Betalain health-promoting effects after ingestion in Caenorhabditis elegans are mediated by DAF-16/FOXO and SKN-1/Nrf2 transcription factors.

Betalain-rich extracts have been used for many years by their nutraceutical potential. However, the study of their bioactivities has always been hampered by their difficult obtention. To explain their mode of action, seventeen pure betalains were tested in vivo using the animal model C. elegans. Four betalains, named indicaxanthin, indoline carboxylic acid-betacyanin, phenylalanine-betaxanthin, and dopaxanthin, behaved as extraordinary in vivo antioxidants and anti-aging compounds, by increasing the lifespan of C. elegans up to 16.82%, 16.65%, 16.53%, and 12.93%, respectively. The first microarrays performed with betalains and biological confirmation with different mutant strains showed that this life extension is due to a reduction of oxidative stress and the activation of the transcription factors DAF-16/FOXO and SKN-1/Nrf2. They are involved in longevity and oxidative stress resistance pathways and lead to overexpression of HSPs genes, involved in resistance to cancer and Alzheimer's, opening novel research lines in the search for effective plant-based treatments.

Light Emission in Betalains: From Fluorescent Flowers to Biotechnological Applications.

The discovery of visible fluorescence in the plant pigments betalains revealed the existence of fluorescent patterns in flowers of plants of the order Caryophyllales, where betalains substitute anthocyanins. The serendipitous initial discovery led to a systemized characterization of the role of different substructures on the photophysical phenomenon. Strong fluorescence is general to all members of the family of betaxanthins linked to the structural property that the betalamic acid moiety is connected to an amine group. This property has led to bioinspired tailor-made probes and to the development of novel biotechnological applications in screening techniques or microscopy labeling. Here, we comprehensively review the photophysics, photochemistry, and photobiology of betalain fluorescence and describe all current applications.

Characterization of betalains, saponins and antioxidant power in differently colored quinoa (Chenopodium quinoa) varieties.

Quinoa was the traditional grain crop used by the prehispanic civilizations in America. Grains are white, black, yellow, and red-violet and plants are cultivated in vast areas of Peru, Bolivia and Ecuador. The recent description of the betacyanin pigment betanin in red-violet varieties is here further analyzed detecting the presence of amaranthin not previously identified in quinoa grains. Yellow-orange grains are characterized for the first time and up to four different betaxanthins are found to be responsible for this coloration. The native fluorescence of the identified betaxanthins makes the surface of the yellow quinoa grains glow with green fluorescent light. The presence of betalains is correlated with high antioxidant and free radical scavenging activities measured under the FRAP, ABTS and ORAC assays in grain extracts of 29 Peruvian varieties. TEAC equivalence is as high as 44.1 and 47.4mmol Trolox/kg for the yellow and red-violet varieties analyzed respectively.

Biological Activities of Plant Pigments Betalains.

Betalains are a family of natural pigments present in most plants of the order Caryophyllales. They provide colors ranging from yellow to violet to structures that in other plants are colored by anthocyanins. These include not only edible fruits and roots but also flowers, stems, and bracts. The recent characterization of different bioactivities in experiments with betalain containing extracts and purified pigments has renewed the interest of the research community in these molecules used by the food industry as natural colorants. Studies with multiple cancer cell lines have demonstrated a high chemopreventive potential that finds in vitro support in a strong antiradical and antioxidant activity. Experiments in vivo with model animals and bioavailability studies reinforce the possible role played by betalains in the diet. This work provides a critical review of all the claimed biological activities of betalains, showing that the bioactivities described might be supported by the high antiradical capacity of their structural unit, betalamic acid. Although more investigations with purified compounds are needed, the current evidences suggest a strong health-promoting potential.

One-step synthesis of betalains using a novel betalamic acid derivatized support.

Betalains are plant pigments with high antioxidant and cancer chemopreventive properties used by the food industry as safe colorants. Betalains are restricted to species of the order Caryophyllales, and difficulty in obtaining individual molecules has limited their structural identification and application. This study was designed to develop a betalamic acid derivatized support generated from a primary amine polymer. The novel material presents color properties of a pseudobetaxanthin, and it is stable for at least 6 months. The bond formed can be displaced at mild conditions by the addition of amines in aqueous solutions over a broad pH range and at 25 °C. This releases the betalamic acid while forming the corresponding pigment. This one-step procedure significantly simplifies the process of obtaining semisynthetic betalains, and it is optimized here for the formation of betaxanthins and betacyanins derived from tyramine, dopamine, pyrrolidine, and indoline. The new method makes access to single betalains available to the entire scientific community and could stimulate research and applications in the field.

Encapsulation of the most potent antioxidant betalains in edible matrixes as powders of different colors.

Betalains are plant pigments with high antioxidant and free radical scavenging activities. While basal activity exists in all betalains, the dihydroxylated molecules present the highest TEAC values of the family of compounds. However, their lability limits possible applications. This work reports the encapsulation of the most active pigments, the yellow miraxanthin V and the violet betanidin in edible matrixes of chitosan and maltodextrin. An appropriate spray-drying procedure is described, with an inlet air temperature of 140 °C. The resulting particles were characterized by scanning electron microscopy, and powder color was analyzed by spectrophotometry using an integrating sphere. Stability of the bioactive compounds was followed by high-performance liquid chromatography, and it was highly promoted by encapsulation, with limited pigment loss after six months' storage. Particles retained the antioxidant and antiradical activities of the soluble pigments measured under the FRAP and ABTS radical assays. A combination of miraxanthin V and betanidin in variable proportions provides a bright palette of encapsulated powders of different colors suitable for food applications.

Purification and antiradical properties of the structural unit of betalains.

Betalamic acid [4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridine-2,6-dicarboxylic acid] is a naturally occurring compound that is normally found condensed with amino acids, amines, cyclo-DOPA, and cyclo-DOPA derivatives to form the betalains. Betalains are the pigments responsible for the yellow to violet color of the fruits and flowers of plants belonging to the order Caryophyllales. Betalamic acid is the structural feature common to all of these pigments and contains the electron resonance system responsible for the spectroscopic properties. Betalamic acid was purified by chromatography and identified by UV-vis spectrophotometry and ESI mass spectrometry. The antioxidant and free radical scavenging capacities of betalamic acid were assessed using the FRAP and ABTS(·+) radical assays. A pK(a) of 6.8 was found for the deprotonation equilibrium involved in the nucleophilic activity of betalamic acid; this pK(a) explains the observed pH effect on the free radical scavenging capacity of these pigments.

Stabilization of the bioactive pigment of opuntia fruits through maltodextrin encapsulation.

Betalains are water-soluble, nitrogen-containing pigments of growing interest in the food industry. They are present in most plants belonging to the order Caryophyllales, where they fulfill the role of anthocyanins, and are divided into two groups: violet betacyanins and yellow betaxanthins. They are bioactive molecules that account for health-promoting properties, recently described for cactus pears (Opuntia). In this work, the characteristic betalain of cactus pears, indicaxanthin, is obtained purely, and its stability is highly promoted by its encapsulation in a maltodextrin matrix. A suitable spray-drying procedure for encapsulation is described, and a bright yellow powder is obtained. The stability is analyzed under different conditions. In the absence of light, pure encapsulated pigment can be stored at 20 °C for months without appreciable loss of the bioactive substance and color variation. Furthermore, free radical scavenging and antioxidant properties of the pigment are studied under the ABTS(•+) radical and ferric reducing antioxidant power assays, in the presence and in the absence of maltodextrins. The stabilization of pure betalain pigments may boost the use of these bioactive and natural coloring molecules.

Structural implications on color, fluorescence, and antiradical activity in betalains.

Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors on flowers and fruits of most plants of the order Caryophyllales. They are classified as betacyanins, exhibiting a violet coloration, and betaxanthins, which exhibit yellow coloration. Traditionally, betalains have been defined as condensation products of betalamic acid with different amines and amino acids, but the implication of the pigment structure for their properties has not been investigated. This paper explores different structural features of the betalains, revealing the clues for the switch from yellow to violet color, and the loss of fluorescence. A relevant series of 15 betalain-related compounds (both natural and novel semisynthetic ones) is obtained and characterized by chromatography, UV-vis spectrophotometry, fluorescence, and electrospray ionization mass spectroscopy. Antiradical properties of individual pure compounds in a broad pH range are studied under the ABTS(*+) radical assay. Relevance of specific bonds is studied, and differences between betaxanthins and betacyanins are used to explore in depth the structure-antiradical activity relationships in betalains.

The role of phenolic hydroxy groups in the free radical scavenging activity of betalains.

Free radical scavenging compounds play important roles as health-protecting factors. Betalains are natural water-soluble pigments present in most plant families belonging to the order Caryophyllales. They are the subject of increasing attention following the discovery of their antiradical capacity, but a systematic analysis of the structural features involved in the activity is necessary. In this paper, both natural and previously unconsidered betaxanthins were obtained in order to study the role of phenolic hydroxy groups in the high free radical scavenging activity of betalains. Pigments were characterized spectrophotometrically, chromatographically, and by ESI-MS, and their antiradical and antioxidant properties were studied under the ABTS(*+) radical and FRAP assays. A high intrinsic activity is described that is not linked to the presence of hydroxy groups or aromaticity in the pigment structure. In addition, the presence of phenolic hydroxy groups implies an enhancement of the antiradical activity, reaching a TEAC value in the ABTS(*+) assay of 5.8 +/- 0.2 for the pure compound with two hydroxy groups.

Fluorescence detection of tyrosinase activity on dopamine-betaxanthin purified from Portulaca oleracea (common purslane) flowers.

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is one of the key enzymes for the biosynthesis of natural pigment betalains. These are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In this work, dopamine-betaxanthin (also known as miraxanthin V) is reported as the pigment responsible for the bright coloration in yellow flowers of Portulaca oleracea (common purslane). The natural pigment is purified, and used as a substrate for the catecholase (diphenolase) activity of the enzyme tyrosinase. A new, continuous method to follow the activity is developed based on the fluorescent properties of the betaxanthin. Fluorescence of the enzyme activity derived products is reported for the first time. Relevance of the fluorescent phenomenon is discussed based on fluorescence images and the description of a physiological inner filter effect present in flowers of P. oleracea. The first description of the betalain content in flower pistils is also provided.

Partial purification of latent polyphenol oxidase from peach (Prunus persica L. Cv. Catherina). Molecular properties and kinetic characterization of soluble and membrane-bound forms.

This paper analyzes the kinetic and structural characteristics of polyphenol oxidase (PPO) from peach cv. Catherina. The PPO was obtained in a latent state in both the soluble and membrane-bound forms, and both forms were activated by acid shock and the detergent SDS. Plant defense is the main function assigned to PPO, which would be activated by the acid environment resulting from tissue damage. On the other hand, it has been suggested that, physiologically, the role played by SDS may be fulfilled by lipids. Native isoelectric focusing identified two acid isoforms of pI 5.7 and 5.8 for the soluble form and one isoform with pI 5.7 for the membrane-bound form. A partially denaturing SDS-PAGE revealed two very close bands of activity in both cases, but the Western blot performed on a totally denaturing SDS-PAGE, using polyclonal antibodies against bean PPO, revealed a single band in the membrane-bound fraction with a molecular mass of 60 kDa.

Characterization of the activity of tyrosinase on betanidin.

Betalains are an important class of water-soluble pigments, with radical scavenging capacity, which is characteristic of the order Caryophyllales. The structural unit of the violet betacyanins, betanidin is reported as a substrate for the enzyme tyrosinase (EC 1.14.18.1), which plays a key role in the betalains biosynthetic scheme. The compound was identified in Lampranthus productus violet flowers, from which it was extracted and purified. The tyrosinase-mediated oxidation of betanidin was characterized in depth and followed by high-performance liquid chromatography and spectrophotometry. The addition of ascorbic acid reversed the reaction product, betanidin-quinone, to the original pigment. Kinetic analysis revealed a Km = 0.66 mM. Betanidin degradation kinetics was also studied in the absence of the enzyme and demonstrated that pH values over 6.0 and high ionic strength reduce the pigment stability.

Development of a protocol for the semi-synthesis and purification of betaxanthins.

A method for the analytical and semi-preparative chromatographic purification of betaxanthins is described together with an improved procedure for the semi-synthesis of these compounds from betalamic acid. Standard conditions for obtaining preparative amounts of betaxanthins free of the precursor amino acids are provided. Following this procedure, 14 pure betaxanthins were obtained with yields of up to 100%. A simple reversed-phase HPLC protocol for pigment identification and quantification is also provided. Calibration for betaxanthins is reported for the first time using the synthesised and purified pigments as standards. Structures were confirmed by UV-vis spectroscopy, HPLC retention times and electrospray ionization mass spectrometry. Betaxanthins can be obtained pure, and in sufficient amounts for further studies, which opens up new perspectives in the research and applications of these pigments.

Oxidation of the flavonoid eriodictyol by tyrosinase.

A pathway is proposed for the oxidation of the flavonoid eriodictyol by mushroom tyrosinase. In it, the enzymatic oxidation of eriodictyol leads to the formation of eriodictyol-o-quinone, which undergoes the nucleophilic attack of another eriodictyol unit to yield a dimer. This dimer is then oxidized by the eriodictyol-o-quinone. The reaction was followed by recording the time course of formation of this second o-quinone at 475 nm. Progress curves at this wavelength showed the appearance of a lag, the length of which varied with enzyme and substrate concentrations, and which must have been caused by the chemical reactions taking place after the enzymatic reaction. When eriodictyol oxidation was studied in the presence of 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH), which competes with the substrate in the reaction with eriodictyol-o-quinone, the lag disappeared. The kinetic parameters were similar with and without MBTH. Eriodictyol oxidation was inhibited by tropolone, which behaved as a slow-binding inhibitor.

Characterization of the activity of tyrosinase on betaxanthins derived from (R)-amino acids.

The activity of tyrosinase (EC 1.14.18.1) on selected (R)-betaxanthins is characterized in depth, demonstrating that the activity of the enzyme is not restricted to betaxanthins derived from (S)-amino acids. Conversion of (R)-tyrosine-betaxanthin [(R)-portulacaxanthin II] to the pigment (R)-dopaxanthin and its further oxidation to a series of products is described. Compound identity was studied by high performance liquid chromatography and electrospray ionization-mass spectrometry. The reaction rate on the (R)-isomer of dopaxanthin is 1.9-fold lower than that obtained for the (S)-isomer in previous studies. Tyrosinase showed stereospecificity in its affinity toward betaxanthins. The characterization of the activity of tyrosinase on (R)-betaxanthins reinforces the role of the enzyme in the biosynthetic scheme of betalains.

Betaxanthins as pigments responsible for visible fluorescence in flowers.

Betalains are water-soluble nitrogen-containing pigments present in flowers and fruits of plants of the order Caryophyllales, where they replace anthocyanins. This article describes how flowers containing yellow betaxanthins are fluorescent. Betaxanthins exhibit spectra with excitation maxima between 463 nm and 474 nm and emission maxima between 509 nm and 512 nm. Thus, betaxanthins are able to absorb blue light and emit green light. Relations between fluorescence and the structural properties of the pigments are discussed. For the first time, pictures of flowers naturally emitting light are presented. Yellow flowers of the ornamental plant Portulaca grandiflora were chosen as a model for the studies in fluorescence due to the existence of the white phenotype, which was used as a control. Studies were also performed in Lampranthus productus flowers, which contain dopaxanthin as a single pigment. The visible fluorescence of betaxanthins inside the petal cells was detected in a confocal microscope after laser excitation.

Botany: floral fluorescence effect.

The way flowers appear to insects is crucial for pollination. Here we describe an internal light-filtering effect in the flowers of Mirabilis jalapa, in which the visible fluorescence emitted by one pigment, a yellow betaxanthin, is absorbed by another, a violet betacyanin, to create a contrasting fluorescent pattern on the flower's petals. This finding opens up new possibilities for pollinator perception as fluorescence has not previously been considered as a potential signal in flowers.

Differential activation of a latent polyphenol oxidase mediated by sodium dodecyl sulfate.

A kinetic study of the activity of soluble and membrane-bound latent polyphenol oxidase (PPO) extracted from beet root (Beta vulgaris) was carried out. For the first time, two types of behavior (hyperbolic and sigmoid) are reported in the same enzyme for PPO activation by the surfactant sodium dodecyl sulfate (SDS), depending on substrate nature. A kinetic model based on cooperative systems is developed to describe the activation effect of SDS, enabling the determination of the number of surfactant molecules binding to the enzyme in the activation process. The results indicate that the active site of the enzyme is not affected by SDS and that a stepwise conformational change favors the access of hydrophobic substrates compared to hydrophilic ones. Differential activation of PPO mediated by SDS may be of relevance in the control of PPO activity since the enzyme is able to express activity toward a specific substrate while remaining latent to others.

Evidence for a common regulation in the activation of a polyphenol oxidase by trypsin and sodium dodecyl sulfate.

Polyphenol oxidase (PPO) was extracted from beet root, in both soluble and membrane fractions, and in both cases the enzyme was in a latent state. PPO from the membrane fraction showed no diphenolase activity unless it was activated by trypsin or sodium dodecyl sulfate (SDS). The kinetics of the activation process of latent PPO by trypsin was studied and the specific rate constant of active PPO formation, k 3 , showed a value of 0.03 s(-1). The protease-activated form showed a pH optimum (6.5) and kinetic properties identical to those of the SDS-activated enzyme. Evidence is provided for the existence of a common peptide responsible for the regulation of the activity of the enzyme by both proteolysis and SDS detergent. Formation of the active proteolyzate was followed by spectroscopic measurements, Western blotting and partially denaturing SDS-PAGE.