Phytochemical Molecules from the Decarboxylation of Gomphrenins in Violet Gomphrena globosa L.—Floral Infusions from Functional Food

Herein, the generation of decarboxylated derivatives of gomphrenin pigments exhibiting potential health-promoting properties and the kinetics of their extraction during tea brewing from the purple flowers of Gomphrena globosa L. in aqueous and aqueous citric acid solutions were investigated. Time-dependent concentration monitoring of natural gomphrenins and their tentative identification was carried out by LC-DAD-ESI-MS/MS. The high content of acylated gomphrenins and their principal decarboxylation products, 2-, 15-, 17-decarboxy-gomphrenins, along with minor levels of their bidecarboxylated derivatives, were reported in the infusions. The identification was supported by the determination of molecular formulas of the extracted pigments by liquid chromatography coupled with high-resolution mass spectrometry (LCMS-IT-TOF). The influence of plant matrix on gomphrenins’ stability and generation of their derivatives, including the extraction kinetics, was determined by studying the concentration profiles in the primary and diluted infusions. Isolated and purified acylated gomphrenins from the same plant material were used for the preliminary determination of their decarboxylated derivatives. The acylated gomphrenins were found to be more stable than nonacylated ones. Citric acid addition had a degradative influence on natural gomphrenins mainly during the longer tea brewing process (above 15 min); however, the presence of plant matrix significantly increased the stability for betacyanins’ identification.


Introduction
Gomphrena globosa L. commonly known as globe amaranth is an annual herbaceous, edible plant belonging to the family of Amaranthaceae. It is widely cultivated in China and the tropical regions of Central America. G. globosa inflorescences occur basically in three varieties: white, red, and violet.
They are utilized in traditional Chinese medicine in the preparation of cough syrups to treat respiratory system diseases. Dried gomphrena blossoms are characterized by unique fragrance and exhibit numerous pro-health properties. They are commonly used for the preparation of tea infusions. Furthermore, globe amaranth flowers are sold on a commercial scale as tisanes and used as a popular ingredient in blooming teas which are commonly known as flowering tea [1]. Nowadays, the number of people suffering from civilization diseases such as diabetes, hypertension, and cancer, is increasing. Therefore, natural products comprising biologically active compounds attract much attention [2]. These products implemented into a daily diet may play an important role in the prevention and treatment of many health problems [3]. The violet G. globosa inflorescences are a precious source of many antioxidants involving betalains. These compounds are proven to exhibit numerous pro-health properties, owing to the high content of hydroxyl groups that participate in free radical scavenging [3][4][5][6][7]. Their antihemorrhage, antimicrobial, antiinflammatory activity, and analgesic effects have been reported [8][9][10][11][12][13]. Betalains are vacuolar, natural plant pigments. They are present in plants belonging to the Caryophyllales order [14,15]. It is worth noting that betacyanins acylated by hydroxycinnamic acid derivatives are present in gomphrena flowers at relatively high levels. To simplify, they are frequently denominated as gomphrenins. Accordingly, nonacylated gomphrenin I is a common name for betanidin-6-O-β-glucoside, whereas gomphrenin II and III are betanidin-6-O-(6 -O-trans-4-coumaroyl)-β-glucoside and betanidin-6-O-(6 -O-trans-feruloyl)-β--glucoside, respectively [16][17][18]. Their chemical structures have been completely elucidated by nuclear magnetic resonance and mass spectrometry [19,20]. The structure of gomphrenin IV has been tentatively elucidated as a betanidin-6-O-(6 -O-sinapoyl)-β-glucoside because of its coelution with gomphrenin III. Betanidin 6-O-glucosides are very rare compounds and hitherto, they have been identified only in G. globosa inflorescences, B. alba L. fruits and its variety B. rubra L. (the main source of nonacylated gomphrenin I) as well as in B. glabra bracts [19][20][21][22][23][24][25][26]. Previous reports indicated that decarboxylated betacyanin derivatives are formed in preparations subjected to thermal processing as a result of betalains' sensitivity to elevated temperatures [21,24,[27][28][29][30][31][32]. Thermal decarboxylation may occur at different degrees. In general, 2-decarboxy-and 17-decarboxy-betacyanins, which exist in two diastereomeric forms, are formed in the greatest amounts. Furthermore, 15-decarboxy-betacyanins with the lost chiral center at the C-15 carbon are observed, therefore, there are no additional diastereomers of these derivatives. Frequently, after prolonged heating, bidecarboxy-and even tridecarboxy-betacyanins can be formed as well. Decarboxylated betacyanins are less polar than the initial forms of betacyanins, therefore, their retention on reversed-phase in HPLC is greater [21,24]. Despite a few phytochemical studies concerning the presence of phenolic compounds in G. globosa flowers, there is a lack of reports referring to the comprehensive extraction of these relevant compounds during thermal processing close to tea brewing conditions [7,28]. For that reason, our study aimed to evaluate the kinetics of betacyanins' extraction in tea infusion prepared from G. globosa flowers and to determine betacyanin degradation products. Research on the influence of citric acid addition and matrix presence on the stability of the compounds during the preparation of G. globosa infusions as well as the comparative experiments with single purified gomphrenins derived from G. globosa flowers was also performed.

Results and Discussion
Previous studies revealed a typical primary betacyanin profile in the extract of violet G. globosa flowers [12,22]. Chromatograms recorded in selected ion monitoring mode (SIM), and DAD chromatogram registered at 545 nm, obtained for a lyophilized sample of G. globosa extract not subjected to heating are depicted in Figure 1. Additionally, chemical patterns of natural gomphrenins and abbreviation names of the observed decarboxylated derivatives are presented. The presence of sinapoyled diastereomers 20/20 has never been confirmed in natural extracts of G. globose, except for low-resolution mass spectrometry (Table 1) [12,22]; therefore, additional high-resolution measurements and confirmation of the molecular formulas were performed by LCMS-IT-TOF ( Figure 2, Table 2).   The dominant presence of the feruloyl-gomphrenin (Fer-Gp) 6, coumaroyl-gomphrenin (Coum-Gp) 14 and their diastereomers (Fer-IGp) 6 , (Coum-IGp) 14 with significantly lower quantities of sinapoyl-gomphrenin/-isogomphrenin (Sin-Gp/-IGp) 20/20 and nonacylated gomphrenin I/isogomphrenin I (Gp/IGp) 1/1 was confirmed. For the aim of simplification, we propose to refer to the acylated pigments as respective "hydroxycinnamoyled gomphrenins" as well as to their abbreviations (Table 1) instead of selected "gomphrenins II, III, and IV". Accordingly, nonacylated "gomphrenin I" will be referred to as "gomphrenin". In the previous study [12], additional cis-isomers of coumaroyl-gomphrenin (cis-Coum-Gp/-IGp) 13/13 and feruloyl-gomphrenin (cis-Fer-Gp/-IGp) 5/5 were tentatively detected.  For 0.94 g or 0.19 g weighted portions of G. globosa flowers which were brewing in 6 mL of water for 60 min, two series of solutions were obtained, nondiluted, and fivefold diluted, respectively. The temperature (90 °C) of the tea brewing process was high enough for monitoring changes in the compositions of the resulting mixtures and close to home tea brewing conditions. The spectra of the visible range of the obtained extract samples collected during heat processing are shown in Figure 3.
Higher absorbance values were observed for the samples which were extracted in aqueous solutions ( Figure 3A,B) in comparison to aqueous citric acid solutions. It indicates mild extraction conditions  For 0.94 g or 0.19 g weighted portions of G. globosa flowers which were brewing in 6 mL of water for 60 min, two series of solutions were obtained, nondiluted, and fivefold diluted, respectively. The temperature (90 • C) of the tea brewing process was high enough for monitoring changes in the compositions of the resulting mixtures and close to home tea brewing conditions. The spectra of the visible range of the obtained extract samples collected during heat processing are shown in Figure 3.
Higher absorbance values were observed for the samples which were extracted in aqueous solutions ( Figure 3A,B) in comparison to aqueous citric acid solutions. It indicates mild extraction conditions of gomphrenins in water without citric acid addition. A stronger influence of citric acid addition on the stability of these compounds can be observed in Figure 3C,D. After 60 min, for undiluted samples, the absorption maximum was not clearly observed and there was no absorption band for diluted samples after 30 min. of gomphrenins in water without citric acid addition. A stronger influence of citric acid addition on the stability of these compounds can be observed in Figure 3C,D. After 60 min, for undiluted samples, the absorption maximum was not clearly observed and there was no absorption band for diluted samples after 30 min. This resulted from the lower impact of the plant matrix which presumably supported the stability of the pigments. The slight absorption maximum at approximately 480 nm is observed in Figure 3A after 60 min. It indicates the presence of other conversion products, presumably oxidized, of natural gomphrenins. Decarboxylated degradation products of natural gomphrenins formed during the tea brewing process in aqueous and citric acid solutions are listed in Table 1. All the detected degradation products of the pigments were less polar than their corresponding precursors. For the most prominent degradation products of acylated gomphrenins, additional confirmation was obtained by LCMS-IT-TOF analyses ( Table 2). A series of selected chromatograms obtained for the processed flowers in the experiments as well as for selected purified and heated pigments are depicted in Figure 4. This resulted from the lower impact of the plant matrix which presumably supported the stability of the pigments. The slight absorption maximum at approximately 480 nm is observed in Figure 3A after 60 min. It indicates the presence of other conversion products, presumably oxidized, of natural gomphrenins. Decarboxylated degradation products of natural gomphrenins formed during the tea brewing process in aqueous and citric acid solutions are listed in Table 1. All the detected degradation products of the pigments were less polar than their corresponding precursors. For the most prominent degradation products of acylated gomphrenins, additional confirmation was obtained by LCMS-IT-TOF analyses ( Table 2). A series of selected chromatograms obtained for the processed flowers in the experiments as well as for selected purified and heated pigments are depicted in Figure 4.

Extraction of Gomphrenins during Tea Brewing of G. globosa in Aqueous Solutions
The highest concentration of all acylated gomphrenins in undiluted and fivefold diluted samples were obtained after 15 min of extraction, but the degradation of substrates was faster for diluted samples, presumably as a result of the diminished stabilizing effect of the matrix in diluted solutions ( Figure 5). The matrix effect in aqueous solutions was not clearly observed for Gp/-IGp 1/1′, but these

Extraction of Gomphrenins during Tea Brewing of G. globosa in Aqueous Solutions
The highest concentration of all acylated gomphrenins in undiluted and fivefold diluted samples were obtained after 15 min of extraction, but the degradation of substrates was faster for diluted samples, presumably as a result of the diminished stabilizing effect of the matrix in diluted solutions ( Figure 5). The matrix effect in aqueous solutions was not clearly observed for Gp/-IGp 1/1 , but these betacyanins were, in general, less stable during tea brewing than the acylated ones. The greatest signal intensity for Gp/-IGp 1/1 was observed after 10 min of thermal treatment. betacyanins were, in general, less stable during tea brewing than the acylated ones. The greatest signal intensity for Gp/-IGp 1/1′ was observed after 10 min of thermal treatment.

Generation and Identification of 17-Decarboxylated Derivatives of Gomphrenins in Aqueous Solutions
Interpretation of the LC-DAD and LC-MS spectra obtained in the HPLC gradient System 5 revealed that the main products appeared to be mono-decarboxylated derivatives for all gomphrenins due to the loss of CO 2 from the corresponding precursors (Figures 4 and 6). Based on previous studies [24], a group of distinct chromatographic peaks with characteristic absorption maxima at λ max 507, 515, 512, and 517 nm, influenced by the bathochromic effect of the acyl substituents, was attributed to 17-decarboxylated derivatives of nonacylated gomphrenins (17- (Table 1) only in undiluted samples due to very low signal intensities ( Figure 6).   Table 2). The observed fragmentation pathway in the MS 2 mode afforded a signal at m/z 507 (Table 2)

Concentration Profiles of 17-Decarboxylated-Gomphrenins in Aqueous Solutions
The time-dependent concentration profiles for 17-dGp 2 as well as acylated Fer-17-dGp 8, Coum-17-dGp 16, and Sin-17-dGp 21 in all the samples during tea brewing are shown in Figure 8A-D. The maximal concentrations for acylated derivatives 8, 16, and 21 were evidently shifted to 30 min in comparison to their parent substrates 6, 14, and 20 ( Figure 5), for which the maxima were observed at ca. 15 min of the brewing experiment. This indicates that a continuous decarboxylation effect took place in the substrates Fer-Gp 6 and Coum-Gp 14 as a result of their heating in the presence of relatively high quantities of the inflorescence matrix. This effect is less demonstrated for the diluted samples ( Figure 8B,C) presumably as a result of a lower concentration of the pigments and a lower stabilizing effect of the matrix, therefore, faster degradation of the generated pigments was observed. For the Sin-17-dGp 21, these differences were less pronounced ( Figure 8D Figure 8A). This presumably results from a low concentration of Gp 1 in the flowers, but also from its much lower stability (evidenced in Figure 5C,D). The CR value of 17-dGp 2 to nonacylated gomphrenin 1 ( Figure 9F) increased definitely faster than CR for acylated decarboxylated gomphrenins presumably due to lower stability of the initial pigment.  Figure 8A). This presumably results from a low concentration of Gp 1 in the flowers, but also from its much lower stability (evidenced in Figure 5C,D). The CR value of 17-dGp 2 to nonacylated gomphrenin 1 ( Figure 9F) increased definitely faster than CR for acylated decarboxylated gomphrenins presumably due to lower stability of the initial pigment.

Generation and Identification of 2-Decarboxylated Derivatives of Gomphrenins in Aqueous Solutions
Other mono-decarboxylated compound 10 and the pair of 17/17′ were assigned to feruloyl-2decarboxy-gomphrenin (Fer-2-dGp) and coumaroyl-2-decarboxy-gomphrenin/-isogomphrenin (Coum-2-dGp/-dIGp), respectively. These compounds displayed the characteristic absorption maxima at λmax 537 and 539 nm [24,28,29] (A and B), coumaroylated (C and D) and sinapoylated (H) as well as non-acylated (E-G) decarboxylated derivatives to their corresponding natural gomphrenins during G. globosa thermal processing at 90 • C. The ratios obtained for diluted citric acid samples after 60 min are not shown for most compounds due to a very high degradation rate of natural gomphrenins in citric acid solutions.

Concentration Profiles of 2-Decarboxylated-Gomphrenins in Aqueous Solutions
Fer-2-dGp 10 was generated in the concentrated and diluted samples mainly within the first 10-15 min of tea brewing and most probably underwent following chemical changes (by decarboxylation) during the rest of the process, similarly to Fer-17-dGp 8 ( Figure 8B). The diastereomer Fer-2-dIGp 10 was not observed in the aqueous tea infusion samples. The time-dependent concentration profile of Coum-2-dGp 17 was very similar to the profile obtained for Coum-17-dGp 16 during thermal treatment ( Figure 8C). CR values of Coum-2-dGp 17 slowly increased, similarly to Coum-17-dGp 16 ( Figure 9C). For gomphrenin, one chromatographic peak, corresponding to 2-decarboxy-gomphrenin/-isogomphrenin 4/4 , was observed. This signal was characterized by absorption maximum at λ max 533 nm, which is coherent with the previous study [24]. Moreover, these data are close to the results obtained for betanin thermal degradation with 2-decarboxy-betanin/-isobetanin being the products present in heated betanin-rich red beet juice [21,[25][26][27]30]. The concentration profile and the CR value of 2-dGp 4 was comparable to 17-dGp 2 ( Figures 8A and 9E).

Generation and Identification of 15-Decarboxylated Derivatives of Gomphrenins in Aqueous Solutions
The 15-decarboxy-derivatives, which were formed with a loss of the chiral center at carbon C-15 and exhibited only single chromatographic peaks, were tentatively identified for gomphrenin as well as feruloylated and sinapoylated gomphrenins. 15-decarboxy-gomphrenin (15-dGp) 3 and feruloyl-15-decarboxy-gomphrenin (Fer-15-dGp) 12 displayed the characteristic absorption maxima at λ max 530 nm. LC-MS and HRMS LCMS-IT-TOF spectra and the fragmentation ions were analogous to the data for 17-decarboxy-derivatives. The absorption maxima could not be observed for sinapoyl-15-decarboxy-gomphrenin (Sin-15-dGp) 24 due to low signal intensity.

Extraction of Gomphrenins during Tea Brewing of G. globosa in Aqueous Citric Acid Solutions
In general, lower concentrations of extracted natural gomphrenins were determined during tea brewing in aqueous citric acid solutions in comparison to aqueous solutions. Exploration of the tea brewing data obtained for citric acid solutions revealed similar profiles of betacyanin degradation products to aqueous solutions. Cis-Fer-2,17-dGp 9 (m/z 639) previously observed in aqueous solutions was not detected in the citric acid solutions ( Figure 6). The greatest concentration of acylated gomphrenins was obtained after 15 min of tea brewing for undiluted and fivefold diluted samples ( Figure 5E,F), and their fast decline was observed within the next 15 min. In contrast to aqueous solutions, the degradation of substrates, cis-Fer-Gp/-IGp 5/5 , Fer-Gp/-IGp 6/6 , cis-Coum-Gp/-IGp 13/13 , Coum-Gp/-IGp 14/14 , and Sin-Gp/-IGp 20/20 was much faster especially for diluted samples, with no substrates being retained after 60 min of the experiment ( Figure 5F). The pair of Gp/-IGp 1/1 was more labile than acylated gomphrenins, reaching the greatest concentration after 5 min and degrading very quickly during the next 25 min of tea brewing ( Figure 5G,H).

Generation of Decarboxylated Derivatives of Gomphrenins in Aqueous Citric Acid Solutions and Their Time-Dependent Concentration Profiles
Similarly to aqueous tea samples, the pairs of diastereomers of Fer-, Coum-and Sin-17-dGp/-IGp (8/8 , 16/16 , 21/21 , respectively) were detected in citric acid tea infusions. An increase of their concentrations was observed during the first 30 min in the undiluted samples ( Figure 8B-D) with a subsequent slight decline, in contrast to their precursors ( Figure 5E). A similar effect was observed for diluted samples, however, in that case, the decline was observed after the first 15 min of tea brewing ( Figure 8B-D). Interestingly, cis-Fer-17-dGp 7 and cis-Coum-17-dG 15 were detected both in undiluted and diluted citric acid samples, whereas these compounds were not present in diluted aqueous samples ( Figure 6). For Fer-17-dGp 8 ( Figure 9A) and Coum-17-dGp 16 ( Figure 9D), a very strong increase of their time-dependent CR values in all samples extracted in citric acid solutions was observed. An increase of the CR ratio for Sin-17-dGp 21 ( Figure 9H) could be observed during the heating of citric acid undiluted samples, whereas in diluted samples, an increase of the ratio was noticed until 30 min, with a subsequent decrease at 60 min. These changes were not observed in aqueous solutions because natural gomphrenins were more stable which resulted in low and steady CR ratios. For 17-dGp 2 ( Figure 9F), an increase of the CR ratio was observed during the heating of the diluted samples. Other detected mono-decarboxylated derivatives, 17 and 12, were assigned to Coum-2-dGp and Fer-15-dGp, respectively. Their concentration profiles were the same as for Coum-17-dGp 16 and Fer-17-dGp 8, respectively, for undiluted and diluted samples ( Figure 8C). For Coum-2-dGp 17 ( Figure 9C) and Fer-15-dGp 12 ( Figure 9B), very similar signal CR ratios were observed to the CR for Coum-17-dGp 16 ( Figure 9D) and Fer-17-dGp 8 ( Figure 9F). Fer-2-dIGp 10 (m/z 683) was detected (Table 1), while this compound was not observed in aqueous samples (data not shown). The maximum signal intensity for 15-dGp 12 was observed after 5 min and 15 min of heating in undiluted and diluted aqueous solutions of citric acid, respectively. The Signal of Sin-15-dGp 24 started diminishing after 30 min and 15 min for undiluted and diluted samples, respectively ( Figure 8D). The CR ratios obtained for 17-dGp 2 and 2-dGp 4 ( Figures 9F and 9E, respectively) increased faster than the ratios for the acylated derivatives for all diluted samples. In contrast, 15-dGp 3 ( Figure 9G) was the most labile and was not present in citric acid solutions after 60 min of heating.

Studies on Model Acylated Gomphrenins Isolated from G. globosa Extract
For the aim of selective generation of simplified profiles of acylated gomphrenin derivatives for clear referencing of complex mixtures obtained during the tea brewing study, other heating experiments were performed on purified diastereomers isolated from G. globosa extract. These selected pigments were heated for 10-20 min at 90 • C only in aqueous citric acid solutions which were sufficient for providing clear comprehensive profiles of the referential derivatives and enabled finding the absorption maxima for most of the relevant chromatographically studied compounds. In a few cases, some derivatives generated in the model experiments were not detected in the tea brewing products. Selected chromatograms of the reaction mixtures obtained by heating of purified acylated gomphrenins registered at λ 500 nm are depicted in Figure 4C-E.  Table 1). The pair of 22/22 , previously not detected in the tea brewing products, was tentatively assigned in the heating products of Sin-Gp/-IGp (20/20 ) to sinapoyl-2-decarboxy-gomphrenin/-isogomphrenin (Sin-2-dGp/-dIGp), based on the absorption λ max 536 nm and the results of HRMS analyses which confirmed the molecular formula (measured m/z 713.2156 vs. calculated 713.2188, Table 2). LCMS-IT-TOF fragmentation yielded three ions of the same molecular formula, as obtained for the fragmentation ions of the isomeric compound Sin-17-dGp 21. 15-decarboxylated derivatives of all the acylated gomphrenins (Fer-15-dGp 12, Coum-15-dGp 19, and Sin-15-dGp 24) were tentatively detected in the heating products as well (Table 1).

Plant Material
Dried purple flowers of Gomphrena globosa L. were purchased from a China market.

Reagents
Formic acid, acetone, LC-MS grade methanol, and water were received from Sigma Chemical Co. (St. Louis, MO, USA). Citric acid was obtained from POCH (Gliwice, Poland).

Tea Brewing of Purple G. Globosa Flowers
To determine the influence of plant matrix on betacyanins' stability, two (0.940 g) or two fifths (0.188 g) of flowers were brewing at 90 • C in 6 mL of solvent (demi-water or 1% aqueous citric acid). Heated samples were collected subsequently for LC-DAD-ESI-MS analyses after 5, 10, 15, 30, and 60 min, respectively. All of the experiments were performed in triplicate and standard deviation (SD) was calculated. Previously isolated single diastereomers of acylated gomphrenins were used for additional heating experiments in a citric acid solution to preliminarily determine degradation products.

Preparation of Plant Material for Semi-Preparative Chromatography
The extraction of 1 kg of plant material was performed in a 1% aqueous solution of formic acid (v/v). The obtained extract was pumped through the 3 cm layer of a silica gel in a Büchner funnel to separate the solid particles and the suspension. Separation and purification of the extract proceeded onto the ion exchange bed of a strong anion exchanger Sepra TM ZT-SAX with a 30 µm pore size (Phenomenex, The collision energy was in the range of 12-50% independence on the structure of the compounds. The Formula Predictor within LCMS Solution software was used for the elaboration of results obtained in high-resolution mass spectrometry experiments (HRMS). Only an empirical formula with a mass error below 5 ppm was considered.

Conclusions
Herein, it is the first qualitative and quantitative report on gomphrenin derivatives formed during a tea brewing process in aqueous and aqueous citric acid solutions. The presence of gomphrenins in G. globosa flower extract may exhibit a positive impact on human health by regular drinking of its tea infusions. The rich profile of decarboxylated gomphrenins may contribute to their antioxidative and other pro-health activities; however, further research on the group of these biologically active compounds is still needed.