Basella alba L. (Malabar Spinach) as an Abundant Source of Betacyanins: Identification, Stability, and Bioactivity Studies on Natural and Processed Fruit Pigments.

The antioxidant and anti-inflammatory activities of acylated and decarboxylated gomphrenins, as well as Basella alba L. fruit extract, were investigated in relation to gomphrenin, known for its high biological potential. The most abundant natural acylated gomphrenins, namely, 6'-O-E-caffeoyl-gomphrenin (malabarin) and 6'-O-E-4-coumaroyl-gomphrenin (globosin), were isolated from B. alba extract for the studies. In addition, controlled thermal decarboxylation of gomphrenin in the purified B. alba extract at 65-75 °C resulted in the formation of the most prevalent decarboxylated products, including 17-decarboxy-gomphrenin and 2,17-bidecarboxy-gomphrenin, along with their isoforms. The structures of the decarboxylated pigments were confirmed by NMR analyses. Exploring the matrix effect on pigment reactivity revealed a tremendous increase in the stability of all betacyanins after the initial stage of extract purification using a cation exchanger under various conditions. This indicates the removal of a substantial portion of the unfavorable matrix from the extract, which presumably contains reactive species that could otherwise degrade the pigments. Furthermore, the high concentration of citrates played a significant role in favoring the formation of 2-decarboxy-gomphrenin to a considerable extent. In vitro screening experiments revealed that the tested compounds demonstrated strong anti-inflammatory properties in lipopolysaccharide (LPS)-activated human macrophages. This effect encompassed the selective inhibition of cytokine and chemokine release from activated macrophages, modulation of the chemotactic activity of immune cells, and the regulation of tissue remodeling mediators' release.

Unveiling Alternative Oxidation Pathways and Antioxidant and Cardioprotective Potential of Amaranthin-Type Betacyanins from Spinach-like Atriplex hortensis var. 'Rubra'.

A comprehensive oxidation mechanism was investigated for amaranthin-type betacyanins with a specific glucuronosylglucosyl moiety isolated from Atriplex hortensis 'rubra' using liquid chromatography coupled to diode array detection and electrospray ionization tandem mass spectrometry (LC-DAD-ESI-MS/MS) and LC-Quadrupole-Orbitrap-MS (LC-Q-Orbitrap-MS). By employing one-dimensional (1D) and two-dimensional (2D) NMR, this study elucidates the chemical structures of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)-oxidized celosianins for the first time. These findings demonstrate alternative oxidation pathways for acylated betacyanins compared to well-known betanidin, betanin, and gomphrenin pigments. Contrary to previous research, we uncover the existence of 17-decarboxy-neo- and 2,17-bidecarboxy-xanneo-derivatives as the initial oxidation products without the expected 2-decarboxy-xan forms. These oxidized compounds demonstrated potent free radical scavenging properties. Celosianin (IC50 = 23 µg/mL) displayed slightly higher antioxidant activity compared to oxidized forms, 17-decarboxy-neocelosianin (IC50 = 34 µg/mL) and 2,17-bidecarboxy-xanneocelosianin (IC50 = 29 µg/mL). The oxidized compounds showed no cytotoxic effects on H9c2 rat cardiomyoblasts (0.1-100 µg/mL). Additionally, treatment of H9c2 cells with the oxidized compounds (0.1-10 µg/mL) elevated glutathione levels and exhibited protective effects against H2O2-induced cell death. These findings have significant implications for understanding the impact of oxidation processes on the structures and biological activities of acylated betalains, providing valuable insights for future studies of the bioavailability and biological mechanism of their action in vivo.

Betalains isolated from underexploited wild plant Atriplex hortensis var. rubra L. exert antioxidant and cardioprotective activity against H9c2 cells.

Atriplex hortensis var. rubra L. extracts prepared from leaves, seeds with sheaths, and stems were characterized for betalainic profiles by spectrophotometry, LC-DAD-ESI-MS/MS and LC-Orbitrap-MS techniques. The presence of 12 betacyanins in the extracts was strongly correlated with high antioxidant activity measured by ABTS, FRAP, and ORAC assays. Comparative assessment between samples indicated the highest potential for celosianin and amaranthin (IC50 21.5 and 32.2 µg/ml, respectively). The chemical structure of celosianin was elucidated for the first time by complete 1D and 2D NMR analysis. Our findings also demonstrate that betalain-rich A. hortensis extracts and purified pigments (amaranthin and celosianin) do not induce cytotoxicity in a wide concentration range in rat cardiomyocytes model (up to 100 µg/ml for extracts and 1 mg/ml for pigments). Furthermore, tested samples effectively protect H9c2 cells from H2O2-induced cell death and prevent from apoptosis induced by Paclitaxel. The effects were observed at sample concentrations between 0.1 and 10 µg/ml.

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

Covering: 2001 to 2021Betacyanins 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 affecting 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 first part of the review, we present an updated classification of all known betacyanins and their derivatives identified 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 artificial fluorescent betalamic acid conjugates, showing various prospective research directions.

High-speed countercurrent chromatography for isolation and enrichment of betacyanins from fresh and dried leaves of Atriplex hortensis L. var. "Rubra".

Betacyanins and their decarboxylated derivatives from fresh and dried edible leaves of Atriplex hortensis L. var. "Rubra" were fractionated for the first time by high-speed countercurrent chromatography. Pigments present in fresh leaf extract were separated in systems: ethanol - acetonitrile - n-propanol - ammonium sulphate - water (0.5:0.5:0.5:1.2:1.0, v/v/v/v/v) (tail-to-head mode) and tert-butyl methyl ether - n-butanol - acetonitrile - water with 0.7% heptafluorobutyric acid (2:2:1:5, v/v/v/v) (head-to-tail mode). The mobile phase flow rate was 2 mL/min and the retention of the stationary phase was 79.8 and 75.2%, respectively. Pigments from dried leaves were separated in a similar ion-pair system with heptafluorobutyric acid in different volume proportions 1:3:1:5 (head-to-tail mode) and the flow rate of the mobile phase 3 mL/min. The stationary phase retention was 64.0%. The application of the countercurrent chromatography for the fractionation of betacyanins from leaves of Atriplex hortensis enabled to isolate and pre-concentrate the pigments for further low- and high-resolution liquid chromatographic-tandem mass spectrometric detection. This study revealed the presence of 10 betacyanins in fresh and 16 in dried leaves of Atriplex hortensis. Two compounds were not previously identified in the whole Amaranthaceae family. Additionally, instead of (iso)amaranthin, celosianin and its epimer were dominant betacyanins in the Atriplex hortensis.

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.

Separation of betacyanins from Iresine herbstii Hook. ex Lindl. leaves by high-speed countercurrent chromatography in a polar solvent system.

Betacyanins, natural plant pigments, from Iresine herbstii Hook. ex Lindl. leaf extract were separated for the first time by high-speed countercurrent chromatography (HSCCC) in a highly polar solvent system composed of PrOH-ACN-(NH4)2SO4satd.soln-H2O (1.0:0.5:1.2:1.0; v/v/v/v) in the tail-to-head mode. The flow rate of the mobile phase was 2.0 ml/min and the column rotation speed was 860 rpm. The retention of the stationary phase was 81.0%. For the identification of separated betacyanins in the crude extract and in the HSCCC fractions, as well as for the molecular formulas and multi-step fragmentation pattern elucidation, liquid chromatography with tandem mass spectrometry and high-resolution ion-trap time-of-flight mass spectrometry were performed. The innovative application of the HSCCC system for fractionation of the betacyanins present in I. herbstii leaves enabled effective separation as well as preconcentration of the pigments for further low- and high-resolution LC-MS/MS analysis. HSCCC separation enabled identification of 22 betacyanins, of which 18 had not been detected previously in the leaves of I. herbstii, and four of these betacyanins (sinapoyl-gomphrenin and coumaroyl-gomphrenin as well as their epimers) were identified for the first time in the Iresine genus.

Identification of Novel Low-Weight Sulfhydryl Conjugates of Oxidized 5-O- and 6-O-Substituted Betanidin Pigments.

The formation of conjugates of oxidized betacyanin pigments with selected low-weight sulfhydryl scavengers was studied. Short-lived quinonoids, quinone methides, and aminochromes derived from oxidized betacyanins are able to form adducts with different efficiencies. In this report, mass spectrometric and NMR identifications of CS-linked conjugates of cysteine, cysteamine, N-acetylcysteine, and dl-dithiolthreitol with quinonoid forms generated through oxidation of betanidin, betanin, and gomphrenin is presented. An adduct that formed between cysteine and quinonoid generated from betanin by its oxidation and decarboxylation (2-decarboxy-xanbetanin) was detected and reported for the first time. The most stable gomphrenin CS-conjugate, N-acetylcysteinylated gomphrenin, was isolated by semipreparative chromatography and its structure was established by NMR analysis. This enabled to confirm the conjugation position at carbon C-4 and to indicate the presence of a dopachromic intermediate during oxidation of gomphrenin. Conjugation of betacyanins with thiol-bearing moieties may generate new molecules with modified chemical and biological properties. Obtained results confirm that gomphrenin is capable of forming CS-conjugates with higher efficiency than betanin.

Structural Study on Hypochlorous Acid-Mediated Chlorination of Betanin and Its Decarboxylated Derivatives from an Anti-Inflammatory Beta vulgaris L. Extract.

Hypochlorous acid (HOCl) produced by neutrophils is a part of the natural innate immune response system in the human body, but excessive levels of HOCl can ultimately be detrimental to health. Recent reports suggest that betacyanin plant pigments can act as potent scavengers of inflammatory factors and are notably effective against HOCl. In this contribution, chlorination mechanism and position of the electrophilic substitution in betacyanins was studied by high-resolution mass spectrometry and further structural analyses by NMR techniques, which completed the identification of the chlorinated betacyanins. For the study on the influence of the position of decarboxylation on the chlorination mechanism, a comparison of the chlorination position between betanin as well as 17-, and 2,17-decarboxylated betanins was performed. The structural study confirmed that the chlorination position in betanin occurs within the dihydropyridinic moiety at carbon C-18. Therefore, out of the aqueous free chlorine equilibrium species: HOCl, OCl-, Cl2, and Cl2O, the most potent chlorinating agents are HOCl and Cl2O postulated previously and the attack of the Cl⁺ ion on the carbon C-18 with a cyclic intermediate version is considered.