic fragmentation patterns [31]. The position of acylation of the carbohydrate moieties was established by the above procedure with changed first step (permethylation of betacyanins) which was performed according to the standard procedure of Prehm [32] using methyl trifluoromethanesulfonate in trimethyl phosphate. Under these mild conditions the prevailing final products are derived mainly from permethylated carbohydrate units with preserved acyl linkage, however a partial loss of acyl units is also observed. 3. Results and discussion 3.1. HSCCC separation of betacyanins from I. lindenii Van Houtte The HSCCC separation trace of the pigments from extract of I. lindenii leaves monitored at 540 nm is depicted in Fig. 2. The extract was fractionated into fractions 1–13 (Figs. 2–4). The identification process is described in Section 3.2 and the list of identified pigments is presented in Table 1. The HSCCC elution order and separation of betalains was compared to the C18 reversed-phase HPLC profile and numerous
G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
Fig. 2. HSCCC-chromatogram of C18 -enriched pigment extract (1677 mg) I. lindenii Van Houtte leaves. HSCCC conditions: flow rate: 3.0 mL/min; ‘head-to-tail mode’; biphasic solvent system TBME–1-BuOH–ACN–H2 O (0.7% HFBA) 1:3:1:5 (v/v/v/v). Below, HPLC retention times of the separated major betacyanins detected in each HSCCC fraction.
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differences were found suggesting the complementary separation capabilities of both the techniques. After the HSCCC separation, the retained amount of stationary phase in the coil-system was calculated to be 75%. In Table 2, betacyanin chromatographic distribution as well as the HPLC total peak area in the recovered fractions obtained from I. lindenii leaves extract by HSCCC is presented. Fraction 3 obtained during the HSCCC separation of 1677 mg of the concentrated pigment extract contained a bulk of the most polar compounds which were very well separated from betacyanins by HSCCC. The principal betacyanins present in the extract, iresinin I/isoiresinin I (3/3 ), are eluted mainly in the fraction 3 (349 mg) and are separated at a high extent from the preceding amaranthine/isoamaranthine (1/1 ) in fraction 2 as well as the following betanin/isobetanin (2/2 ). Taking into account that the pigments 1/1 , 2/2 and 3/3 are included to the group of the most polar betacyanins for which it is extremely difficult to find an appropriate organic solvent [22,6,24], it can be stated that their separation is satisfactory in the applied solvent system. This could be explained not only by the ion-pair interactions but also by the instant diminishing of the carboxyl dissociation under relatively high acidic conditions leading to the increase of the hydrophobic character of the molecules. A complete separation of the newly reported pigments in Amaranthaceae, hylocerenin/isohylocerenin 4/4 (Fig. 3F), from another 6 -O-3 -hydroxy-3 -methyl-glutarylated pair, iresinin I/isoiresinin I 3/3 (Fig. 3C), is observed in spite of their similar chromatographic properties indicated in the C18 -HPLC system. Pigments 3/3 and 4/4 differ only by the glucuronosyl unit which is the case also for the pigment pairs 1/1 and 2/2 . These significantly different elution profiles of the 3/3 and 4/4 pairs in the HSCCC in comparison to HPLC systems confirm the complementary separation capabilities of both techniques
Table 1 Chromatographic, spectrophotometric and mass-spectrometric data of the analyzed betacyanin pigments in the recovered fractions obtained from I. lindenii Van Houtte leaves extract by HSCCC. No.
Compound
1
Betanidin 5-O-ˇ-glucuronosyl-glucoside (amaranthine) Isobetanidin 5-O-ˇ-glucuronosyl-glucoside (isoamaranthine) Betanidin 5-O-ˇ-glucoside (betanin) Isobetanidin 5-O-ˇ-glucoside (isobetanin) Betanidin 5-O-(6 -O-3 -hydroxy-3 -methylglutaryl)-ˇ-glucuronosyl-glucoside (iresinin I) Betanidin 5-O-(6 -O-3 -hydroxy-3 -methylglutaryl)-ˇ-glucoside (hylocerenin) Isobetanidin 5-O-(6 -O-3 -hydroxy-3 -methylglutaryl)-ˇ-glucuronosyl-glucoside (isoiresinin I) Isohylocerenin (2 -O-E-Feruloyl)-amaranthine (celosianin II) (2 -O-E-Sinapoyl)-amaranthine (2 -O-E-Feruloyl)-isoamaranthine (isocelosianin II) (2 -O-E-Sinapoyl)-isoamaranthine (2 -O-E-Feruloyl)-iresinin I (2 -O-E-Sinapoyl)-iresinin I (2 -O-E-Feruloyl)-isoiresinin I (2 -O-E-Sinapoyl)-isoiresinin I Betanidin 6-O-(6 -O-E-feruloyl)-ˇ-glucoside (gomphrenin III) Isobetanidin 6-O-(6 -O-E-feruloyl)-ˇ-glucoside (isogomphrenin III)
1 2 2 3
4
3
4 5 6 5 6 7 8 7 8 9 9 a b
Rt [min]
max a [nm] I
max b [nm] II
Abs. ratio II:I
m/z [M + H]+
m/z from MS/MS of [M + H]+
8.1
–
536
–
727
551; 389
8.9
–
536
–
727
551; 389
9.8 10.3 11.6
– – –
535 535 540
– – –
551 551 871
389 389 695; 551; 389
11.9
–
537
–
695
551; 389
12.3
–
540
–
871
695; 551; 389
12.4 12.8 13.2 13.6 13.7 16.0 16.3 16.5 16.8 18.5
326 328 326 328 328 330 328 330 322
537 542 543 542 543 542 542 542 542 548
– 0.45 0.46 0.45 0.49 0.46 0.46 0.48 0.44 0.47
695 903 933 903 933 1047 1077 1047 1077 727
19.0
322
548
0.47
727
max I, wavelength of absorption maximum of the hydroxycinnamoyl moiety. max II, wavelength of absorption maximum in the visible range.
551; 389 727; 551; 389 727; 551; 389 727; 551; 389 727; 551; 389 871; 695; 551; 389 871; 695; 551; 389 871; 695; 551; 389 871; 695; 551; 389 551; 389 551; 389
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Fig. 3. HPLC profiles of betacyanins (monitored at 540 nm) analyzed in I. lindenii Van Houtte leaves extract (A) and in the HSCCC fractions 2–6 (B–F).
Table 2 Betacyanin distributions in the recovered fractions obtained from I. lindenii Van Houtte leaves extract by HSCCC evaluated by HPLC-DAD-MS. No.
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9
Compound
Amaranthine Isoamaranthine Betanin Isobetanin Iresinin I Isoiresinin I Hylocerenin Isohylocerenin Celosianin II Isocelosianin II Sinapoyl-amaranthine Sinapoyl-isoamaranthine Feruloyl-iresinin I Feruloyl-isoiresinin I Sinapoyl-iresinin I Sinapoyl-isoiresinin I Gomphrenin III Isogomphrenin III
Relative content of betacyanin pigments in HSCCC fractions analyzed by HPLC-DAD-MS
Total HPLC peak area
2
3
4
5
6
7
8
9
10
11
12
13
(×10−5 )
91 89 – – 10 10 – – – – – – – – – – – –
9 11 12 11 69 73 – – – – – – – – – – – –
– – 69 67 19 17 – – – – – – – – – – – –
– – 19 22 1 1 – – – – – – – – – – – –
– – – – – – 59 52 – – 100 100 – – – – – –
– – – – – – 41 48 100 100 – – – – – 20 – –
– – – – – – – – – – – – 4 9 75 80 – –
– – – – – – – – – – – – 12 26 25 – – –
– – – – – – – – – – – – 29 31 – – – –
– – – – – – – – – – – – 44 25 – – – –
– – – – – – – – – – – – 11 9 – – 32 20
– – – – – – – – – – – – – – – – 68 80
0.8 0.4 3.3 1.9 20.5 14.1 0.8 0.4 0.3 0.3 0.3 0.1 1.8 1.2 1.1 1.8 0.1 0.1
G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
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Fig. 4. HPLC profiles of betacyanins (monitored at 540 nm) analyzed in the fractions 7–13 (A–G) obtained after HSCCC separation of I. lindenii Van Houtte leaves extract.
observed recently [22,24]. In these cases, both the C-15 configurational diastereomers are separated from the other isomeric pair in HSCCC, greatly simplifying the final HPLC chromatograms of the obtained fractions. Another example of such simplifying operation is a fractionation of the 5/5 (celosianin II/isocelosianin II) and 6/6 (sinapoyl-amaranthine/sinapoyl-isoamaranthine) pigment pairs (into fractions 7 and 6, respectively) by HSCCC (Figs. 4A and 3F, respectively). In addition, a reversed elution order of both the pairs in these two separation techniques is obtained. Because pigments 5 and 6 are closely related (feruloylated and sinapoylated amaranthines) and eluted closely in C18 -HPLC (Fig. 3A), complementary HSCCC fractionation is proved to be highly useful for a convenient separation of both the pairs. Similarly, the separation of the pairs 7/7 and 8/8 (feruloylated and sinapoylated iresinins I, respectively) by HSCCC and HPLC also proves the complementarity of the two separations. In this case, the sinapoylated iresinins I 8/8 are eluted faster in HSCCC than the feruloylated derivatives 7/7 which is in opposite to the HPLC elution order. The analyzed fractions 8–12 are now much more simplified in HPLC (Figs. 4B–F) than the starting extract containing completely unresolved mixture of peaks 7/7 and 8/8 (Fig. 3A).
The last pigment pair, gomphrenin III/isogomphrenin III 9/9 , appears as the most hydrophobic betacyanins from I. lindenii fractionated in HSCCC (Fig. 4G). 3.2. LC-DAD-ESI-MS/MS analysis of HSCCC-fractionated betacyanins The detailed LC-DAD betacyanin profiles in the obtained fractions from the HSCCC fractionation of I. lindenii leaves extract are presented in Figs. 3 and 4. The main two pigment pairs of I. lindenii leaves were readily identified in the LC-DAD-ESI-MS/MS chromatograms by their characteristic pattern already observed in Iresine herbstii [9,10,12]. The presence of the known structures of amaranthine 1 (betanidin 5-O-ˇ-glucuronosylglucoside) and iresinin I 3 (betanidin 5-O-(6 -O-3-hydroxy-3-methyl-glutaryl)ˇ-glucuronosyl-glucoside) at the highest concentration levels accompanied by their C-15 isoforms (1 and 3 ) was confirmed by their wavelengths of maximum absorption in the visible range max , their detected protonated molecular ions at m/z 727 and 871, respectively (Table 1), as well as by their retention times (co-elution with authentic standards isolated from Iresine herbstii leaves).
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Fig. 5. ESI-MS/MS spectra and MS/MS-cleavage of substituents (6 -O-3 -hydroxy-3 -methyl-glutaryl, glucosyl, glucuronosyl, feruloyl and sinapoyl units) from the principal betacyanin components (6–8) of I. lindenii Van Houtte.
Other acylated and non-acylated betacyanins were detected by means of mass spectrometry and diode-array detection coupled to HPLC. Two prominent chromatographic peaks of betacyanins, 7 and 8 (with the other two ones of the isoforms, 7 and 8 ) were detected and identified tentatively as acylated iresinin I (3) by ferulic (7) and sinapic acid (8). The peaks were eluted in HPLC close to each other, suggesting a similar structure differing only by a methoxy moiety which was also supported by the similarity of elution orders of the analyzed pigments (7, 8, 7 and 8 ) and the recently identified acylated apiosyl-derived betacyanins [22]. The m/z values of their protonated molecular ions were 1047 and 1077, respectively, suggesting the presence of feruloylated iresinin I (the m/z difference 1047 − 871 = 176) and sinapoylated iresinin I (the m/z difference 1077 − 871 = 206). The presence of the fragmentation ion at m/z 695 (Fig. 5) indicated the positions of acylation at the glucuronosyl rings by these acyl moieties (the m/z differences 1047 − 695 = 176 + 176 and 1077 − 695 = 176 + 206, respectively). Their max 542 nm as well as the second absorption maxima at max-HCA 328 and 330 nm, respectively, (resulting from the presence of a hydroxycinnamoyl moiety in the molecule) with their absorbance ratio of ∼0.5 (Abs at max-HCA : Abs at max ) suggested the presence of one acyl moiety in each molecule. These betacyanins have never been identified in any plant material. The enzymatic essay with -glucuronidase of the finely purified fractions [12] confirmed that no hydrolysis of 7/7 and 8/8 took
place which indicates that the glucuronosyl rings are acylated by the hydroxycinnamic acids. Subsequent linkage between the sugar moiety and the acyl substituents in purified 7/7 and 8/8 were definitely established by mild methylation analysis [32] followed by the procedure of partially methylated alditol acetate preparation [30]. The detection of the prevailing 1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl-glucitol by GC-MS, identified by its retention time and characteristic fragmentation pattern [31], indicated the position of the acyl moiety (6 -O-3 -hydroxy-3 -methyl-glutaryl) which was bound to C-6 carbon of the glucosyl unit. Under the mild conditions of the Prehm derivatization procedure the acyl linkage is generally not lost, however some 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl-glucitol was detected, indicating partial destruction of the acyl linkage during the methylation. Parallel methylation analysis of 7/7 and 8/8 in basic conditions [30], under which all acyl residues are lost, resulted in detection of sole 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl-glucitol, confirming the presence of the glucosyl moiety in the structure of 7/7 and 8/8 . At the same conditions, a detection of 1,5-di-O-acetyl-2,3,4-tri-Omethyl-glucuronic acid methyl ester by GC-MS, identified by its characteristic fragmentation pattern, which was also exhibited for similarly analyzed iresinin 3 standard, showed the terminal position of the glucuronic acid bound to C-2 of the first glucopyranosyl moiety. Under the mild conditions of the Prehm derivatization [32]
G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
when the acyl linkage was behaved, 1,2,5-tri-O-acetyl-3,4-di-Omethyl-glucuronic acid methyl ester was detected which indicated the presence of the acyl moiety (feruloyl or sinapoyl, respectively) at the C-2 position. Further inspection of the chromatograms and mass spectra revealed the acylated forms of betanin and amaranthine (feruloylated or sinapoylated) existing in the plant. Two pairs of chromatographic peaks, 5/5 and 6/6 , were assigned to protonated molecular ions of the m/z at 903 and 933, respectively, suggesting the presence of feruloylated amaranthine (the m/z difference 903 − 727 = 176) and sinapoylated (Fig. 5) amaranthine (the m/z difference 933 − 727 = 206). The max 542 and 543 nm and the second absorption maxima at max-HCA 326 and 328 nm, respectively, (the absorbance ratio of ∼0.5) as well as the same chromatographic elution order as in the case of 7/7 and 8/8 suggested the presence of one acyl moiety in each molecule differing only by a methoxy group (the m/z difference 933 − 903 = 30). For 5/5 and 6/6 , no hydrolytic activity of ˇ-glucuronidase [12] could be detected which confirms that the glucuronosyl rings are acylated by the hydroxycinnamic acids. The linkage between the sugar moiety and the acyl substituents in purified 5/5 and 6/6 were definitely established as in the case of in 7/7 and 8/8 by mild methylation analysis [32] followed by the procedure of partially methylated alditol acetate preparation [30] as well as by parallel methylation of 5/5 and 6/6 in basic conditions [30]. The detection of the 1,2,5-tri-O-acetyl-3,4,6-tri-O-methylglucitol by GC-MS [31] indicated the linkage of the glucosyl unit between the aglycone and the terminal glucuronic moiety as well as no acylation of the glucosyl unit. This was confirmed by a further detection of 1,2,5-tri-O-acetyl-3,4-di-O-methyl-glucuronic acid methyl ester and 1,5-di-O-acetyl-2,3,4-tri-O-methyl-glucuronic acid methyl ester by GC-MS after their preparation from 5/5 and 6/6 under the mild [32] and rigid [30] conditions, respectively, as in the case of 7/7 and 8/8 linkage analysis. Thus, the terminal glucuronic moiety position as well as its acylation at C-2 by feruloyl (5/5 ) and sinapoyl (6/6 ) substituents was confirmed. The presence of 6/6 in plant material has never been acknowledged, however, the feruloylated amaranthine (celosianin II) (5/5 ) has been reported in the Amaranthaceae species [9,10,12]. In addition, the presence of celosianin II was unambiguously confirmed by a co-chromatography experiment with an authentic standard obtained from extract of Celosia argentea var. cristata [27–29]. Further analysis of the data resulted in identification of betanin 2/2 (the most known betacyanin (betanidin 5-O-ˇ-glucoside) and hylocerenin/isohylocerenin 4/4 (betanidin 5-O-(6 -O-3-hydroxy3-methyl-glutaryl)-ˇ-glucoside) which was readily confirmed by co-elution with the authentic standards derived from fruits of Hylocereus polyrhizus [6] and the spectral data ([M + H]+ m/z 551 and 695, respectively; max 537 nm). Hylocerenin and its epimer are reported for the first time in the Amaranthaceae species. The most lipophilic betacyanin 9/9 was detected at the m/z 727 of protonated molecular ions with the max 548 nm and the second absorption maximum at max-HCA 322 nm (the absorbance ratio of ∼0.5), indicating the presence of an acyl moiety (feruloylated glucosyl: the m/z difference 727 − 551 = 176). The bathochromic shift of the max value supported the presence of 6-O-glucosyl-derived betacyanin, therefore, this compound was identified as gomphrenin III (betanidin 6-O-(6 -O-E-feruloyl)-ˇ-glucoside), reported in the Amaranthaceae species [27–29] which was unambiguously confirmed by a co-chromatography experiment with an authentic standard obtained from extract of Gomphrena globosa flowers [27–29].
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4. Conclusions Using heptafluorobutyric acid as the ion-pair forming additive to the HSCCC solvent system improves highly the affinity of betacyanins to the organic stationary phase. This effect is so strong that the fractionation of the most polar betacyanins (amaranthine, betanin and iresinin I) results in their good resolution. Betacyanin-pigments are highly enriched in a single chromatography step by IP-HSCCC. This is recognized by HPLC-DAD analysis of the HSCCC fractions and indicates the principal compounds 3/3 (iresinin I/isoiresinin I) in HSCCC fraction 3 (Fig. 3). Fraction 5 consists mainly of 2/2 (betanin/isobetanin) and in fraction 8 highly pure is the pigment pair 8/8 (sinapoyl-iresinin I/sinapoylisoiresinin). Fractions 10–12 contain the pigment pair 7/7 (feruloyl-iresinin I/feruloyl-isoiresinin) in purified form (Fig. 4). HSCCC fraction 13 contains the most apolar pigments 9/9 (gomphrenin III/isogomphrenin III) enriched. Closely related diastereomeric pairs (feruloylated and sinapoylated derivatives of amaranthine and iresinin I) are readily separated in the HSCCC mode simplifying the HPLC pigment profiles. This confirms the complementarity between HSCCC and HPLC techniques in chromatography of betacyanins. In addition, in comparison to C18 reversed-phase HPLC, during countercurrent chromatography (e.g. HSCCC), completely different physicochemical separation effects are working, such as rapid changes in centrifugal force-fields causing strong mixing/demixing processes of the biphasic system and distribution of analytes of different polarities. Whereas the separation between the most polar betacyanins required improvement in previously reported solvent systems [22,6,24], in this study, the resolution between polar betacyanins is now satisfactory for the adequate enrichment of the concentrated pigments for further studies. The identification of eighteen betacyanins/isobetacyanins of a wide range of polarities in the I. lindenii Van Houtte was accomplished. Three new acylated betacyanins were detected in I. lindenii Van Houtte leaves for the first time and they are also new for the whole Amaranthaceae family [9–11].
References [1] D. Strack, T. Vogt, W. Schliemann, Phytochemistry 62 (2003) 247. [2] D. Strack, W. Steglich, V. Wray, in: P.M. Dey, J.B. Harborne, P.G. Waterman (Eds.), Methods in Plant Biochemistry, vol. 8, Academic Press, London, 1993, p. 421. [3] S. Wybraniec, Anal. Bioanal. Chem. 389 (2007) 1611. [4] B. Nemzer, Z. Pietrzkowski, A. Spórna, P. Stalica, W. Thresher, T. Michałowski, S. Wybraniec, Food Chem. 127 (2011) 42. [5] F.C. Stintzing, J. Conrad, I. Klaiber, U. Beifuss, R. Carle, Phytochemistry 65 (2004) 415. [6] S. Wybraniec, P. Stalica, G. Jerz, B. Klose, N. Gebers, P. Winterhalter, A. Spórna, M. Szaleniec, Y. Mizrahi, J. Chromatogr. A 1216 (2009) 6890. [7] F.C. Stintzing, A. Schieber, R. Carle, J. Agric. Food Chem. 50 (2002) 2302. [8] S. Wybraniec, B. Nowak-Wydra, J. Agric. Food Chem. 55 (2007) 8138. [9] Y. Cai, M. Sun, H. Corke, J. Agric. Food Chem. 49 (2001) 1971. [10] Y. Cai, M. Sun, H. Corke, Trends Food Sci. Technol. 16 (2005) 370. [11] Y. Cai, J. Xing, M. Sun, H. Corke, J. Agric. Food Chem. 54 (2006) 6520. [12] L. Minale, S. Piattelli, S. De Stefano, R.A. Nicolaus, Phytochemistry 5 (1966) 1037. [13] L. Tesoriere, D. Butera, M. Allegra, M. Fazzari, M.A. Livrea, J. Agric. Food Chem. 53 (2005) 1266. [14] J. Escribano, M.A. Pedreno, F. Garcia-Carmona, R. Munoz, Phytochem. Anal. 9 (1998) 124. [15] G.J. Kapadia, M.A. Azuine, R. Sridhar, Y. Okuda, A. Tsuruta, E. Ichiishi, T. Mukainake, M. Takasaki, T. Konoshima, H. Nishino, H. Tokuda, Pharmacol. Res. 47 (2003) 141. [16] J. Kanner, S. Harel, R. Granit, J. Agric. Food Chem. 49 (2001) 5178. [17] F. Gandía-Herrero, J. Escribano, F. García-Carmona, Planta 232 (2010) 449. [18] S. Wybraniec, T. Michałowski, J. Agric. Food Chem. 59 (2011) 9612. [19] D.A. Moreno, C. Garcia-Viguera, J.I. Gil, A. Gil-Izquierdo, Phytochem. Rev. 7 (2008) 261. [20] Y. Ito, W.D. Conway (Eds.), High-Speed Countercurrent Chromatography (Chemical Analysis, vol. 132), Wiley-Interscience, New York, 1996. [21] Y. Ito, J. Chromatogr. A 1065 (2005) 145.
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[22] G. Jerz, T. Skotzki, K. Fiege, P. Winterhalter, S. Wybraniec, J. Chromatogr. A 1190 (2008) 63. [23] G. Jerz, S. Wybraniec, N. Gebers, P. Winterhalter, J. Chromatogr. A 1217 (2010) 4544. [24] S. Wybraniec, G. Jerz, N. Gebers, P. Winterhalter, J. Chromatogr. B 878 (2010) 538. [25] S. Wybraniec, J. Chromatogr. A 1127 (2006) 70. [26] W. Schliemann, Y. Cai, T. Degenkolb, J. Schmidt, H. Corke, Phytochemistry 58 (2001) 159.
[27] [28] [29] [30] [31]
L. Minale, S. Piattelli, S. De Stefano, Phytochemistry 6 (1967) 703. S. Heuer, V. Wray, J.W. Metzger, D. Strack, Phytochemistry 31 (1992) 1801. S. Heuer, V. Wray, J.W. Metzger, D. Strack, Phytochemistry 33 (1993) 1553. K.R. Anumula, P.B. Taylor, Anal. Biochem. 203 (1992) 101. C.J. Biermann, G.D. McGinnis, Analysis of Carbohydrates by GLC and MS, CRC, Boca Raton, USA, 1989. [32] P. Prehm, Carbohydr. Res. 78 (1980) 372.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Separation of amaranthine-type betacyanins by ion-pair high-speed countercurrent chromatography Gerold Jerz a , Nadine Gebers a , Dominika Szot b , Maciej Szaleniec c , Peter Winterhalter a , Slawomir Wybraniec b,∗ a
Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany Faculty of Analytical Chemistry, Institute C-1, Department of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24, Cracow 31-155, Poland c Joint Laboratory of Biotechnology and Enzyme Catalysis at Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, 30-239 Cracow, Poland b
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 29 March 2014 Accepted 31 March 2014 Available online 12 April 2014 Keywords: Acylated betacyanins Betanin Betalains Ion-pair countercurrent chromatography IP-HSCCC Iresinin
a b s t r a c t Betacyanins, red-violet plant pigments, were fractionated by ion-pair high-speed countercurrent chromatography (IP-HSCCC) from leaves extract of Iresine lindenii Van Houtte, an ornamental plant of the family Amaranthaceae. An HSCCC solvent system consisting of TBME–1-BuOH–ACN–H2 O (1:3:1:5, v/v/v/v) was applied using ion-pair forming heptafluorobutyric acid (HFBA). Significantly different elution profiles of betacyanin diastereomeric pairs (derivatives based on betanidin and isobetanidin) observed in the HSCCC in comparison to HPLC systems indicate a complementarity of both techniques’ fractionation capabilities. The numerous diastereomeric pairs can be selectively separated from each other using the HSCCC system simplifying the pigment purification process. Apart from the three well known highly abundant pigments (amaranthine, betanin and iresinin I) together with their isoforms, three new acylated (feruloylated and sinapoylated) betacyanins as well as known pigment hylocerenin (previously isolated from cacti fruits) were characterized in the plant for the first time and they are new for the whole Amaranthaceae family. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Betacyanins, erroneously named as nitrogenous anthocyanins before their basic structure was disclosed in the 60s of the last century, are distinctively different from the anthocyanin plant pigments by the characteristic diazaheptamethine chromophoric system [1,2]. Betacyanins can be considered as condensation products of betalamic acid with cyclodopa or its O-glycosylated (in most cases 5-O-glucosylated) derivatives. Further esterification of the Oglycosides with acids such as ferulic, p-coumaric or malonic acid is very common [1,2]. Betalains exist in two diastereomeric forms differing by the configuration of the C-15 carbon (Fig. 1) [1–3]. Betanin, the simplest 5-O-glucosylated betacyanin and its C-15 isoform are frequently derived from red beet root (Beta vulgaris L.) [1–4]. Another edible rich source of betacyanins are recently investigated cacti species of Hylocereus containing betanin, phyllocactin
∗ Corresponding author. Tel.: +48 12 628 3074; fax: +48 12 628 2036. E-mail address: [email protected] (S. Wybraniec). http://dx.doi.org/10.1016/j.chroma.2014.03.085 0021-9673/© 2014 Elsevier B.V. All rights reserved.
and hylocerenin as the most abundant pigments [5,6] as well as Opuntia [7] or Mammillaria [8]. Betacyanin’s early investigations resulted in publication of a series of their chemical structures present in many plants, however, due to the lack of appropriate techniques, many of the these red-violet pigments remained unknown [1,2]. One of the most investigated species was Iresine herbstii, an ornamental plant of the family Amaranthaceae [9–11]. In spite of extensive studies performed, only two highly abundant betacyanins were fully characterized in the plant by chemical means [12] and mass spectrometry [9–11]. Minale et al. published in 1966 their comprehensive report on acylated betacyanins present in Iresine herbstii leaves [12] and Cai et al. surveyed the pigments in a broad range of Amaranthaceae species [9–11]. A series of betacyanins was assigned with the trivial names as ‘iresinin I–IV’ from which only ‘iresinin I’ as well as its C-15 diastereoisomer (‘isoiresinin I’) had been structurally identified completely [12]. According to comprehensive studies on betacyanins of the species of the Amaranthaceae family, their pigments have high potential for use as colorants in food products [9,10]. Some Amaranthus genotypes produce high biomass and contain more betacyanins than red beet [9,10].
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Choosing a suitable solvent system for betalains purification is challenging due to their low solubility in organic solvents as a result of their ionization. The addition of ion-pair reagents results in a different chromatographic behaviour of betalains, e.g. longer retention times of some betalains in RP-HPLC [25]. This is due to a formation of much more lipophilic ion-pairs between the counterions and betalains and thereby a better solubility of the compounds in the organic stationary phase. The addition of ion-pair reagents shifts the partition coefficient of betalains and creates a new possibility for CCC separation of these ionic plant pigments, including betanin and its diastereomer [22,23,6,24]. Except of the ion-pairing action, the acidification suppresses the carboxylic group’s dissociation, further enhancing the demanded hydrophobicity of the pigments. The most challenging is the separation of the most polar betacyanins for which appropriate organic phases dissolving the pigments should be elaborated. This contribution reports on the first results of HSCCC fractionation of betacyanins from Iresine lindenii leaves including a group of amaranthine-type pigments for their structural characterization.
2. Experimental 2.1. Reagents Formic acid, heptafluorobutyric acid (HFBA), HPLC-grade acetonitrile, TMBE, 1-butanol, methanol and HPLC-grade water were obtained from Merck (Darmstadt, Germany).
2.2. Preparation of crude pigment extract
Fig. 1. Chemical structures of amaranthine-, betanin- and gomphrenin-type betacyanins identified in I. lindenii Van Houtte leaves (for the full list of the pigments; see Table 1).
Betalains can be easily oxidized enzymatically and nonenzymatically [13–18] and having strong antioxidant properties are arising as interesting chemopreventive natural compounds. Therefore, in connection to their good colorant properties, a growing interest in betalains has been noticed during the past decade [19] and some of these compounds have not been identified yet. High-speed countercurrent chromatography (HSCCC) has been proved to be a very useful technique for preparative isolation of natural compounds from plant extracts [20,21]. Preparative isolation of betalains is problematic, therefore, new separation methods such as countercurrent chromatography create an important possibility of obtaining pure pigments. In addition, new interesting betalain structures are usually present in plant materials at low concentration and their isolation requires tedious and time consuming procedures. Hitherto, successful isolation and purification of betalains for further analytical investigations (structure elucidation) was carried out only in the HSCCC technique in a solvent system consisting either of TBME–1-BuOH–ACN–water (acidified with TFA or HFBA) [22,23,6,24].
For semipreparative pigment isolation, the leaves of I. lindenii Van Houtte (100 g) were extracted with 1000 mL of water in a blender and subsequently filtered through a 0.2 m i.d. pore size filter (Millipore, Bedford, MA). The extract was concentrated using a freeze-drier. For the co-injection experiments, the extracts of Hylocereus polyrhizus fruits from a previous study [6], Iresine herbstii leaves [9,10,12], Celosia argentea var. cristata flowers [26] as well as Gomphrena globosa flowers [27–29] were processed by a similar procedure. The pigment extract was chromatographically concentrated by solid phase extraction on C18 cartridges (Merck, Darmstadt, Germany) according to the procedure of Stintzing et al. [7]. After rinsing with water and acetonitrile the betacyanin fraction was eluted with acidified methanol (methanol/3% TFA, 95:5, v/v). The eluates were pooled and concentrated using a rotary evaporator under reduced pressure at 25 ◦ C and freeze-dried. The freeze-dried residue was submitted to semipreparative HPLC for isolation of betacyanins.
2.3. Reference compounds For structure confirmation, completely elucidated reference betacyanins as well as C-15 diastereomers (mostly by ESI-MS/MS and 2D-NMR) were derived from extracts of fruits, flowers or leaves of the following plants:betanidin 5-O-ˇ-glucuronosyl-glucoside (amaranthine) from Iresine herbstii [9,10,12], betanidin 5-O-(6 (hylocerenin) O-3 -hydroxy-3 -methyl-glutaryl)-ˇ-glucoside from Hylocereus polyrhizus [6], (2 -O-E-feruloyl)-amaranthine (celosianin II) from Celosia argentea var. cristata [26], betanidin 5-O(6 -O-3 -hydroxy-3 -methyl-glutaryl)-ˇ-glucuronosyl-glucoside (iresinin I) from Iresine herbstii [9,10,12], and betanidin 5-O-(6 -OE-feruloyl)-ˇ-glucoside (gomphrenin III) from Gomphrena globosa [27–29].
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2.4. Apparatus The HSCCC separation was carried out on a triple-coil highspeed countercurrent chromatograph (HSCCC) model CCC-1000 (Pharma-Tech. Res. Corp., USA) equipped with three preparative coils made of PTFE tubing (2.6 mm × 160 m i.d.), which were connected in a series (850 mL total volume). A manual sample injection valve with a 25 mL loop was used to introduce the sample into the coil system. All solvents were delivered by a Biotronik HPLC pump BT 3020 (Jasco, Gross-Umstadt, Germany). A Gynkotek HPLC system with UVD340U, Gynkotek HPLC Pump Series LPG-3400A and thermostat (Gynkotek Separations, H.I. Ambacht, The Netherlands) was used for the chromatographic analysis. For the data acquisition, the software package Chromeleon 6.0 (Gynkotek Separations) was used. For the separation of betalains, an ONYX monolithic column 100 mm × 4.6 mm i.d., protected by a guard column (Phenomenex, Torrance, CA, USA) was used. The positive ion electrospray mass spectra were recorded on ThermoFinnigan LCQ Advantage (electrospray voltage 4.5 kV; capillary 250 ◦ C; sheath gas: N2 ) coupled to ThermoFinnigan LC Surveyor pump utilizing the HPLC gradient. The MS was controlled and total ion chromatograms and mass spectra were recorded using ThermoFinnigan Xcalibur software (San Jose, CA, USA). The relative collision energies for the CID experiments were set at 30% (according to a relative energy scale). Helium was used to improve trapping efficiency and as the collision gas for the CID experiments. 2.5. Selection of the two-phase solvent system For the evaluation of suitable solvent systems with ion-pair forming capacity, the previously published solvent systems containing differently concentrated perfluorinated acids (aqueous TFA or HFBA solutions) were compared in a system TBME–1BuOH–ACN–H2 O (0.7% or 1% perfluorinated acid) 1:3:1:5 (v/v/v/v). Investigation of polar solvent systems suitable for preparative HSCCC separations of betalains clearly indicated that acetonitrile cannot be replaced by methanol because of a strong decomposition of the pigments in the latter solvent. The partition of pigment components was determined in the systems after shaking of 100 L TBME, 500 L 1-BuOH, 100 L ACN and 600 L acidified H2 O (0.4% or 0.7% perfluorinated acid) I. lindenii crude betacyanin extract in 2 mL vials. The estimation of the pigment distribution was performed by LC-DAD. For systems containing HFBA, a very good distribution of the polar as well as more lipophilic pigments between the organic (stationary) and aqueous (mobile) phase (head-to-tail mode) was observed, therefore, the systems containing 0.7% and 1% HFBA were chosen for further experiments in HSCCC apparatus. Because of the highest retention of its stationary phase [6], the system with 0.7% HFBA was elaborated further, enabling much better separation of the most polar betacyanins. 2.6. HSCCC separation of I. lindenii Van Houtte leaves extract The separation of the freeze-dried I. lindenii leaves crude betacyanin extract (C18 -SPE-cleaned, 1677 mg) was performed in the HSCCC system operated in the ‘head-to-tail’ mode applying the novel biphasic solvent system TBME–1-BuOH–ACN–H2 O (0.7% HFBA) 1:3:1:5 (v/v/v/v). The flow rate was 3.0 mL/min and the rotation velocity was set to 850 rpm. After separation, the solvent in the coil column was ejected with nitrogen gas. Analysis of CCC fractions was performed by HPLC-DAD and HPLC-ESI-MS/MS. Recovered fractions (No. 2–14) from the IP-HSCCC separation (injection amount 1677 mg of I. lindenii crude pigment extract): F2 (257 mg), F3 (349 mg), F4 (139 mg), F5 (195 mg), F6 (256 mg), F7 (138 mg), F8 (43.5 mg), F9 (7.4 mg), F10 (27.1 mg), F11 (17.7 mg),
F12 (3.2 mg), F13 (1.9 mg), F14-coil (with no betacyanins, 190 mg); sum of recovered betacyanin yields from HSCCC-fractions: 1435 mg (∼86%). 2.7. Analytical HPLC system For the separation of the analytes, the following gradient system was used: 95% A with 5% B at 0 min; gradient to 70% A with 30% B at 40 min (Solvent A: 1% HCOOH, Solvent B: acetonitrile). The injection volume was 10 L, and the flow rate was maintained at 0.5 mL/min. The detection of analytes was performed typically at 538, 505, 480, 450 and 310 nm. For the UV–vis spectra acquisition, the detection was performed in the DAD (diode-array detection) mode. The column was thermostated at 35 ◦ C. The same chromatographic conditions were applied for the HPLC-ESI-MS/MS analyses. 2.8. Enzymatic hydrolysis of I. lindenii pigments For the identification of the main structural units in the analyzed betacyanins, enzymatic hydrolysis for 90 min of the purified I. lindenii pigments by -glucuronidase from Helix pomatia (Sigma–Aldrich) at pH 5.0 and 37 ◦ C was performed [12]. The assays were performed for the newly identified pigments in comparison to the known betacyanins, amaranthine 1 and iresinin I 3 in which the glucuronosyl moiety is hydrolytically detached and the deglucuronosylated betacyanins (betanin and hylocerenin, respectively) can be detected by LC-MS. Acylation of the glucuronosyl ring prevents betacyanins from the action of the enzyme [12]. 2.9. Carbohydrate linkage analysis The linkage between the carbohydrate moieties was established by GC-MS analysis of partially methylated alditol acetates prepared by permethylation of betacyanins in basic conditions (dispersed NaOH in DMSO) using methyl iodide with subsequent hydrolysis, reduction and peracetylation as described by Anumula and Taylor [30]. Any existing acyl-linked organic residues are lost under these conditions. The GC-MS analyses were performed on a Finnigan gas chromatograph equipped with a 30-m Rtx-5 capillary column (Restek Co., Bellefonte, PA) connected to a Finnigan GCQ ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA) running in the 70 eV electron-impact mode. The gas chromatographic program used was 80 ◦ C (1 min)–10 ◦ C/min–300 ◦ C. The analytes were identified by their retention times and characteristic fragmentation patterns [31]. The position of acylation of the carbohydrate moieties was established by the above procedure with changed first step (permethylation of betacyanins) which was performed according to the standard procedure of Prehm [32] using methyl trifluoromethanesulfonate in trimethyl phosphate. Under these mild conditions the prevailing final products are derived mainly from permethylated carbohydrate units with preserved acyl linkage, however a partial loss of acyl units is also observed. 3. Results and discussion 3.1. HSCCC separation of betacyanins from I. lindenii Van Houtte The HSCCC separation trace of the pigments from extract of I. lindenii leaves monitored at 540 nm is depicted in Fig. 2. The extract was fractionated into fractions 1–13 (Figs. 2–4). The identification process is described in Section 3.2 and the list of identified pigments is presented in Table 1. The HSCCC elution order and separation of betalains was compared to the C18 reversed-phase HPLC profile and numerous
G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
Fig. 2. HSCCC-chromatogram of C18 -enriched pigment extract (1677 mg) I. lindenii Van Houtte leaves. HSCCC conditions: flow rate: 3.0 mL/min; ‘head-to-tail mode’; biphasic solvent system TBME–1-BuOH–ACN–H2 O (0.7% HFBA) 1:3:1:5 (v/v/v/v). Below, HPLC retention times of the separated major betacyanins detected in each HSCCC fraction.
45
differences were found suggesting the complementary separation capabilities of both the techniques. After the HSCCC separation, the retained amount of stationary phase in the coil-system was calculated to be 75%. In Table 2, betacyanin chromatographic distribution as well as the HPLC total peak area in the recovered fractions obtained from I. lindenii leaves extract by HSCCC is presented. Fraction 3 obtained during the HSCCC separation of 1677 mg of the concentrated pigment extract contained a bulk of the most polar compounds which were very well separated from betacyanins by HSCCC. The principal betacyanins present in the extract, iresinin I/isoiresinin I (3/3 ), are eluted mainly in the fraction 3 (349 mg) and are separated at a high extent from the preceding amaranthine/isoamaranthine (1/1 ) in fraction 2 as well as the following betanin/isobetanin (2/2 ). Taking into account that the pigments 1/1 , 2/2 and 3/3 are included to the group of the most polar betacyanins for which it is extremely difficult to find an appropriate organic solvent [22,6,24], it can be stated that their separation is satisfactory in the applied solvent system. This could be explained not only by the ion-pair interactions but also by the instant diminishing of the carboxyl dissociation under relatively high acidic conditions leading to the increase of the hydrophobic character of the molecules. A complete separation of the newly reported pigments in Amaranthaceae, hylocerenin/isohylocerenin 4/4 (Fig. 3F), from another 6 -O-3 -hydroxy-3 -methyl-glutarylated pair, iresinin I/isoiresinin I 3/3 (Fig. 3C), is observed in spite of their similar chromatographic properties indicated in the C18 -HPLC system. Pigments 3/3 and 4/4 differ only by the glucuronosyl unit which is the case also for the pigment pairs 1/1 and 2/2 . These significantly different elution profiles of the 3/3 and 4/4 pairs in the HSCCC in comparison to HPLC systems confirm the complementary separation capabilities of both techniques
Table 1 Chromatographic, spectrophotometric and mass-spectrometric data of the analyzed betacyanin pigments in the recovered fractions obtained from I. lindenii Van Houtte leaves extract by HSCCC. No.
Compound
1
Betanidin 5-O-ˇ-glucuronosyl-glucoside (amaranthine) Isobetanidin 5-O-ˇ-glucuronosyl-glucoside (isoamaranthine) Betanidin 5-O-ˇ-glucoside (betanin) Isobetanidin 5-O-ˇ-glucoside (isobetanin) Betanidin 5-O-(6 -O-3 -hydroxy-3 -methylglutaryl)-ˇ-glucuronosyl-glucoside (iresinin I) Betanidin 5-O-(6 -O-3 -hydroxy-3 -methylglutaryl)-ˇ-glucoside (hylocerenin) Isobetanidin 5-O-(6 -O-3 -hydroxy-3 -methylglutaryl)-ˇ-glucuronosyl-glucoside (isoiresinin I) Isohylocerenin (2 -O-E-Feruloyl)-amaranthine (celosianin II) (2 -O-E-Sinapoyl)-amaranthine (2 -O-E-Feruloyl)-isoamaranthine (isocelosianin II) (2 -O-E-Sinapoyl)-isoamaranthine (2 -O-E-Feruloyl)-iresinin I (2 -O-E-Sinapoyl)-iresinin I (2 -O-E-Feruloyl)-isoiresinin I (2 -O-E-Sinapoyl)-isoiresinin I Betanidin 6-O-(6 -O-E-feruloyl)-ˇ-glucoside (gomphrenin III) Isobetanidin 6-O-(6 -O-E-feruloyl)-ˇ-glucoside (isogomphrenin III)
1 2 2 3
4
3
4 5 6 5 6 7 8 7 8 9 9 a b
Rt [min]
max a [nm] I
max b [nm] II
Abs. ratio II:I
m/z [M + H]+
m/z from MS/MS of [M + H]+
8.1
–
536
–
727
551; 389
8.9
–
536
–
727
551; 389
9.8 10.3 11.6
– – –
535 535 540
– – –
551 551 871
389 389 695; 551; 389
11.9
–
537
–
695
551; 389
12.3
–
540
–
871
695; 551; 389
12.4 12.8 13.2 13.6 13.7 16.0 16.3 16.5 16.8 18.5
326 328 326 328 328 330 328 330 322
537 542 543 542 543 542 542 542 542 548
– 0.45 0.46 0.45 0.49 0.46 0.46 0.48 0.44 0.47
695 903 933 903 933 1047 1077 1047 1077 727
19.0
322
548
0.47
727
max I, wavelength of absorption maximum of the hydroxycinnamoyl moiety. max II, wavelength of absorption maximum in the visible range.
551; 389 727; 551; 389 727; 551; 389 727; 551; 389 727; 551; 389 871; 695; 551; 389 871; 695; 551; 389 871; 695; 551; 389 871; 695; 551; 389 551; 389 551; 389
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G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
Fig. 3. HPLC profiles of betacyanins (monitored at 540 nm) analyzed in I. lindenii Van Houtte leaves extract (A) and in the HSCCC fractions 2–6 (B–F).
Table 2 Betacyanin distributions in the recovered fractions obtained from I. lindenii Van Houtte leaves extract by HSCCC evaluated by HPLC-DAD-MS. No.
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9
Compound
Amaranthine Isoamaranthine Betanin Isobetanin Iresinin I Isoiresinin I Hylocerenin Isohylocerenin Celosianin II Isocelosianin II Sinapoyl-amaranthine Sinapoyl-isoamaranthine Feruloyl-iresinin I Feruloyl-isoiresinin I Sinapoyl-iresinin I Sinapoyl-isoiresinin I Gomphrenin III Isogomphrenin III
Relative content of betacyanin pigments in HSCCC fractions analyzed by HPLC-DAD-MS
Total HPLC peak area
2
3
4
5
6
7
8
9
10
11
12
13
(×10−5 )
91 89 – – 10 10 – – – – – – – – – – – –
9 11 12 11 69 73 – – – – – – – – – – – –
– – 69 67 19 17 – – – – – – – – – – – –
– – 19 22 1 1 – – – – – – – – – – – –
– – – – – – 59 52 – – 100 100 – – – – – –
– – – – – – 41 48 100 100 – – – – – 20 – –
– – – – – – – – – – – – 4 9 75 80 – –
– – – – – – – – – – – – 12 26 25 – – –
– – – – – – – – – – – – 29 31 – – – –
– – – – – – – – – – – – 44 25 – – – –
– – – – – – – – – – – – 11 9 – – 32 20
– – – – – – – – – – – – – – – – 68 80
0.8 0.4 3.3 1.9 20.5 14.1 0.8 0.4 0.3 0.3 0.3 0.1 1.8 1.2 1.1 1.8 0.1 0.1
G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
47
Fig. 4. HPLC profiles of betacyanins (monitored at 540 nm) analyzed in the fractions 7–13 (A–G) obtained after HSCCC separation of I. lindenii Van Houtte leaves extract.
observed recently [22,24]. In these cases, both the C-15 configurational diastereomers are separated from the other isomeric pair in HSCCC, greatly simplifying the final HPLC chromatograms of the obtained fractions. Another example of such simplifying operation is a fractionation of the 5/5 (celosianin II/isocelosianin II) and 6/6 (sinapoyl-amaranthine/sinapoyl-isoamaranthine) pigment pairs (into fractions 7 and 6, respectively) by HSCCC (Figs. 4A and 3F, respectively). In addition, a reversed elution order of both the pairs in these two separation techniques is obtained. Because pigments 5 and 6 are closely related (feruloylated and sinapoylated amaranthines) and eluted closely in C18 -HPLC (Fig. 3A), complementary HSCCC fractionation is proved to be highly useful for a convenient separation of both the pairs. Similarly, the separation of the pairs 7/7 and 8/8 (feruloylated and sinapoylated iresinins I, respectively) by HSCCC and HPLC also proves the complementarity of the two separations. In this case, the sinapoylated iresinins I 8/8 are eluted faster in HSCCC than the feruloylated derivatives 7/7 which is in opposite to the HPLC elution order. The analyzed fractions 8–12 are now much more simplified in HPLC (Figs. 4B–F) than the starting extract containing completely unresolved mixture of peaks 7/7 and 8/8 (Fig. 3A).
The last pigment pair, gomphrenin III/isogomphrenin III 9/9 , appears as the most hydrophobic betacyanins from I. lindenii fractionated in HSCCC (Fig. 4G). 3.2. LC-DAD-ESI-MS/MS analysis of HSCCC-fractionated betacyanins The detailed LC-DAD betacyanin profiles in the obtained fractions from the HSCCC fractionation of I. lindenii leaves extract are presented in Figs. 3 and 4. The main two pigment pairs of I. lindenii leaves were readily identified in the LC-DAD-ESI-MS/MS chromatograms by their characteristic pattern already observed in Iresine herbstii [9,10,12]. The presence of the known structures of amaranthine 1 (betanidin 5-O-ˇ-glucuronosylglucoside) and iresinin I 3 (betanidin 5-O-(6 -O-3-hydroxy-3-methyl-glutaryl)ˇ-glucuronosyl-glucoside) at the highest concentration levels accompanied by their C-15 isoforms (1 and 3 ) was confirmed by their wavelengths of maximum absorption in the visible range max , their detected protonated molecular ions at m/z 727 and 871, respectively (Table 1), as well as by their retention times (co-elution with authentic standards isolated from Iresine herbstii leaves).
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G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
Fig. 5. ESI-MS/MS spectra and MS/MS-cleavage of substituents (6 -O-3 -hydroxy-3 -methyl-glutaryl, glucosyl, glucuronosyl, feruloyl and sinapoyl units) from the principal betacyanin components (6–8) of I. lindenii Van Houtte.
Other acylated and non-acylated betacyanins were detected by means of mass spectrometry and diode-array detection coupled to HPLC. Two prominent chromatographic peaks of betacyanins, 7 and 8 (with the other two ones of the isoforms, 7 and 8 ) were detected and identified tentatively as acylated iresinin I (3) by ferulic (7) and sinapic acid (8). The peaks were eluted in HPLC close to each other, suggesting a similar structure differing only by a methoxy moiety which was also supported by the similarity of elution orders of the analyzed pigments (7, 8, 7 and 8 ) and the recently identified acylated apiosyl-derived betacyanins [22]. The m/z values of their protonated molecular ions were 1047 and 1077, respectively, suggesting the presence of feruloylated iresinin I (the m/z difference 1047 − 871 = 176) and sinapoylated iresinin I (the m/z difference 1077 − 871 = 206). The presence of the fragmentation ion at m/z 695 (Fig. 5) indicated the positions of acylation at the glucuronosyl rings by these acyl moieties (the m/z differences 1047 − 695 = 176 + 176 and 1077 − 695 = 176 + 206, respectively). Their max 542 nm as well as the second absorption maxima at max-HCA 328 and 330 nm, respectively, (resulting from the presence of a hydroxycinnamoyl moiety in the molecule) with their absorbance ratio of ∼0.5 (Abs at max-HCA : Abs at max ) suggested the presence of one acyl moiety in each molecule. These betacyanins have never been identified in any plant material. The enzymatic essay with -glucuronidase of the finely purified fractions [12] confirmed that no hydrolysis of 7/7 and 8/8 took
place which indicates that the glucuronosyl rings are acylated by the hydroxycinnamic acids. Subsequent linkage between the sugar moiety and the acyl substituents in purified 7/7 and 8/8 were definitely established by mild methylation analysis [32] followed by the procedure of partially methylated alditol acetate preparation [30]. The detection of the prevailing 1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl-glucitol by GC-MS, identified by its retention time and characteristic fragmentation pattern [31], indicated the position of the acyl moiety (6 -O-3 -hydroxy-3 -methyl-glutaryl) which was bound to C-6 carbon of the glucosyl unit. Under the mild conditions of the Prehm derivatization procedure the acyl linkage is generally not lost, however some 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl-glucitol was detected, indicating partial destruction of the acyl linkage during the methylation. Parallel methylation analysis of 7/7 and 8/8 in basic conditions [30], under which all acyl residues are lost, resulted in detection of sole 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl-glucitol, confirming the presence of the glucosyl moiety in the structure of 7/7 and 8/8 . At the same conditions, a detection of 1,5-di-O-acetyl-2,3,4-tri-Omethyl-glucuronic acid methyl ester by GC-MS, identified by its characteristic fragmentation pattern, which was also exhibited for similarly analyzed iresinin 3 standard, showed the terminal position of the glucuronic acid bound to C-2 of the first glucopyranosyl moiety. Under the mild conditions of the Prehm derivatization [32]
G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
when the acyl linkage was behaved, 1,2,5-tri-O-acetyl-3,4-di-Omethyl-glucuronic acid methyl ester was detected which indicated the presence of the acyl moiety (feruloyl or sinapoyl, respectively) at the C-2 position. Further inspection of the chromatograms and mass spectra revealed the acylated forms of betanin and amaranthine (feruloylated or sinapoylated) existing in the plant. Two pairs of chromatographic peaks, 5/5 and 6/6 , were assigned to protonated molecular ions of the m/z at 903 and 933, respectively, suggesting the presence of feruloylated amaranthine (the m/z difference 903 − 727 = 176) and sinapoylated (Fig. 5) amaranthine (the m/z difference 933 − 727 = 206). The max 542 and 543 nm and the second absorption maxima at max-HCA 326 and 328 nm, respectively, (the absorbance ratio of ∼0.5) as well as the same chromatographic elution order as in the case of 7/7 and 8/8 suggested the presence of one acyl moiety in each molecule differing only by a methoxy group (the m/z difference 933 − 903 = 30). For 5/5 and 6/6 , no hydrolytic activity of ˇ-glucuronidase [12] could be detected which confirms that the glucuronosyl rings are acylated by the hydroxycinnamic acids. The linkage between the sugar moiety and the acyl substituents in purified 5/5 and 6/6 were definitely established as in the case of in 7/7 and 8/8 by mild methylation analysis [32] followed by the procedure of partially methylated alditol acetate preparation [30] as well as by parallel methylation of 5/5 and 6/6 in basic conditions [30]. The detection of the 1,2,5-tri-O-acetyl-3,4,6-tri-O-methylglucitol by GC-MS [31] indicated the linkage of the glucosyl unit between the aglycone and the terminal glucuronic moiety as well as no acylation of the glucosyl unit. This was confirmed by a further detection of 1,2,5-tri-O-acetyl-3,4-di-O-methyl-glucuronic acid methyl ester and 1,5-di-O-acetyl-2,3,4-tri-O-methyl-glucuronic acid methyl ester by GC-MS after their preparation from 5/5 and 6/6 under the mild [32] and rigid [30] conditions, respectively, as in the case of 7/7 and 8/8 linkage analysis. Thus, the terminal glucuronic moiety position as well as its acylation at C-2 by feruloyl (5/5 ) and sinapoyl (6/6 ) substituents was confirmed. The presence of 6/6 in plant material has never been acknowledged, however, the feruloylated amaranthine (celosianin II) (5/5 ) has been reported in the Amaranthaceae species [9,10,12]. In addition, the presence of celosianin II was unambiguously confirmed by a co-chromatography experiment with an authentic standard obtained from extract of Celosia argentea var. cristata [27–29]. Further analysis of the data resulted in identification of betanin 2/2 (the most known betacyanin (betanidin 5-O-ˇ-glucoside) and hylocerenin/isohylocerenin 4/4 (betanidin 5-O-(6 -O-3-hydroxy3-methyl-glutaryl)-ˇ-glucoside) which was readily confirmed by co-elution with the authentic standards derived from fruits of Hylocereus polyrhizus [6] and the spectral data ([M + H]+ m/z 551 and 695, respectively; max 537 nm). Hylocerenin and its epimer are reported for the first time in the Amaranthaceae species. The most lipophilic betacyanin 9/9 was detected at the m/z 727 of protonated molecular ions with the max 548 nm and the second absorption maximum at max-HCA 322 nm (the absorbance ratio of ∼0.5), indicating the presence of an acyl moiety (feruloylated glucosyl: the m/z difference 727 − 551 = 176). The bathochromic shift of the max value supported the presence of 6-O-glucosyl-derived betacyanin, therefore, this compound was identified as gomphrenin III (betanidin 6-O-(6 -O-E-feruloyl)-ˇ-glucoside), reported in the Amaranthaceae species [27–29] which was unambiguously confirmed by a co-chromatography experiment with an authentic standard obtained from extract of Gomphrena globosa flowers [27–29].
49
4. Conclusions Using heptafluorobutyric acid as the ion-pair forming additive to the HSCCC solvent system improves highly the affinity of betacyanins to the organic stationary phase. This effect is so strong that the fractionation of the most polar betacyanins (amaranthine, betanin and iresinin I) results in their good resolution. Betacyanin-pigments are highly enriched in a single chromatography step by IP-HSCCC. This is recognized by HPLC-DAD analysis of the HSCCC fractions and indicates the principal compounds 3/3 (iresinin I/isoiresinin I) in HSCCC fraction 3 (Fig. 3). Fraction 5 consists mainly of 2/2 (betanin/isobetanin) and in fraction 8 highly pure is the pigment pair 8/8 (sinapoyl-iresinin I/sinapoylisoiresinin). Fractions 10–12 contain the pigment pair 7/7 (feruloyl-iresinin I/feruloyl-isoiresinin) in purified form (Fig. 4). HSCCC fraction 13 contains the most apolar pigments 9/9 (gomphrenin III/isogomphrenin III) enriched. Closely related diastereomeric pairs (feruloylated and sinapoylated derivatives of amaranthine and iresinin I) are readily separated in the HSCCC mode simplifying the HPLC pigment profiles. This confirms the complementarity between HSCCC and HPLC techniques in chromatography of betacyanins. In addition, in comparison to C18 reversed-phase HPLC, during countercurrent chromatography (e.g. HSCCC), completely different physicochemical separation effects are working, such as rapid changes in centrifugal force-fields causing strong mixing/demixing processes of the biphasic system and distribution of analytes of different polarities. Whereas the separation between the most polar betacyanins required improvement in previously reported solvent systems [22,6,24], in this study, the resolution between polar betacyanins is now satisfactory for the adequate enrichment of the concentrated pigments for further studies. The identification of eighteen betacyanins/isobetacyanins of a wide range of polarities in the I. lindenii Van Houtte was accomplished. Three new acylated betacyanins were detected in I. lindenii Van Houtte leaves for the first time and they are also new for the whole Amaranthaceae family [9–11].
References [1] D. Strack, T. Vogt, W. Schliemann, Phytochemistry 62 (2003) 247. [2] D. Strack, W. Steglich, V. Wray, in: P.M. Dey, J.B. Harborne, P.G. Waterman (Eds.), Methods in Plant Biochemistry, vol. 8, Academic Press, London, 1993, p. 421. [3] S. Wybraniec, Anal. Bioanal. Chem. 389 (2007) 1611. [4] B. Nemzer, Z. Pietrzkowski, A. Spórna, P. Stalica, W. Thresher, T. Michałowski, S. Wybraniec, Food Chem. 127 (2011) 42. [5] F.C. Stintzing, J. Conrad, I. Klaiber, U. Beifuss, R. Carle, Phytochemistry 65 (2004) 415. [6] S. Wybraniec, P. Stalica, G. Jerz, B. Klose, N. Gebers, P. Winterhalter, A. Spórna, M. Szaleniec, Y. Mizrahi, J. Chromatogr. A 1216 (2009) 6890. [7] F.C. Stintzing, A. Schieber, R. Carle, J. Agric. Food Chem. 50 (2002) 2302. [8] S. Wybraniec, B. Nowak-Wydra, J. Agric. Food Chem. 55 (2007) 8138. [9] Y. Cai, M. Sun, H. Corke, J. Agric. Food Chem. 49 (2001) 1971. [10] Y. Cai, M. Sun, H. Corke, Trends Food Sci. Technol. 16 (2005) 370. [11] Y. Cai, J. Xing, M. Sun, H. Corke, J. Agric. Food Chem. 54 (2006) 6520. [12] L. Minale, S. Piattelli, S. De Stefano, R.A. Nicolaus, Phytochemistry 5 (1966) 1037. [13] L. Tesoriere, D. Butera, M. Allegra, M. Fazzari, M.A. Livrea, J. Agric. Food Chem. 53 (2005) 1266. [14] J. Escribano, M.A. Pedreno, F. Garcia-Carmona, R. Munoz, Phytochem. Anal. 9 (1998) 124. [15] G.J. Kapadia, M.A. Azuine, R. Sridhar, Y. Okuda, A. Tsuruta, E. Ichiishi, T. Mukainake, M. Takasaki, T. Konoshima, H. Nishino, H. Tokuda, Pharmacol. Res. 47 (2003) 141. [16] J. Kanner, S. Harel, R. Granit, J. Agric. Food Chem. 49 (2001) 5178. [17] F. Gandía-Herrero, J. Escribano, F. García-Carmona, Planta 232 (2010) 449. [18] S. Wybraniec, T. Michałowski, J. Agric. Food Chem. 59 (2011) 9612. [19] D.A. Moreno, C. Garcia-Viguera, J.I. Gil, A. Gil-Izquierdo, Phytochem. Rev. 7 (2008) 261. [20] Y. Ito, W.D. Conway (Eds.), High-Speed Countercurrent Chromatography (Chemical Analysis, vol. 132), Wiley-Interscience, New York, 1996. [21] Y. Ito, J. Chromatogr. A 1065 (2005) 145.
50
G. Jerz et al. / J. Chromatogr. A 1344 (2014) 42–50
[22] G. Jerz, T. Skotzki, K. Fiege, P. Winterhalter, S. Wybraniec, J. Chromatogr. A 1190 (2008) 63. [23] G. Jerz, S. Wybraniec, N. Gebers, P. Winterhalter, J. Chromatogr. A 1217 (2010) 4544. [24] S. Wybraniec, G. Jerz, N. Gebers, P. Winterhalter, J. Chromatogr. B 878 (2010) 538. [25] S. Wybraniec, J. Chromatogr. A 1127 (2006) 70. [26] W. Schliemann, Y. Cai, T. Degenkolb, J. Schmidt, H. Corke, Phytochemistry 58 (2001) 159.
[27] [28] [29] [30] [31]
L. Minale, S. Piattelli, S. De Stefano, Phytochemistry 6 (1967) 703. S. Heuer, V. Wray, J.W. Metzger, D. Strack, Phytochemistry 31 (1992) 1801. S. Heuer, V. Wray, J.W. Metzger, D. Strack, Phytochemistry 33 (1993) 1553. K.R. Anumula, P.B. Taylor, Anal. Biochem. 203 (1992) 101. C.J. Biermann, G.D. McGinnis, Analysis of Carbohydrates by GLC and MS, CRC, Boca Raton, USA, 1989. [32] P. Prehm, Carbohydr. Res. 78 (1980) 372.
