Alternative Mechanisms of Betacyanin Oxidation by Complexation and Radical Generation.

The use of natural pigments such as betalains in food and health-related products is often limited by said pigments' relative oxidative stabilities in the products or physiological matrices. Determination of the mechanism of oxidation may inform future development and delivery of better stabilized molecules for improved outcomes. In order to best determine the oxidation mechanism of betanin, a natural food colorant, our efforts were directed toward structural elucidation (LCMS-IT-TOF and NMR) of previously tentatively identified key dehydrogenation products that had been generated as a result of betanin, decarboxylated betanin, and neobetanin oxidation by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radicals. The resultant oxidation products, the neo-derivatives, were the most stable and survived the preparative isolation and purification process. Structural analyses subsequently confirmed that these compounds, as well as neobetanin, were also the key products of alternative pathways of betanin and 2-decarboxy-betanin oxidation when catalyzed by Cu2+ cations in aqueous solutions at pH close to neutral. Therefore, the structures of the following five neo- or xanneo-derivatives (14,15- or 2,3,14,15-dehydrogenated derivatives, respectively) were confirmed: neobetanin, 2-decarboxy-neobetanin, 2-decarboxy-xanneobetanin, 2,17-bidecarboxy-xanneobetanin, and 2,15,17-tridecarboxy-xanneobetanin. This research confirmed that Cu2+-catalyzed oxidation of betanin and 2-decarboxy-betanin results in generation of neo-derivatives of betanin. In contrast, Cu2+-catalyzed oxidation of 17-decarboxy-betanin and 2,17-bidecarboxy-betanin resulted mostly in formation of betanin xan-derivatives. A relevant mechanism of Cu2+-catalyzed oxidation of the pigments is proposed herein that suggests that the oxidation of betanin can possibly occur in the region of the dihydropyridinic ring and can omit the stage of methide quinone formation in the dihydroindolic system.

Separation of betacyanins from flowers of Amaranthus cruentus L. in a polar solvent system by high-speed counter-current chromatography.

Betacyanin extract of Amaranthus cruentus L. flowers was fractionated by semi-preparative high-speed counter-current chromatography in a highly polar solvent system: propan-1-ol/acetonitrile/(NH4 )2 SO4satd. soln /H2 O (1.0:0.5:1.2:1.0, v/v/v/v) in tail-to-head mode with 76% retention of the stationary phase. The crude extract as well as the fractions containing betacyanins were analyzed by liquid chromatography with tandem mass spectrometry as well as by high-resolution ion-trap time-of-flight mass spectrometry detection technique for the molecular formulae and multi-step fragmentation pattern elucidation. Four betacyanins; namely, amaranthin, betanin, 6'-O-formyl-amaranthin, and 6'-O-malonyl-amaranthin as well as their diastereomeric forms differing in the configuration of the C-15 carbon atom were identified in the fractions. Amaranthin was the dominant pigment in the extract and was additionally analyzed by nuclear magnetic resonance correlation techniques after the counter-current chromatographic and high-performance liquid chromatographic isolation. Betacyanins were highly enriched during a single high-speed counter-current chromatographic step; therefore, the tentative identification of new compounds for the whole Amaranthaceae family, 6'-O-formyl-amaranthin and 6'-O-malonyl-amaranthin was possible. Different elution profiles of the pigments observed in the counter-current chromatographic system in comparison to high-performance liquid chromatography system confirm a complementarity of both the techniques especially in the separation of diastereomeric pairs of betacyanins.

Conjugation of Oxidized Betanidin and Gomphrenin Pigments from Basella alba L. Fruits with Glutathione.

Formation of glutathionic conjugates with quinonoid forms generated through oxidation of betanidin and gomphrenin obtained from fruits of Basella alba L. was studied by high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) and ion-trap time-of-flight high-resolution mass spectrometry (LCMS-IT-TOF). The conjugates were studied for the aim of trapping the formed quinonoids by glutathione which would indicate a presence of specific quinonoid structures in reaction products of the pigments with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radicals. The structure of betanidin conjugate, which was formed with high efficiency, was established by NMR analysis. In the case of gomphrenin conjugate, its structure was tentatively indicated as analogous to betanidin conjugate by MS n fragmentation paths. In contrast, no detectable glutathionic conjugate of betanin quinonoid (quinone methide) was present in similar betanin reaction mixtures. As a result of additional experiments performed during oxidation of gomphrenin by ABTS cation radicals in the absence of glutathione, except for decarboxylated and dehydrogenated gomphrenin derivatives, generation of betanidin and its derivatives was observed which indicated that the subsequent dopachromic intermediate rearrangement affected hydrolysis of the glucosidic bond. This is in contrast to betanin which is not deglucosylated in the same conditions during the oxidation. The obtained results shed some light on the oxidation pathways of various glycosylated betacyanins with gomphrenin being presumably the most potent antioxidant ascertained in this group of pigments.

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

Betacyanins and betaxanthins were characterized and determined in an intensely pigmented red-colored callus culture of Celosia cristata L. (Amaranthaceae). A new malonyl derivative, 6'- O-malonyl-amaranthin (celoscristatin) was isolated and identified by spectroscopic and mass spectrometric techniques. Its stereoisomer, 4'- O-malonyl-amaranthin (celoscristatin acyl-migrated), as well as its 15 R diastereomer were also detected in the callus as a result of the malonyl group migration in celoscristatin/isoceloscristatin, respectively. Amaranthin occurs in the callus as the major betacyanin, followed by celoscristatin, betanin, phyllocactin, and other minor betacyanins. The effect of different carbon sources on the growth rates of the Celosia callus as well as on betalains profiles in the callus cultures was studied. High dopamine content in the callus culture was determined and compared with the content in C. cristata inflorescences. The dopamine-based betalain (miraxanthin V) was detected as the main betaxanthin in the callus, however, at a concentration level much lower than that of the identified betacyanins. The studied callus culture of C. cristata can accumulate betalains in amounts which approach the quantities produced by most known high-yielding plant species.

Studies on polar high-speed counter-current chromatographic systems in separation of amaranthine-type betacyanins from Celosia species.

Betacyanins, natural plant pigments exhibiting antioxidant and chemopreventive properties, were extracted from Celosia spicata (Thouars) Spreng. inflorescences and separated by high-speed counter-current chromatography (HSCCC) in two polar solvent systems composed of: TBME - 1-BuOH - ACN - H2O (0.7% HFBA, 2:2:1:5, v/v/v/v) (system I) and EtOH - ACN - 1-PrOH - (NH4)2SO4satd.soln - H2O (0.5:0.5:0.5:1.2:1, v/v/v/v/v) (system II). The systems were used in the head-to-tail (system I) and tail-to-head (system II) mode. The flow rate of the mobile phase was 2.0 ml/min and the column rotation speed was 860 rpm. The retention of the stationary phase was 73.5% (system I) and 80.0% (system II). For the identification of separated betacyanins in the crude extract as well as in the HSCCC fractions, LC-DAD-ESI-MS/MS analyses were performed. Depending on the target compounds, each of the systems exhibit meaningfully different selectivity and applicability. For the pairs of amaranthines (1/1') and betanins (2/2'), the best choice is the system II, but the acylated amaranthine pairs (3/3' and 4/4') can be resolved only in the ion-pair system I. For the indication of the most suitable solvent system for Celosia plumosa hort., Celosia cristata L. and Celosia spicata (Thouars) Spreng. species, the profiles of betacyanins in different plant parts were studied.

Thermal Degradation of Major Gomphrenin Pigments in the Fruit Juice of Basella alba L. (Malabar Spinach).

Generation of decarboxylated and dehydrogenated gomphrenins during heating of Basella alba L. fruit juice containing high levels of betacyanin pigments was monitored by LC-DAD-ESI-MS/MS. The presence of principal decarboxylation products, 2-, 17-, and 2,17-decarboxy-gomphrenins, their diastereomers, as well as minor levels of their dehydrogenated derivatives are reported. In addition, determination of molecular masses of decarboxylated gomphrenins by high-resolution mass spectrometry (LCMS-IT-TOF) was performed. Enzymatic deglucosylation of decarboxylated and dehydrogenated gomphrenins resulted in the generation of betanidin diagnostic derivatives for further identification processes. In addition, experiments were conducted to prove that the position of glucosylation of the chromophoric part of betacyanins (betanidin part) has decisive influence on different chromatographic properties of their decarboxylated derivatives.