Josefa Escribano

Characterization of the antiradical activity of betalains from Beta vulgaris L. roots

Betalains (betacyanins and betaxanthins), the main pigments of red beet (Beta vulgaris) roots, showed an antiradical effect when measured by the destruction of the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) free radical generated by the horse-radish peroxidase/hydrogen peroxide-mediated oxidation of ABTS. The antiradical activity of betacyanins was greater than that of the betaxanthins and increased with the pH of the reaction medium. The different antiradical properties shown by both types of betalain is discussed in light of the respective ease with which it is possible to withdraw one electron from their molecules and the stability of their corresponding radicals.

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.

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.

Betaxanthins as Substrates for Tyrosinase. An Approach to the Role of Tyrosinase in the Biosynthetic Pathway of Betalains

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis and in the enzymatic browning of fruits and vegetables. The role of tyrosinase in the secondary metabolism of plants still remains unclear, but its implication in betalain biosynthesis has been proposed. Betalains are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In this article, the betaxanthins, tyrosine-betaxanthin (portulacaxanthin II) and dopaxanthin, are reported to be physiological substrates for tyrosinase. The direct activity of tyrosinase on selected betaxanthins is characterized in depth, and conversion of tyrosine-betaxanthin to dopaxanthin and its further oxidation to a series of compounds are described. Identity of the reaction products was studied by high-performance liquid chromatography and electrospray ionization-mass spectrometry. Masses determined for the reaction products were the same in all cases, 389 m/z ([M + H]+) and equal to that determined for betanidin. Data indicate that dopaxanthin-quinone is obtained and evolves to more stable species by intramolecular cyclization. Kinetic parameters for tyrosinase acting on dopaxanthin were evaluated, showing a high affinity for this substrate (Km = 84.3 μm). The biosynthetic scheme of betalains is reviewed and a branch is proposed based on the description of physiological substrates for tyrosinase. Lampranthus productus, Glottiphylum oligocarpum, and Glottiphylum pigmaeum are described as sources of stereopure (2S/S)-dopaxanthin.

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.

A continuous spectrophotometric assay for phospholipase A2 activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).

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.

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.

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

Abstract 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.

Kinetic study of the suicide inactivation of latent polyphenoloxidase from iceberg lettuce (Lactuca sativa) induced by 4-tert-butylcatechol in the presence of SDS

In plants, polyphenoloxidase undergoes an irreversible inactivation during the oxidation of o-diphenol to o-quinones. In the present paper, using latent polyphenoloxidase from iceberg lettuce (Lactuca sativa), in the presence of an activating agent, SDS, the kinetic parameters that characterize the enzyme during its action on the suicide substrate 4-tert-butylcatechol have been determined. The effect of pH has also been considered. It was seen that the presence of SDS in the reaction medium changed the kinetic parameters of the enzyme during suicide inactivation, this phenomenon depended on the SDS concentration up to saturating concentrations. Variations were also observed in the kinetic parameters at pH values below 5 where SDS provoked inactivation of the enzyme. This differing kinetic behaviour during suicide inactivation in the presence of SDS may be caused by the conformational changes provoked by SDS in the enzyme in latent state. Thus, polyphenoloxidases showing suicide inactivation would present an enzymatic activity resulting from the balance between the activation process and suicide inactivation.