Multi-colored shades of betalains: recent advances in betacyanin chemistry

2021 Betacyanins cover a class of remarkable natural red-violet plant pigments with prospective chemical and biological properties for wide-ranging applications in food, pharmaceuticals, and the cosmetic industry. Betacyanins, forming the betalain pigment group together with yellow betaxanthins, have gained much attention due to the increasing social awareness of the positive impact of natural products on human health. Betalains are commercially recognized as natural food colorants with preliminarily ascertained, but to be further investigated, health-promoting properties. In addition, they exhibit a remarkable structural diversity based on glycosylated and acylated varieties. The main research directions for natural plant pigments are focused on their structure elucidation, methods of their separation and analysis, biological activities, bioavailability, factors a ﬀ ecting their stability, industrial applications as a plant-based food, natural colorants, drugs, and cosmetics as well as methods for high-yield production and stabilization. This review covers period of the last two decades of betacyanin research. In the ﬁ rst part of the review, we present an updated classi ﬁ cation of all known betacyanins and their derivatives identi ﬁ ed by chemical means as well as by mass spectrometric and NMR techniques. In the second part, we review the current research reports focused on the chemical properties of the pigments (decarboxylation, oxidation, conjugation, and chlorination reactions as well as the acyl group migration phenomenon) and describe the semi-synthesis of natural and arti ﬁ cial ﬂ uorescent betalamic acid conjugates, showing various prospective research directions.

One step semi-synthesis of betalains from betalamic acid-derivatized support 4. 3 Semi-synthetic betalain pigments with extended conjugated systems 4.3.1 Semi-synthesis of blue pigments from betanin 4.3.2 Semi-synthesis of a pseudo-natural betalain-nitrone OxiBeet pigment 4.3.3 Semi-synthesis of N-methyl phenyl-betaxanthin and Naryl phenyl-betaxanthin pigments 4.3.4 Semi-synthesis of articial coumarinic betalains 5 Conclusions 6 Conicts of interest 7 Acknowledgements 8 References form a group of betalain pigments. 1 Simultaneously with anthocyanins, carotenoids, and chlorophylls, betalains are one of the most common plant pigments found in nature. 2,3 These compounds share betalamic acid as the main chromogenic structural unit condensed with cyclo-DOPA, forming betanidin or glycosylated cyclo-DOPA in other betacyanins as well as different amino acids or amines in betaxanthins. 4,5 So far, betacyanins (Table 1) have been assigned to four structural groups comprising betanin-type, gomphrenin-type, amaranthin-type, and Bougainvillea-r-I-type, 6,7 the latter was recently renamed as melocactin-type. 8 However, for a convenient view on a wide spectrum of pigments, an expanded division with three additional betacyanin groups is proposed based on the sugar linkage: oleracin-type 9 (a very early result, not indepedently conrmed), apiocactin-type, [10][11][12] and Bougainvillea-v-type (here, we propose to name it as glabranin-type). In particular, the latter group of the pigments with the structures based on gomphrenin is important in the division system not only because of its less common 6-O-glycosylation pattern but also because of its unique and extremely complex prole of ca. 146 betacyanins tentatively detected in only one plant species, Bougainvillea glabra Choisy, 11 including nine denitively iden-tied betacyanins by LC-ESI-MS and NMR. 13 The schematic division of betacyanins into seven main structural groups based on their chemical structures is shown in Fig. 1. Betacyanins assigned to particular types differ in the attachment of the glucosyl moiety to the oxygen atom in the ortho position of cyclo-DOPA as well as the position of the substitution with additional glycosyl or glucuronosyl moieties. 6 The esterication of sugar moieties with organic acids such as ferulic, pcoumaric, caffeic, sinapic, and malonic acids is very common and leads to the formation of various acylated derivatives. The 15R diastereomers (the isoforms, epimers) of betacyanins are found in plants at much lower concentrations than the 15S forms and are regarded rather as artifacts due to epimerization, which takes place during the preparation of extracted plant samples. 14 The rst studies on isomerization (as well as decarboxylation) mechanism in betacyanins were performed by Dunkelblum et al. 15 In more recent reports, the chromatographic differences in the 15S/15R diastereomers of decarboxylated betacyanins 16,17 as well as in the acyl migration products 18 were investigated.
Betanin (betanidin 5-O-b-D-glucopyranoside) is the main representative and most common betacyanin pigment found in the plant kingdom ( Fig. 1 and 2A). In addition, betanin isolated from Beta vulgaris L. (beetroot, red beet) is the most studied pigment in the context of the antioxidant properties of betacyanins. Structurally, betanin and most of its derivatives are composed of aglycone (betanidin) linked by the b-glucosidic bond with the glucose unit at the C-5 carbon atom. 19,20 The chemical structures of additional compounds belonging to betanin-type betacyanins are shown in Fig. 2

. Such compounds share
TomaszŚwiergosz is a researcher at the Department of Chemical Technology and Environmental Analysis, Faculty of Chemical Engineering and Technology (Cracow University of Technology, Poland). His research interests include natural compounds, active organic and macromolecular materials, in particular, supramolecular chemistry of functional dyes and p-conjugated systems, molecular self-assemblies, nanostructures and molecular probes, synthesis and characterization of polymers, photoinitiators for cationic polymerization processes and the investigation of their effectiveness and efficiency in photopolymerization, along with the synthesis of uorescent compounds as uorescent probes for special applications. Katarzyna Sutor is a second year PhD student at the Cracow University of Technology (Faculty of Chemical Engineering and Technology). She graduated from MA studies in the eld of biotechnology. During her BSc and MSc studies, she focused on the inuence of reactive oxygen species on the properties of cancer cells studied by AFM and uorescence microscopy. This research was carried out in cooperation with the Department of Biophysical Microstructures, H. Niewodniczański Institute of Nuclear Physics of Polish Academy of Sciences (Cracow, Poland), where she was on internship. Currently, she focuses on the chemical and bioactive properties of acylated betacyanins from plant sources. Agnieszka Kumorkiewicz-Jamro received her PhD in 2021 from AGH University of Science and Technology (Cracow, Poland). She graduated with a BSc in the eld of biotechnology and MSc in analytical chemistry. She is a research and teaching assistant at the Cracow University of Technology. Her main research interests are in phytochemical analysis, and chromatographic methods for natural compounds' separation, purication, and identication. Her current work focuses on (bio)chemical research on betalain pigments isolated from versatile plant sources. a common betanidin backbone in their structures with an additional glucose moiety (betanin) along with (3-methyl-3-hydroxymethyl)glutaryl-, malonyl-, E-4-coumaroyl-, and E-feruloylmoieties as well as the half sulphate ester in hylocerenin, phyllocactin, lampranthin I, lampranthin II, and prebetanin, respectively, but can be also partially oxidized (neobetanin) (Fig. 2).
Gomphrenin (betanidin 6-O-b-D-glucopyranoside), which is the isomeric pigment to betanin, is found at high concentrations in Basella alba L. fruits as well as in the leaves of its variety Basella alba var. 'Rubra' L. 21,22 According to our recent recommendation, we propose to rename gomphrenin I as "gomphrenin" in order to simplify the naming of its derivatives, especially generated by gomphrenin decarboxylation and oxidation. 16 In contrast to betanins, gomphrenins are characterized by the presence of a glucosyl moiety attached at the carbon C-6 ( Fig. 1 and 3A). 21,22 Furthermore, the acylation of gomphrenin enables the formation of its derivatives such as E-4-coumaroyl-gomphrenin (former gomphrenin II, globosin), Eferuloyl-gomphrenin (former gomphrenin III, basellin), and Esinapoyl-gomphrenin (gomphrenin IV, gandolin) ( Table 1, Fig. 3B-D). 12 Gomphrenin pigments are of interest because their glucosylation position at the carbon C-6 should promote an interaction of the acylated residues with the carboxyl groups by increasing the intramolecular stabilization (by intramolecular stacking) of globosin and basellin 23 or other types of multiple acylated sophorosyl residues observed in glabranins. Betacyanins were also detected in red and purple Gomphrena globosa L. inorescences. The most dominant betacyanins present in red G. globosa cultivars are amaranthin and celosianin but in violet species, gomphrenin and especially the acylated derivatives, globosin and basellin, are present. 24,25 In addition, other betacyanins were initially identied, namely, Z-4-coumaroyl-gomphrenin and Z-feruloyl-gomphrenin isomers as well as gandolin. 25,26 Plant pigments belonging to the amaranthin-type group have a characteristic glucuronosylglucosyl moiety attached to the carbon C-5 in their structure. The most common example of these pigments is amaranthin [betanidin 5-O-b-(2 0 -O-b-glucuronosyl)glucoside] (Fig. 4A) isolated from plants of the Amaranthaceae family. 27,28 The other frequently detected pigments in the plants are betanin and 6 0 -O-malonyl-amaranthin. 29 Furthermore, sinapoyl-amaranthin has been tentatively identi-ed in G. globosa petals. 30 Iresinin I [6 0 -O-(3 00 -hydroxy-3 00 -methyl) glutaryl-2 0 -O-glucuronosyl-betanin] and amaranthin are the most abundant pigments detected in Iresine herbstii Hook. ex Lindl leaves (Table 1). 27 Acylated amaranthin-based pigments, argentianin and celosianin, were also found in Celosia species 31 and the purple leaves of I. herbstii (Fig. 4). 25 Most of the pigments from the two Bougainvillea groups have sophorosyl moieties linked to carbons C-5 or C-6 of the basic skeleton of betanidin, expanded further by glucosyl, rhamnosyl, apiosyl, or xylosyl moieties. 13,26,[32][33][34][35] A complex mixture of betacyanins that differs in acyl-oligoglycoside units and exists mostly in the 6-O-glycosylated forms of betanidin was initially identied in the purple bracts of B. glabra. 13 However, due to the complex mixture of a huge number of betacyanins present in B. glabra, 34 its prole has not been completely characterized. The examples of bougainvillein-type betacyanins are bougainvilleinr-I (melocactin-type) and bougainvillein-v (glabranin-type) ( Table 1, Fig. 5A and B). Similar betacyanins were recently found within Melocactus species. 8 The most abundant pigment, melocactin, previously named 'bougainvillein-r-I', was identi-ed as betanidin 5-O-b-sophoroside (Fig. 5A). The presence of feruloylated and sinapoylated melocactins as well as melocactin's malonylated derivative, mammillarinin, was also noted (Fig. 5C). 8 In some Mammillaria species, mammillarinin [betanidin 5-O-(6 0 -O-malonyl)-b-sophoroside] was reported as the dominant pigment. 36 Two red-violet acylated betacyanins, oleracins I and II, have been found in Portulaca oleracea L. Upon hydrolysis in alkaline conditions, oleracins gave ferulic acid and two newly detected pigments that were identied as 5-O-b-cellobiosides of  (1998)(1999)(2000) at the Institutes for Applied Research-Ben-Gurion University of the Negev (Beer-Sheva, Israel), he started research on betalain pigments, which he has continued at the Cracow University of Technology to date, exploring the chemistry and bioactivity of betalains.     betanidin and isobetanidin (Fig. 1E). 9 Five other yellow oleracins were also detected in P. oleracea; however, in contrast to oleracins I and II, they did not possess betalamic acid in their structures. 37 Betacyanins belonging to the apiocactin-type group contain the rare branched pentose, apiose, bound to the carbon C-2 0 of betanin. Pigments such as 2 0 -O-b-apiosyl-betanin (apiocactin) and/or its acylated derivatives ( Table 1, Fig. 6A-C) have been found in fruits of pokeberry and its corresponding suspension and callus cultures (Phytolacca americana L.), Christmas cactus owers (Schlumbergera x buckleyi), and Hylocereus species. 10,11,38 The chemical structure of the basic apiocactin-type betacyanin present in Hylocereus species was conrmed as 2 0 -O-b-apiosylbetanin by Wybraniec et al. 11 In addition, in the same study, acylated compounds with sinapoyl and feruloyl residues attached to C-5 00 of the apiose moiety were identied as 5 00 -O-Esinapoyl-apiocactin and 5 00 -O-E-feruloyl-apiocactin. 11 Similarly, 5 00 -O-E-feruloyl-apiocactin was identied earlier in P. americana cultures by Schliemann et al. 10 Another betacyanin, 2 0 -O-b-(5 00 -O-E-feruloyl)-apiosyl-phyllocactin, which is the rst betacyanin example containing both an aliphatic and an aromatic acyl residue, was tentatively detected in Christmas cactus ower extracts together with other apiocactin derivatives 38 and later in Hylocereus species. 11 2 Characterization of betacyanins

Sources of betacyanin pigments
There are several known edible sources of betacyanins (Table 1). These include red and yellow beetroots (B. vulgaris ssp. vulgaris), 1 B. alba fruits, 39 G. globosa owers, 13,30 grainy amaranth (Amaranthus sp.), 27 grains of Chenopodium quinoa, 40 leaves of A. hortensis var. rubra, 41 and fruits/owers of cacti genera (Opuntia, Hylocereus, Mammillaria, Melocactus, and Myrtillocactus spec.). 8,36,[42][43][44][45] Pokeberry (Phytolacca americana L.) is another source of betacyanins but it has been forbidden as a food colorant due to the presence of toxic saponins and lectins. 46 Compounds tentatively identied as betacyanin-like are also found in some higher fungi such as Amanita muscaria (y agaric). 70,71 The less common edible sources are Ulluco tubers (Ullucus tuberosus) 64,72 as well as fruits and berries of pigeonberry (Rivina humilis). 73 Table 1 presents a detailed summary of the identied betacyanins in different plant sources along with the methods of their identication. Most of the chemical methods were applied in the early decades of betalain research based on the typical reactions of hydrolysis and derivatization, such as permethylation and partial degradation, resulting in the generation of diagnostic products, which can be identied and can conrm certain parts of the chemical structure of a starting compound. The chemical structures of 31 naturally occurring betacyanins were denitively identied by NMR methods, whereas a huge group of over 187 betacyanins (including pigments from their richest source, B. glabra) were detected by LC-MS techniques and still await the nal conrmation. In addition, structures of 18 betacyanin derivatives were also     conrmed by NMR and 61 tentatively were identied by LC-MS methods (Fig. 7).
Furthermore, it was noted that betacyanins protect low-density lipoproteins (LDL) against oxidative damage by reacting with the LDL polar groups. 78 In addition, betanin present in red beets prevents DNA from damage in lymphocytes and hepatocytes. 79 Betanin also shows neuroprotective effects, improves cognitive functions and reduces oxidative stress caused by D-galactose in the brain of mice by increasing the level of antioxidant enzymes and reducing lipid peroxidation. 80 Some betacyanin pigments have been shown to have higher antioxidant activity compared to typical natural antioxidants such as ascorbic acid, 81 rutin, 82 catechin, 83 b-carotene, 84 and atocopherol. 6,85 In a test of the antioxidant activity expressed in TROLOX equivalents, betacyanins extracted from red beet exhibited 1.5-2.0 times higher free radical scavenging activity than some anthocyanins such as cyanidine-3-O-glucoside and cyanidine above pH 4. 86,87 The effect of the hydroxyl group position on the antioxidant activity of the betacyanins bearing the conjugated phenol moiety was studied. Three non-natural regioisomeric phenolic betacyanins (common name: o-, m-, p-OH-pBeets) were semisynthesized by coupling betalamic acid with appropriate aminophenols in water according to a procedure described by Schliemann and co-authors 88 and their antioxidant activity was studied. 89,90 The results showed that the meta isomer exhibits greater antiradical activity than most of the betalains, avonoids, and anthocyanins. This result may be explained by the fact that the phenolic moiety is not conjugated with the diazapolymethine system but both groups are prone to further oxidation and can stabilize the radicals by resonance. In addition, the N-H imine bond present in the meta regioisomeric structure is the preferred site for oxidation, whereas 1e À oxidation of the phenolic groups in p-and o-OH-pBeet results in the formation of semi-quinone and leads to lower values of the potential energy (Ep) compared to m-OH-pBeet. 89  Betacyanins have been shown to actively participate in free radical scavenging and consequently, their consumption may lead to chemoprevention and delay of the development of cancer tissues. 75 Puried betanin shows strong inhibition of melanoma cancer cell proliferation 91 and excellent growth inhibition of MCF-7 (breast), HCT-116 (colon), AGS (stomach), SF-268 (CNS), and NCI-H460 (lungs) cancer cell lines with IC 50 values of 162, 142, 158, 164, and 147 mg mL À1 , respectively. 92 It has also been found that betanin induces dose-and timedependent apoptosis of cells in the human chronic myelogenous leukemia (K562) cell line. 93 In vitro experiments revealed that betalains extracted from B. vulgaris juice show pro-apoptotic effects on activated neutrophils and inhibit the neutrophil oxidative metabolism. 94 In addition, pilot clinical trials have shown that short-term treatment with red beet extract improves joint function in people with knee joint discomfort. 76,95

Applications of betacyanin pigments
In Fig. 8, betacyanin applications are schematically presented. Natural plant extracts containing betacyanin pigments are increasingly used in food technology as a safe alternative to synthetic food colorants. According to Title 21 of the Code of  Union, beetroot powder rich in betanin is permitted as a natural red food colorant. 3 Food preparations obtained in different industrial conditions can signicantly vary in their stability and coloration due to the presence and the composition of betacyanin degradation products. Betalains are stable at pH ranging from 3 to 7 and they are suitable for dyeing low acidic and neutral foods. In addition, they may be stabilized by ascorbic acid. In contrast, due to instability at pH over 3, the application of anthocyanins in the coloration of such foods is not possible. Furthermore, the use of ascorbic acid also facilitates the degradation of anthocyanins. For this reason, the utilization of betalain pigments instead of anthocyanins for coloring food with a high amount of vitamin C or vitamin C-supplemented products seems to be more favorable. 96,97 Beetroot extracts are utilized to emphasize the redness of dairy products such as tomato soups, sauces, pastes, desserts, jams, sweets, and jelly beans. They are also used to protect meat from discoloration and to extend its shelf-life. 98 However, due to the unpleasant avor of beetroot extracts due to their geosmin and pyrazine derivatives content, a membrane process during juice concentration is needed to be applied for commercial red beet application. 14 Due to health-related benets, betalains are also regarded as natural dietary supplements. 99 The effect of food supplements containing betalain-rich extracts (a betalain-rich supplement of red beetroot and a betacyanin-rich supplement of Opuntia stricta) on different atherosclerotic risk factors were tested among coronary artery disease patients. The results showed that the levels of glucose, total cholesterol, homocysteine, triglyceride, and low-density lipoprotein (LDL) of 48 male patients were decreased. Furthermore, betalain-rich supplements taken at a safe dose of 50 mg betalain/betacyanins for 2 week interventions reduced the systolic and diastolic blood pressures. 100 In the past 20 years, a number of food and nutraceutical preparations have been developed from quinoa (Chenopodium quinoa Willd.) as well. Several clinical studies have demonstrated that quinoa supplementation exerts prominent effects on the cardiovascular, gastrointestinal, and metabolic health of humans. 101 Natural plant pigments were also tested in the context of solar cell applications as a cheaper, faster, low energy, and environment-friendly alternative to dyes based on ruthenium complexes. 102,103 Betanin, 2,17-bidecarboxy-betanin, vulgaxanthin I, extracted from red beetroot extract and betanidin-6-O-(6 0 ,6 00 -di-O-E-4-coumaroyl)-b-sophoroside from Bougainvillea were also tested toward the application as dye-sensitized solar cells (DSSC). The construction of betalain-based DSSC is possible due to the carboxylic groups in betalains, which serve as anchors aer the immersion of TiO 2 electrodes in betalain aqueous solutions at pH z 3. The quantum yield of electron injection in betacyanin-based and betaxanthin-based DSSC is about 50% and 70%, respectively. In the case of betaxanthinbased DSSC, the total sunlight conversion efficiency was however smaller because of the blue-shied absorption band. 104 The electron injection from betanin to TiO 2 is a two-electron, one-proton process. 105,106 Enhanced light-harvesting and the photoconversion efficiency of nanocrystalline TiO 2 sensitized with betanin was observed and revealed the self-assembly of betanin on the surface. Due to the fact that an aggregated betanin sensitizer improved the performance in DSSC, the mechanism of improved electron injection and collection in DSSCs with more aggregation as compared to monomeric betanin need to be regarded. 107 Betanin is capable of injecting up to two electrons per photon absorbed into the ZnO conduction band in less than 15 ps. 108 Betanin was also used for the light-harvesting process in ultra-stable ZnO nanocrystals modied with a carboxylate oligoethylene glycol shell system studied for utilization in H 2 production under visible light irradiation. 109 The photophysical properties of betanin in aqueous and alcoholic solutions were determined and formation of betanin electronically excited species was studied. Betanin has a short S 1 state lifetime (p, p*) (6.4 ps in water), mainly determined by the efficient S 1 / S 0 radiationless relaxation, which probably requires a strong geometry change. Other processes, such as photoproduct formation or S 1 / T 1 intersystem crossing have  been reported as virtually absent. In the absence of triplet excited state formation, the fast light-to-heat conversion was observed, supporting the consideration that betanin is a photoprotector in vivo. 110,111 Due to the presence of the phenolic moiety within the betanin structure, which may react with 1 O 2 , the colorimetric detection of reactive oxygen species is possible. Efficient singlet oxygen quenching by betanin in deuterated water was reported. The capacity of betanin to quench 1 O 2 supports the photoprotective role of betanin in vivo. 112 In another study, a red beetroot pigment-based colorimetric sensor for detecting copper ions in drinking water was developed. In addition, an application for quantitative and visual colorimetric analysis was designed for the Android system smartphone. When Cu 2+ concentration in drinking water increased, the red beet pigment solution gradually changed from bright purple to orange-red as it formed a complex by chelating Cu 2+ . The linear range of this detection system ranged from 4 to 20 mM and the limit of detection was 0.84 mM. 113 Betanin can also compete with synthetic dyes in terms of the depth and color stability. The possibility of dying of modied acrylic fabrics with betanin was investigated. It was found that the optimal conditions during 45 min dyeing of acrylic fabrics with betanin were 50 C and pH 5. The modied materials were resistant to color loss under the inuence of water and the addition of cobalt(II) sulphate(VI) (CoSO 4 ) ensured light resistance. 114 Attempts are being made to develop cheap, green, and easyto-use betalain-based biosensors. A good example is a need for the early detection of Bacillus species. The highly stable and virulent B. anthracis has been considered as a dangerous bioterrorism agent aer the anthrax attack took place in 2001. 115 Another threat is pathogenic B. cereus that grows on food. 116 In order to detect the Bacillus species early, research on the application of betanin as a ligand in the new europium(III) complex, sensitive to calcium dipicolinate, CaDPA (the major component of the bacterial spore core) was conducted. In the presence of Eu(III) ions, betanin was converted into a watersoluble, non-luminescent orange complex. The addition of CaDPA changes the orange color of the [Eu(betanin) + ] aqueous solution to magenta. The limit of detection of CaDPA is about 2.06 Â 10 À6 mol L À1 . In addition, the complex is sensitive to CaDPA but not to other structurally similar aromatic, pyridinic, and acidic ligands. 117,118 Beetroot extracts are under consideration as a mild alternative to organometallic reductants, such as NaBH 4 and N 2 H 4 , and as stabilizing (growth-limiting) agents. Beetroot extract was exploited for the bottom-up synthesis of metal nanoparticles, leading to broad size and shape distributions. 119 3 Chemistry of betacyanins and betalamic acid derivatives

Overview of betacyanin chemical reactions
Taking into account the pro-health nature of betacyanin pigments, their diversity, structural characteristics, and lightabsorption properties, there is a growing need to supplement and broaden the knowledge on their chemistry. Understanding the reactions that betacyanins undergo may contribute to the determination of their fate and distribution in the human body, increase their stability, the development of betalain-based biosensors, screening assays and tailor-made probes, the preparation of betalain-based potential medicinal substances or cosmetics. In the following part of this contribution, we sum up the current knowledge obtained during the last decade on the chemical properties of betacyanins as well as new perspectives of the semi-synthesis of betalamic acid derivatives with extended chromophoric systems. The list of betacyanin derivatives semi-synthesized from plant sources and betacyanin degradation or chemical conversion products, with their structural identication by LC-MS and NMR methods, is presented in Table 2.
3.1.1 Thermal decarboxylation and dehydrogenation of betacyanins. Considering the signicant impact of organoleptic characteristics on food selection and consumer acceptance, color is one of the most important attributes of foods as it is considered as a quality indicator. 120,121 Nowadays, the complex processing of most foodstuffs may cause some unwanted changes in their visual appearance, contributing to the fading  effect in their coloration. During the degradation of betacyanins, they may undergo deglucosylation, decarboxylation, dehydrogenation, and isomerization processes (Fig. 9). The rst heat-degradation structural studies on betanin from B. vulgaris, as well as betacyanins isolated from H. polyrhizus (betanin, phyllocactin, and hylocerenin), were performed by Herbach et al. [122][123][124] and Wybraniec et al. [125][126][127] Betanin was proven to be the most stable pigment, while the stability of phyllocactin and hylocerenin was seemingly enhanced due to the formation of red degradation products, exhibiting improved color retention in comparison to their precursors. Betanin degradation proceeded through hydrolytic cleavage, while hylocerenin dominated in decarboxylation and dehydrogenation. The degradation of phyllocactin involves the decarboxylation of the malonic acid moiety and the formation of betanin by demalonylation along with subsequent betanin degradation. Aer prolonged heating, dehydrogenation at C2-C3 carbons was also noticed. [123][124][125]127 Decarboxylated and dehydrogenated betanin derivatives occurring at the highest concentrations in a betacyanin-rich red beetroot extract (RBE) were also characterized ( Table 2). 128 The main pigments in the RBE are betanin and its isoform with 17decarboxy-betanin/-isobetanin, 15-decarboxy-betanin, and small quantities of 2-decarboxy-betanin/-isobetanin as well as 2,17-bidecarboxy-betanin/-isobetanin. 128 Furthermore, mixtures of mono-, bi-, and tridecarboxylated betanins together with their corresponding neo-derivatives obtained from the RBE were identied as the heating degradation products. 129 In addition, new isomeric decarboxylated derivatives, namely, 2,15-bidecarboxy-betanin and 15,17-bidecarboxy-betanin, were detected for the rst time. These newly detected derivatives were also formed during heating experiments on red beetroot extract. Furthermore, the presence of 2,15,17-tridecarboxy-betanin was also acknowledged. 129 Presumably, the presence of these additional betanin derivatives results from enhanced decarboxylation, which is an integral process during RBE preparation. It was also reported that the higher concentration of acetic acid during the thermal treatment of RBE contributes to a selective formation of 2-decarboxy-betanin/-isobetanin. The latter pigments are present in the RBE at a very low level. However, according to the report, the most decisive factors are the concentrations of the substrate and acetic acid. Higher RBE concentration favors the formation of 2,17-bidecarboxybetanin/-isobetanin over 2,15-bidecarboxy-betanin. 129 An effective method for the separation of newly formed derivatives was also proposed. A bunch of decarboxylated and dehydrogenated betacyanins obtained during mild thermal treatment of B. vulgaris juice was subjected to high-performance counter-current chromatographic (HPCCC) preparative fractionations. 130 The mixtures composed of betacyanins with different polarity and physicochemical properties were separated in highly polar solvent systems containing a high concentration of ammonium sulfate and ion-pairs, aqueousorganic solvent systems including ion-pair reagents (triuoroacetic acid, heptauorobutyric acid). The application of both the solvent systems enabled the separation and purication of 2-decarboxy-betanin, 2,17-bidecarboxy-betanin, their corresponding isoforms, and neobetanin from a mixture composed mainly of betacyanins, neobetanin, and their decarboxylated derivatives (Table 2), as well as 17-decarboxyneobetanin, 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2,17-bidecarboxy-2,3-dehydro-neobetanin, 2,15,17tridecarboxy-neobetanin, and 2-decarboxy-neobetanin from another mixture consisting of decarboxy-and dehydrobetacyanins. The results also show that the utilization of heptauorobutyric acid as an ion-pair reagent is more suitable than a polar solvent system with triuoroacetic acid because it contributes to the formation of more hydrophobic betacyanin ion-pairs. 130 The separation of betacyanins from a processed B. vulgaris juice (betanin, isobetanin, neobetanin, and decarboxylated betacyanins) was also demonstrated in a food-grade, gradient solvent system consisting of sodium chloride, butanol, water, as well as different volumes of phosphoric acid and/or ethanol. The quality of isolation was dependent on the ethanol and acid concentrations with the lowest volume gradient, providing the best separation conditions. Betacyanins were eluted in the organic, upper mobile phase, which has a low salt content compared to the aqueous lower phase. 131 The rst study on the thermal decomposition of gomphrenin pigments present in the fruit juice of B. alba was carried out and the chromatographic proles, as well as fragmentation pathways of decarboxylation and dehydrogenation products of gomphrenin (Table 2), were determined by LC-DAD-ESI-MS/MS and LC-MS-IT-TOF. 16 The short-term treatment of B. alba juice revealed the proles of mono-and bi-decarboxylated gomphrenins analogous to betacyanins from heated beetroot juice.
Prolonged heating of B. alba extract acidied with acetic acid led to the formation of dehydrogenated derivatives as a result of gomphrenin derivative oxidation. The presence of neogomphrenin was detected at a low concentration level in the heated juice, which is opposite to RBE rich in neobetanin. In addition, in contrast to the results of the RBE heating experiments, 129 the isomeric 2,15and 15,17-decarboxylated derivatives were not detected in the heated samples. The above experiments indicated that the chromatographic differences between gomphrenin and betanin derivatives arose from the position of glucosylation in betanidin at carbon atoms C-5 or C-6. 16 Sawicki and Wiczkowski investigated the impact of boiling and spontaneous fermentation on the betalain prole and content in beetroot. The boiling and fermentation of beetroot decreased the content of the betalains by 51-61% and 61-88%, respectively. Processes occurring during boiling and spontaneous fermentation such as heating, soening, leaching, and acidication together with the matrix effect may be responsible for the changes. In addition, the microbial activity and matrix soening occurring during fermentation caused a release of betalains, which is responsible for the potent antioxidant capacity of the formed beet juice. 132 Sawicki et al. determined the impact of three technological processes (fermentation, boiling, and microwavevacuuming treatment) and in vitro digestion on betalain proles. The content of betalains in the products was reduced by 42-70%. Microwave-vacuuming treatment has contributed to the degradation of betanin at the lowest level and may be regarded as the best treatment method. In addition, the content of betalains released from the beetroot products aer in vitro digestion reached the range of 0.001-0.10%, and the number of compounds identied in the digestion phases was decreased. 133 3.1.2. Oxidation of betacyanin pigments. Preliminary studies on the enzymatic and non-enzymatic oxidation of betanidin, betanin, neobetanin, as well as decarboxylated betanins along with the research on intermediate and nal reaction products, were carried out. [134][135][136] Betanidin is the basic structure of all the betacyanins and the only one with the catechol moiety (5,6-dihydroxyl moiety). Studies show that during the oxidation of betanidin, the pigment is most likely converted into three tautomeric quinoid derivatives (Fig. 10). Interconvertions, which occur slowly at pH 4-7 and quickly in a more acidic environment, are likely to result in the formation of dehydrogenated and decarboxylated derivatives. Interconversions between the three tautomeric quinoid forms of oxidized betanidin are based on the proven oxidation pathways of DOPA and dopamine. 137,138 The initial oxidation of betanidin should generate the transient radical cation, which rapidly loses a proton and forms a neutral phenoxy radical; it is equivalent to the donation of a hydrogen atom by betanidin or to the loss of a proton with further electron transfer. In the next step, the loss of a second proton and electron results in the formation of a two-electron  oxidized form, i.e., one of the three quinoid tautomers. Presumably, not only the betanidin o-quinone with its characteristic absorption spectrum (l max at 550 and 400 nm) is formed on the rst oxidation stage but it is assumed that the two other quinoid derivatives are also possible: dopamine derivative and/ or quinone methide. The discussion of the balance between the three tautomeric forms of quinoid remains open. 135 As a result, 2,17-bidecarboxy-xanbetanidin (synonym of 2,17-bidecarboxy-2,3-dehydro-betanidin) and 2-decarboxy-xanbetanidin (synonym of 2-decarboxy-2,3-dehydro-betanidin) are postulated as the principal oxidation products formed ( Table 2). The recently proposed xan is derived from the hypsochromic shi toward the yellow color of the resulting compounds.
The further oxidation of 2,17-bidecarboxy-xanbetanidin again involves the formation of one of the two possible quinoid forms as the oxidized intermediates, e.g., the dopachromic derivative of the indolic system. Subsequent rearrangement of the conjugated system of the originated structures into the neo-forms (14,15-dehydrogenated derivatives) is possible which nally leads to 2,17-bidecarboxyxanneobetanidin (2,17-bidecarboxy-2,3-dehydro-neobetanidin), i.e., a compound doubly dehydrogenated at the positions C-2,3 and C-14,15. [134][135][136] In contrast to betanidin oxidation, no compound that could be regarded as the betanin oxidation intermediate (quinone methide) was detected. 139 This could be a consequence of its instability and fast rearrangement combined with decarboxylation. The main compound formed during the rst step of betanin oxidation is 2-decarboxy-xanbetanin (Table 2), which exhibits a characteristic protonated molecular ion [M + H] + at m/ z 505 and an absorption maximum at l max ¼ 446 nm (Fig. 11). It is not excluded, however, that the generated 2-decarboxyxanbetanin rearranges to a more stable structure of 2decarboxy-neobetanin (synonym of 2-decarboxy-14,15-dehydrobetanin). According to the alternative pathway proposed by Wybraniec et al., 135 the formation of 2-decarboxy-neobetanin can result from a rearrangement of the quinone methide generated from 2-decarboxy-betanin. This alternate pathway does not include the presumably more stable product, 2decarboxy-xanbetanin. Further, the decarboxylation of 2decarboxy-xanbetanin presumably results in the formation of 2,17-bidecarboxy-xanbetanin. The most hydrophobic product of betanin degradation/oxidation is 2-decarboxy-xanneobetanin, characterized by a protonated molecular ion at m/z 503 and maximum absorption at l max 422 nm, followed by 2,15,17-tridecarboxy-xanneobetanin.
In Fig. 12, the schematic formation of dehydrogenated betanin derivatives is presented along with 2,3-dehydro-and 14,15-dehydro-positions marked on particular pigment structures, together with their abbreviations (xan-and neo-, respectively). 52 In the case of gomphrenin oxidation by the ABTS cation radical, except for its decarboxylated and dehydrogenated derivatives, the formation of betanidin as well as betanidin derivatives was detected. It is assumed that the rearrangement of dopachromic derivative affects the hydrolysis of the glucosidic bond. This is opposite to betanin, which is not deglucosylated during oxidation in the same reaction conditions. Therefore, it is important to notice that the position of betanidin glucosylation at carbon C-5 or C-6 has a signicant inuence on the differences in the reactivity of both the 5-O-and 6-O-glucosides. 140 It was reported that metal cations, e.g., copper, aluminum, tin, and iron, accelerate the degradation of betanin even at trace amounts. It is worth noting that the release of metals from food packaging materials may also catalyze the unwanted betacyanin degradation. 141 The results of high-resolution mass spectrometry experiments (HRMS) and NMR structural studies on key dehydrogenation products generated during the oxidation of betanin, neobetanin, and decarboxylated derivatives by ABTS cation radical as well as by Cu 2+ complexation conrmed that Cu 2+ catalyzed oxidation of betanin and 2-decarboxy-betanin leads to the formation of neo-derivatives (14,15-dehydrogenated betanins). It has been proven for the rst time that betanin oxidation is possible in the dihydropyridine ring and this can be achieved presumably by omitting the stage of quinone methide formation in the dihydroindole system (Fig. 13). The additional results obtained for Cu 2+ -catalyzed oxidation of 17-decarboxy-betanin and 2,17-bidecarboxybetanin suggest the possibility of formation of xan-derivatives, similar to the results obtained for oxidation by the ABTS cation radical. In this case, the factor determining the oxidation direction is presumably the absence of the carboxyl moiety at carbon C-17, clearly changing the geometry of the complex formed with Cu 2+ . In addition, the exclusive effect of 2,3-dehydrogenation in 17-decarboxy-betanin is probably supported by the concurrent occurrence of decarboxylation at carbon C-2. 52 3.1.3 Conjugation of oxidized betacyanin pigments with sulydryl scavengers. A well-developed approach for the direct detection of reactive, short-lived intermediates generated during the oxidation processes is to utilize trapping agents capable of forming stable adducts with the reactive species. The adducts may be detected and characterized using LC-MS/MS and NMR techniques. 142 There are many reports on the formation of adducts between nucleophilic thiols and a variety of compounds. [143][144][145][146][147][148] Research on the conjugation of quinones generated during betacyanin oxidation in the presence of thiols seems essential and may broaden the knowledge on betacyanin functions in biological systems.
The rst studies on the possibility of conjugation of betacyanin reactive quinoids with sulydryl scavengers such as glutathione (GSH), cysteine (Cys), N-acetylcysteine (NAC), cysteamine (CSH), and DL-ditiothreitol (DTT) were performed recently ( Table 2, Fig. 14). 140,149 The formation of glutathionylated quinoids, following betanidin and gomphrenin oxidation, conrmed the presence of quinoid forms in the products of pigment oxidation. The chemical structure of glutathionylated betanidin was conrmed by NMR studies. It was also reported that glutathione reacts with quinones at the C-4 carbon of the betanidin ring. On the contrary, the conjugation of the betanin quinoid intermediate (quinone methide) with glutathione was not observed, presumably because of a steric hindrance at the C-7 carbon of betanin, which presumably conrms that the quinoid form generated during betanin oxidation is a quinone methide. 140 In order to determine the possibility of attaching thiols to the C-7 carbon of the betanin quinoid, additional research was performed using low molecular weight thiols (Cys, CSH, NAC, DTT). Furthermore, since the conjugation of betacyanins with the thiol groups may generate new molecules with the modied chemical and biological properties, the conjugation reactions with quinones derived from betanidin and gomphrenin were also performed (Fig. 14). The chemical structure of Nacetylcysteinyl-gomphrenin, generated with the highest yield, was determined by the NMR method. The dopachrome derivative from gomphrenin oxidation undergoes conjugation at the C-4 carbon atom. In addition, cysteine conjugate with the oxidized betanin derivative was produced for the rst time. The results suggest that the formation of quinone methide during the oxidation of betanin and the production of dopachrome derivative during the oxidation of gomphrenin is highly possible. 149 The results conrm so far that gomphrenin is able to form conjugates with a higher yield than betanin but the ability to form stable conjugates with dehydrogenated betanin derivatives produced during the oxidation of betanin or its intermediate conjugates may also favor this pigment as a valuable food protection component. 149 From the perspective of the food industry, this aspect appears to be relevant as the biological activity of gomphrenin and betanin in combination with the possibility of adduct formation between the thiols and betacyanin quinoids may contribute to the increased quality and durability of food products such as meat. 146 From another perspective, adduct research can signicantly contribute to the development of detection methods for betacyanin quinoids formed in the complex food matrices as well as to research the bioavailability and distribution of betacyanins in humans. The sulydryl part of the molecules may modulate their ability to penetrate the biological membranes as well as their absorption and metabolism. Many attempts have been made in the food chemistry to inhibit the formation of oxidative cross-linking proteins, affecting the quality, water content, and red color of meat products. In this respect, CS-conjugates of betacyanins may play a signicant role in blocking the thiol groups in food proteins, contributing to improving the food quality. [150][151][152] Whether betacyanins act as effective antioxidants without adverse pro-oxidative effects depends on how quickly their oxidized derivatives gradually degrade through decarboxylation and dehydrogenation as well as through other transformations, including hydrolysis and the nal polymerization of betalamic acid and cyclo-DOPA to inactive forms. 134,136 So far, no research on the toxicity and stability of quinones produced from betacyanins has been conducted. The literature data are lacking on the pro-oxidative properties of betalains. In addition, there are questions whether betalains are susceptible to undesired metabolic activation and if the blocking of the sites prone to the formation of reactive forms in molecules is required for betalain application as potential therapeutic agents.
3.1.4 Chlorination of betacyanins. The literature reports suggest that betacyanins may be regarded as potent scavengers of inammatory factors such as hypochlorous acid (HOCl). In 2007, Allegra et al. reported that betanin and indicaxanthin are effective in the scavenging of hypochlorous acid. 77 In addition, both the betalains reduce the myeloperoxidase activity and oxidation, and are therefore regarded as the two key components of the anti-inammatory processes, leading to the reduction of hypochlorous acid generation. 153 In 2016, Wybraniec et al. 154 reported that in the reaction between betanin/betanidin/neobetanin and sodium hypochlorite or myeloperoxidase (MPO)/H 2 O 2 /Cl À systems, the chlorination of betanin and betanidin took place within the aglycone unit. In addition, the possible reaction with another potent chlorinating agent, Cl 2 O, which co-exists with HOCl in equilibrium, especially in acidic conditions, was suggested according to a similar mechanism. For neobetanin, no chlorinated products in the reaction mixtures were detected. The fact that the pyridinic ring within the neobetanin structure cannot be chlorinated to form the detectable pigment products indicates that only the unsaturated bond is attacked, preferably at C-18, due to its partial negative charge. 154 Monochloro-betanin and monochlorobetanidin were formed with the highest yield at pH 3-5 while at higher Cl À concentrations, the efficiency was dramatically decreased, suggesting that the generated Cl 2 is not the chlorinating agent in the presence of sodium hypochlorite. The low activity of Cl 2 during betanin and betanidin chlorination compared to HOCl and/or Cl 2 O was explained by a special position (C-18) of the attack by HOCl and/or Cl 2 O. In the case of the MPO/H 2 O 2 /Cl À system, the highest efficiency of monochloro-betanin/-betanidin generation was observed at pH 5.
In another study, 155 additional chlorination experiments were extended to decarboxylated betacyanins ( Table 2). The comparison of the chromatographic proles of the chlorinated decarboxylated betanins and betanidins generated under the activity of hypochlorous acid revealed two different directions of retention changes in relation to the corresponding precursors. The chlorination of betacyanins decarboxylated at C-17 (17-, 2,17-bi-, and 2,15,17-tri-decarboxy derivatives) resulted in higher retention times in comparison to the corresponding substrates. In contrast, the non-decarboxylated betacyanins as well as their 2-and 15-decarboxy-derivatives exhibited lower chromatographic retention aer chlorination. 155 The identication of the chlorinated betacyanins was completed by a further study on the chlorination mechanism and the position of the electrophilic substitution in betacyanins by high-resolution mass spectrometry and further structural analyses by NMR techniques, 156 which conrmed that the chlorination position in betanin and decarboxylated betanins occurs within the dihydropyridinic moiety at the C-18 carbon. 156 According to an electrophilic mechanism based upon the leaving group ability from Cl + in HOCl (-OH À ) and in Cl 2 O (-OCl À ), the reaction paths between betanin and HOCl/Cl 2 O were proposed (Fig. 15). 157 The mechanism was supported by the NMR elucidation results as well as by the inactivity of neobetanin toward chlorination. 154 Chlorination version A shows the position of Cl + attack on C-18 carbon within the betanin structure. The failure in the chlorination of betanin with Cl 2 , which is an active oxidizing agent, conrmed that Cl + leaving from HOCl or Cl 2 O can be the chlorinating factor. Also, the hypothesis that H 2 OCl + may be regarded as a chlorinating factor carrying Cl + is intriguing but has not been conrmed for decades. 158 In the chlorination version B, a cyclic intermediate 159 is formed during the electrophilic attack of the whole HOCl or Cl 2 O molecules as a result of the polarization of Cl-O bond along with the stabilizing effect of hydrogen bonding. The oxidation of C-18 carbon in betanin did not take place because of its negative charge, making the nucleophilic attack of oxygen 159 impossible.
Since uorescent probes have been promising analytical tools for the rapid and specic detection of HOCl/OCl À , the HCSe and HCS probes were tested in the determination of the betalains' anti-hypochlorite activity. The comparison of the in vitro antihypochlorite activities of a betalain-rich red beetroot extract (RBE) with its pure betalainic pigments revealed that the extract had the highest anti-hypochlorite activity, far exceeding the activity of all of the betalainic derivatives and selected reference antioxidants. 160 In addition, the pilot clinical studies showed that the short-term treatment with RBE improved the function and comfort of knee joints in individuals with knee distress resulting from increased hypochlorous acid formation. 76 3.1.5 The acyl migration phenomenon. The acyl migration phenomenon in betacyanins isolated from Mammillaria and Hylocereus fruits was discovered by Wybraniec et al. 11,18,36,56 The driving force behind the research on the acyl migration effect was the fact that 4 0 -O-malonyl betanin isolated from the cacti fruits of Hylocereus species isomerized to 6 0 -O-malonyl betanin (phyllocactin) (Fig. 16). It was proved that acyl migration is intramolecular; hence, hydrolysis and re-esterication mechanisms are not relevant in that process. 161 For that reason, free carboxylic groups in the acyl moieties present in hylocerenin, phyllocactin, and their derivatives are not involved in the rearrangement. The acyl migration mechanism is based on the interaction of the adjacent group with an ortho acid ester being an intermediate. In many cases, the distance between the acyl migrating group and the free hydroxyl is appropriate for creating of a cyclic intermediate. [161][162][163] The formation of the six-membered cyclic intermediate between the glucosidic O-6 0 and O-4 0 hydroxyls may be responsible for the intramolecular rearrangement in phyllocactin. The signicant factor that provides this phenomenon is the alkaline reaction environment; however, the effect is also observed in acidic conditions. A series of new isomers of various 6 0 -O-acylated betacyanins as well as decarboxylated betacyanins formed by intramolecular pH-dependent acyl migration between adjacent hydroxy groups on polyhydroxy compounds in aqueous solutions was chromatographically characterized. 18 The migration rate was markedly accelerated under alkaline conditions at pH 10.5, however, always favoring the 6 0 -O-position. The phenomenon of acyl migration was less apparent at pH below 7.0. Partial rearrangement may result in the formation of 3 0 -O-and 4 0 -O-acylated compounds. In malonylated betacyanins and 17decarboxy-betacyanins, the 3 0 -O-acylated forms were presumably the most polar isomers and were eluted rst in RP-HPLC, whereas the 4 0 -O-forms were characterized by retention higher than the 6 0 -O forms. In contrast, in 2-decarboxy-and 2,17bidecarboxy-betacyanins, both 3 0 -O-and 4 0 -O-acyl-betacyanins were eluted before the 6 0 -O-acylated forms. The study on the acyl migration effect in 4 0 -O-malonyl-betanin revealed a strong tendency to reverse acyl migration (4 0 / 6 0 ) and also partial rearrangement (4 0 / 3 0 ), leading to the monoester regioisomeric distribution in the proportion 87 : 7 : 6 [%] in relation to 6 0 -O-, 4 0 -O-, and 3 0 -O-, respectively.
A new malonyl betacyanin, 6 0 -O-malonyl-amaranthin (celoscristatin), as well as 4 0 -O-malonyl-amaranthin, formed as a result of the malonyl group migration in celoscristatin, were also identied in Celosia cristata Linn. callus culture. 29 3.1.6 NMR elucidation of betacyanin pigments. First NMR structural investigations on betacyanins performed at low pH resulted in the quick degradation of the substances, preventing longer in-depth structural analysis of the betacyanins. 53 Nevertheless, key experiments were performed, e.g., conrming the E/ Z-stereoisomerism at one of the partial double bonds (C-11,12) in betanidin/isobetanidin. 50 As a consequence, only the 13 C NMR spectrum of neobetanin was properly registered due to the stability of this dehydrogenated derivative in a highly acidic environment in contrast to other betacyanins. In Fig. 17, an overview of the solvents used for NMR analysis of particular Fig. 17 The summary of the solvents used for NMR spectra registration of particular betacyanins.  164 The key products formed aer betanin decomposition were identied and their structures were conrmed by NMR analysis. The structural elucidation of 2-, 17-, and 2,17-decarboxybetacyanins generated during the decarboxylation of betanin, phyllocactin, and hylocerenin isolated from H. polyrhizus fruits was performed by Wybraniec et al. 127 A variety of coupled spin systems of all the analyzed compounds were successfully distinguished and assigned by 1 H NMR, 1 D TOCSY, gCOSY, and NOESY spectra in D 2 O. Due to gHSQC correlations, the 13 C chemical shis for all the carbons directly bound to the protons were unambiguously assigned, while the gHMBC (the 1 H-13 C long-range) correlations enabled the establishment of the chemical shis of the quaternary carbons. In addition, the rst 1 H NMR spectra of 2-decarboxy-hylocerenin together with all the studied 17-decarboxy-and 2,17-bidecarboxy-betacyanins were received. Furthermore, the 13 C NMR spectra of the decarboxylated betacyanins were obtained for the rst time. 127 The NMR spectra of amaranthin and iresinin I were recorded in triuoroacetic acid. 61 The structures of the two other acylated betacyanins and lampranthin II from the petals of Lampranthus peersii and L. sociorum as well as celosianin (previously named as celosianin II) from the cell suspension cultures of Chenopodium rubrum were elucidated as 6 0 -O-E-feruloyl-betanin and 2 00 -O-E-feruloyl-b-(1 00 ,2 0 )-glucuronosyl-betanin, respectively, by 1 H NMR. All 1D (normal and NOE difference) and 2D spectra (COSY) were registered in acidic DMSO-d6 containing traces of DCl. 58 A low eld shi of the protons H-6 0 A and H-6 0 B of the glucosylic moiety in lampranthin II caused by acylation indicated an attachment of the feruloyl moiety at the C-6 0 carbon. For celosianin II, the NOE difference spectra indicated that the b-glucuronosyl moiety was bound to C-2 0 of the b-glucosyl moiety. The presence of the feruloyl moiety attached at C-2 00 of b-glucuronosyl was evident from the low eld shi of H-2 00 . 58 In addition to betanin and phyllocactin in H. polyrhizus fruits, another characteristic pigment, hylocerenin [6 0 -O-(3 00 -hydroxy-3 00methyl-glutaryl)-betanin], was also characterized by 1D and 2D (COSY) NMR techniques in deuterated methanol (CD 3 OD) containing traces of deuterium chloride (DCl). 56 Other unknown pigments, 2 0 -O-b-apiofuranosyl-betanin and 2 0 -O-b-apiofuranosylphyllocactin, were also elucidated by 1 H and 13 C NMR techniques in non-acidied D 2 O. 11 The structure of a new malonyl derivative, celoscristatin (6 0 -O-malonyl-amaranthin), isolated from C. cristata callus culture was conrmed by NMR analysis in D 2 O. 29 In order to determine the oxidation mechanism of betanin, structures of tentatively identied key dehydrogenation products of betanin, decarboxylated betanin, and neobetanin oxidation by the ABTS cation radical were elucidated by NMR. 52 The 1 H and 2D (COSY, TOCSY, HSQC, HMBC, and NOESY) measurements were performed in DMSO-d6 for all the studied compounds except for 2-decarboxy-neobetanin, which was analyzed in CD 3 OD acidied with 0.01% deuterated TFA. The structures of ve neo-and xanneo-derivatives, namely, neobetanin, 2-decarboxy-neobetanin, 2-decarboxy-xanneobetanin, 2,17-bidecarboxy-xanneobetanin, and 2,15,17-tridecarboxyxanneobetanin, were conrmed. 52 The structures of gomphrenin as well as its acylated derivatives, globosin and basellin, (gomphrenin I, II, and III, respectively) were elucidated by NMR spectroscopy by Heuer et al. 24 The 1 H and 2D COSY NMR spectra were recorded in CD 3 OD containing traces of DCl. The presence of acyl (4-coumaroyl-and feruloyl-) moieties at the C-6 0 carbon in globosin and basellin, respectively, was indicated by a low eld shi of the corresponding protons H-6 0 A and H-6 0 B. Non-typical 1 H chemical shi differences between gomphrenin I and globosin and basellin suggested intramolecular stacking occurring in the latter two compounds. 24 In another attempt, gomphrenin was identied by means of 1 H, selective NOESY, and TOCSY as well as 2D NMR (COSY, NOESY, HSQC, and HMBC) techniques in D 2 O. 69 By NOESY-correlation spectral analysis, it was demonstrated that the glucopyranosyl group is linked to the phenolic group at the C-6 carbon of gomphrenin, which differentiated it from betanin.
The structures of glutathionylated conjugate of betanidin 140 and N-acetylcysteinylated gomphrenin 149 were established by NMR analysis, conrming the conjugation position at the C-4 carbon, thus indicating the presence of a dopachromic intermediate during gomphrenin oxidation. All 1 H and 2D NMR (COSY, HSQC, HMBC, TOCSY, and NOESY) analyses were accomplished in non-acidied D 2 O. The attachment position of the glutathionyl moiety was primarily indicated by the strong long-range correlation of the H-12a/b 0 protons to quaternary C-4 and C-9 carbons as well as a weak correlation to C-8. The lack of the H-4 signal in the 1 H spectrum unambiguously conrmed this position. 140,149 In the case of N-acetylcysteinylated gomphrenin, the attachment position of the N-acetyl-cysteinyl moiety was initially suggested by a strong, long-range correlation of H-6a/b 00 protons to quaternary carbon C-4. In addition, the lack of the H-4 signal in the 1H spectrum denitely conrmed this position. Furthermore, the CS-conjugation position in gomphrenin was supported by the downeld chemical shi of the H-6a/b 00 proton signal compared to the signal obtained for free N-acetyl-cysteine. 149 In order to prove the position of chlorination in betanin and its decarboxylated derivatives, structural studies were also performed. 156 The structure elucidation of 18-chloro-betanin, 18chloro-17-decarboxy-betanin, and 18-chloro-2,17-bidecarboxybetanin conrmed that the chlorination position in betanin occurs within the dihydropyridine moiety at carbon C-18. The results of 1 H NMR, COSY, and TOCSY analyses obtained for 18chloro-betanin in non-acidied D 2 O enabled to distinguish and characterize several diagnostic, conjugated spin systems characteristic for betanin (H-2, H-3ab; H-11, H-12; H-14ab, H-15) in D 2 O. The dihydroindolic system was indicated by the HSQC correlations of H-2, H-3ab, H-4, and H-7 with their respective carbons. In the dihydropyridine system, the correlations of H-14ab and H-15 with their respective carbons in the HSQC spectra were visible. 156 The structural studies of the newly semi-synthesized pseudonatural pigments based on betanin hydrolysis were also reported. The structures of various compounds semi-synthesized by coupling betalamic acid with N-methyl anilines, N-aryl anilines, and blue solid named BeetBlue as well as 7-amino-4-methylcoumarin-and 7amino-4-triuoromethylcoumarin-betacyanins (cBeet120 and cBeet151) were accomplished by NMR analysis in D 2 O. [165][166][167] 4 Chemistry of betacyanins and their semi-synthetic derivatives based on betanin hydrolysis

Hydrolysis of betanin
Betanin is susceptible to acid-and base-catalyzed hydrolysis, leading to the formation of colorless cyclo-DOPA-5-O-b-D-glucoside and fairly stable bright yellow betalamic acid (Fig. 18). Recently, new light was shed on betanin hydrolysis mechanism. 169 The impact of temperature and pH on the rate of betanin hydrolysis was examined. The mechanism of betanin decomposition was studied in the phosphate solution at pH ranging from 2 to 11 and temperature of 60-85 C. It was postulated that the fully protonated betanin form is reactive toward the nucleophilic attack of water and the intramolecular hydrogen bond between the carboxylic group at C-2 carbon and N-1 nitrogen stabilizes the transition state, leading to the hydrated betanin. The N-1 nitrogen atom was expected to be protonated in the acidic media and to form a leaving group. The protonation of the N-16 nitrogen atom enhances the electrophilicity of C-11, C-13, and C-17 carbons and catalyzes betanin hydrolysis, nally contributing to the opening of the 2,6dicarboxy-1,2,3,4-tetrahydropyridine ring of betalamic acid. It was indicated that the hydrogen phosphate ion may act as a general base at pH 8.2, even at a low buffer concentration. The formation of cyclo-DOPA and 2,6-dicarboxy-4-methylpyridine as the products of betanin degradation in an alkaline environment at 130 C identied by IR spectroscopy was reported in the pioneering work of Wyler and Dreiding on betanin purication by crystallization. 47,170 In contrast to that study, 2,6-dicarboxy-4methylpyridine was not detected. 169 However, the presence of cyclo-DOPA, betalamic acid, and decarboxylation/oxidation products was observed.
Based on theoretical calculations, the double protonation of the nitrogen atom within the tetrahydropyridine ring cannot be excluded from what was observed for the amino dicarboxylic acids of simpler structures. 169 Next to hydrolysis, betacyanins are also sensitive to watercatalyzed isomerization. A study on the hydrolytic stability of betanin Bn, indicaxanthin BtP, and an articial betalainic coumarin BtC soluble hydrogen bond donating 2,2,2-tri-uoroethanol (TFE) was conducted. 171 The hydrolytic stability in water was as follows: BtP > Bn > BtC. Improved hydrolytic stability of the abovementioned betalains in hydroalcoholic solutions was noticed with an increase in the TFE co-solvent. The hydrolysis rate of betalainic coumarin in water was 700fold greater than in TFE, whereas the use of TFE as a co-solvent reduced the hydrolysis of betanin and indicaxanthin by 20 and 100-times, respectively. The proportion of water and TFE in the co-solvent mixture determined the hydrogen-bond donating capacity, polarity, and ionization power. However, the most important factor of the enhanced hydrolytic stability of the betacyanins is probably due to the low nucleophilicity of TFE. In addition, the uorescence quantum yields of betaxanthins were also increased with the presence of TFE. 171

One step semi-synthesis of betalains from betalamic acid-derivatized support
So far, all the proposed methods of the de novo synthesis of betalains achieve very low efficiency; therefore, the best way to obtain individual betalain derivatives is to isolate them from the biological material and purify. However, this makes the whole process highly time-consuming due to the plant matrix that is rich in various compounds, especially polysaccharides. The process of betalain formation was simplied using a novel betalamic acid-derivatized support (Fig. 19). 172 Betalamic acid was produced during betanin hydrolysis from B. vulgaris extract in the presence of cross-linked polystyrene resin at room temperature and nitrogen atmosphere. Aer the release of betalamic acid and neutralization with acetic acid, the reaction of imine formation between the aldehyde group of betalamic acid and the free primary amine groups present in the matrix surface was performed, resulting in the immobilization of betalamic acid in the resin. The derivatization of the resin with betalamic acid was indicated by the resin color change from pale to intense yellow. Aer washing and drying at room temperature, the novel solid support was generated and characterized by SEM. The material exhibited the spectroscopic properties of a pseudobetaxanthin. Then, the resin from the betalamic acid-derivatized support was tested toward the production of betalains. As model amines, individual dopamine, tyramine, indoline, and pyrrolidine were added to the support turning the color of the solutions from colorless into yellow in the case of dopamine, tyramine, and pyrrolidine, as well as into purple in the case of indoline. Then, a Schiff's condensation between the amine and betalamic acid anchored in the support provided the dopamine-, tyramine-, pyrrolidine-, and indoline-betaxanthins. 172 4.3 Semi-synthetic betalain pigments with extended conjugated systems 4.3.1 Semi-synthesis of blue pigments from betanin. Naturally-occurring blue pigments are rare, especially among plants. A metal-free photostable blue pigment was designed by extending the p-system of betalains (Table 2). 166 The dye was semi-synthesized from betalamic acid, which may be obtained from the hydrolysis of red beetroot juice or by the enzymatic oxidative cleavage of S-DOPA. The 1,11-diazaundecamethinium chromophore of the dye is formed by the irreversible dehydrative C-C coupling of betalamic acid with the carbon nucleophile 2,4-dimethylpyrrole in acidied ethyl acetate (Fig. 20). The reaction lasted less than 30 min and it was performed at room temperature under air condition. The 1,11-diazaundecamethinium chromophore induces a redshi of the absorption and uorescence spectra in water in relation to standard betanin and indicaxanthin. The product was puried by ash gel permeation chromatography with water used as eluent. This dye exhibits high solubility in polar solvents, e.g., water, and does not produce singlet oxygen upon photoexcitation. 166 4.3.2 Semi-synthesis of a pseudo-natural betalain-nitrone OxiBeet pigment. A prototypical betalain-nitrone pigment (OxiBeet) based on the N-oxide 1,7-diazaheptamethinium scaffold was semi-synthesized for the rst time by Capistrano Pinheiro et al. (Table 2). 173 Betalamic acid was produced by the hydrolysis of the betalains present in red beetroot juice and coupled with N-phenylhydroxylamine in acidied water, giving rise to OxiBeet pigment (Fig. 21). The resulting product was Fig. 20 Semi-synthesis of a blue dye from betanin derived from beetroot juice. 166 Fig. 19 Betalamic acid-derivatized support and the surface reaction leading to pigment isolation, based on dopamine-derived betaxanthin: (A) starting material containing primary amine groups; (B) betalamic acid-derivatized support; (C) amine attack to betalamic acid; (D) synthesis and concomitant release of miraxanthin V. 172 puried and isolated with 50% yield, which is twice as high as the values reported for other betalains.
This bio-based molecule exhibits low reduction potential and even higher radical scavenging activity than typical antioxidants such as ascorbic acid and gallic acid. Furthermore, it is non-cytotoxic toward human hepatic cell line HepaRG up to 1 mM concentrations. This pseudo-natural product is resistant to hydrolysis under neutral and slightly alkaline conditions and it may also be converted into a persistent N-oxide 1,7-diazaheptamethinium radical cation in an aqueous solutions via autoxidation. These ndings open up new perspectives for utilizing betalain pigments and betalain derivatives as therapeutics against various human diseases.
4.3.3 Semi-synthesis of N-methyl phenyl-betaxanthin and N-aryl phenyl-betaxanthin pigments. The semi-synthesis of Nmethyl phenyl-betaxanthin (common name: mepBeets) and Naryl phenyl-betaxanthin (dipBeets) pigments (Table 2) was described by Pioli et al. and the inuence of the structure on the betalain derivatives' hydrolytic stability and electronic properties were compared. 165 Eight compounds were formed by the derivatization of betalamic acid and N-methyl-anilines as well as N-aryl-anilines in ethyl acetate in the presence of p-toluenesulfonic acid used as a catalyst (Fig. 22). The stabilization of the positive charge density at the nitrogen atom N-9 by the N-methyl group reduces the rate of hydrolysis by decreasing charge delocalization through the 1,7-diazaheptamethinium system as compared to the non-methylated phenyl-betaxanthin. On the contrary, the N-aryl moiety increases charge delocalization while improving the hydrolytic stability. The electronwithdrawing substituents within the aryl moiety increase the uorescence quantum yields of semi-synthesized betaxanthins, accelerate hydrolysis, and lower the anodic potential compared to the corresponding unsubstituted betalains. On the other hand, the presence of electron-donating substituents improves their hydrolytic stability. The uorescence of N-aryl phenylbetaxanthins is strongly inuenced by the polarity and viscosity of the medium which enables the design of the "turnon" uorescent (bio)sensors. 165 4.3.4 Semi-synthesis of articial coumarinic betalains. Betalains may be modied to produce water-soluble two-photon uorophores. 167 Coumarinic betalains cBeet120 and cBeet151 ( Table 2) were obtained in the reaction between betalamic acid and aminocoumarins in acidied ethyl acetate (Fig. 23). Betalamic acid underwent Schiff condensation (aldimine formation) with 7-amino-4-methylcoumarin c120 and 7-amino-4-triuoromethylcoumarin c151 in the presence of p-toluenesulfonic acid used as a catalyst. The coupling of the betalamic acid chromophore to the uorescent hydrophobic chromophore via the 1,7-diazeheptamethine p-bridge results in a D-p-A molecular arrangement. Coumarinic betalain containing  a triuoromethyl electron-withdrawing substituent within the coumarin moiety is more uorescent than the one with a methyl group but it has a lower two-photon absorption cross-section, which is attributed to the destabilization of the charge transfer character of the excited state affecting the transition dipole moment. 167 In another study, an articial coumarinic betalain (BtC) semi-synthesized via the aldimine coupling of 7-amino-4methylcoumarin to betalamic acid was used for the fabrication of the uorescent probe for the live-cell imaging of erythrocytes infected with Plasmodium falciparum responsible for malaria disease in humans. 168 In order to test the capacity of BtC for labeling living Plasmodium-infected red blood cells, erythrocytes with parasites were incubated with BtC and examined by uorescence microscopy. BtC was accumulated within the infected cells selectively and uorescence imaging microscopy has shown that only the parasite was stained. On the contrary, no stain was observed in the infected erythrocytes incubated with an indicaxanthin (control probe) and uninfected cells. 168

Conclusions
During the last two decades, the research on betalains, more specically on betacyanins, has experienced signicant acceleration due to their health-protective properties as well as applications in the food industry. Before 2000, there were no reports on any pro-health activities of betacyanins; from 2020 onwards, this is the main direction of projects and research on betacyanins. Furthermore, studies on the oxidation mechanism of betacyanins are highly relevant because pigments with different chemical structures seem to have different inuence on their oxidation pathways. Considering the fairly simple semi-synthetic methods for additional betacyanin derivatives summarized in this review as well as the rst promising results of various studies, the perspectives for further investigations on the bioactivity of betacyanins are inexhaustible. For the time being, the main obstacle that limits the research is the lack of highly efficient and inexpensive methods of betacyanin isolation and purication as well as methods of de novo synthesis. The constantly growing number of potential applications of betalains as natural colorants of food, fabrics, and dietary supplements but also their utilization in sensors, photoprotectors, and solar cells set promising directions of the investigations on these still not fully-explored plant pigments in the near future. In spite of the most recognizable betanin-based betacyanins present in a variety of betalainic plant sources, gomphrenin-like pigments also represent a promising group of potentially more stable and more active compounds at once. There are still many unexplored plants with presumably new interesting betacyanin proles. The chemical structures of only 31 betalain pigments isolated from the plant sources were denitively identied by the NMR method, which is relatively small in comparison to the other groups of plant pigments. Furthermore, a huge group of over 187 betacyanins (including pigments from their richest source, B. glabra) detected by LC-MS techniques still awaits the nal conrmation of their structures. Particular attention should also be paid to the chemical modications of betacyanins that result in the generation of an increasing number of new derivatives with completely unknown bioactivities. The aspects discussed in this review show that the eld of betalain chemistry is a goldmine and ideas for further research are still limitless.

Conflicts of interest
The authors declare no competing nancial interest. Semi-synthesis of artificial coumarinic betalain pigments with an extended conjugated system. 167