Purification and characterization of betacyanin monomers from Hylocereus polyrhizus peel: A comparative study of their antioxidant and antidiabetic activities with mechanistic insights.

Betacyanins have garnered escalating research interest for their promising bioactivities. However, substantial challenges in purification and separation have impeded a holistic comprehension of the distinct bioactivities of individual betacyanins and their underlying mechanisms. Herein, betanin and phyllocactin monomers with purity exceeding 95% were successfully obtained from Hylocereus polyrhizus peel using a feasible protocol. These monomers were subsequently employed for comparative bioactivity assessments to uncover underlying mechanisms and illuminate structure-activity relationships. Interestingly, phyllocactin exhibited superior antioxidant activities and 36.1% stronger inhibitory activity on α-glucosidase compared to betanin. Mechanistic studies have revealed that they function as mixed-type inhibitors of α-amylase and competitive inhibitors of α-glucosidase, with interactions predominantly driven by hydrogen bonding. Notably, phyllocactin demonstrated a greater binding affinity with enzymes than betanin, thereby substantiating its heightened inhibitory activity. Overall, our results highlight novel bioactivities of betacyanin monomers and provide profound insights into the intricate interplay between structures and properties.

Dehydrogenation of Betacyanins in Heated Betalain-Rich Extracts of Red Beet (Beta vulgaris L.).

Betacyanins are a group of water-soluble red-violet compounds containing nitrogen in their structure. These are biosynthesized in red beetroot (Beta vulgaris L.), a widely consumed vegetable that contains significant amounts of nutritious and bioactive compounds which are also found in dietary supplements. This contribution presents results of betacyanin thermal oxidation (resulting in dehydrogenation) interrelated with decarboxylation in selected acetate/phosphate buffers at pH 3-8 and at 85 °C, which may be of particular significance for formulation and performance of foods. Most of the reaction products were detected at the highest concentrations in the acidic solutions (pH 3-4). The main dehydrogenation reaction pathways were monitored by LC-DAD-MS/MS and were associated with decarboxylation of the principal extract pigments, betanin/isobetanin and neobetanin, at carbon positions C-2 and C-17. Additional reactions are accompanied by the 2,15-decarboxylation processes at different dehydrogenation levels with 15-decarboxy-betanin and 2,15-bidecarboxy-betanin, structurally elucidated by NMR analysis, as the distinct indicators of this route type. For other novel pigments detected, 2,15-bidecarboxy-xanbetanin, 2,15-bidecarboxy-xanneobetanin and 2,15,17-tridecarboxy-neobetanin, additional high resolution mass spectrometric analyses were performed and confirmed their molecular formulas.

Synthesis of Ammonium-Based Ionic Liquids for the Extraction Process of a Natural Pigment (Betanin).

The use of new synthesized ammonium-based ionic liquids was explored as an alternative to the current process implemented in the betanin extraction from red beet juice, resulting in high yields: 70% and 82%. Betanin is a vegetal pigment that has been applied to a large variety of products in the food industry, which is important, for it can work as a substitute for the red synthetic dyes used nowadays. Additionally, the use of the kosmotropic salt sodium acetate was explored in order to separate the complex formed by the ionic liquid and pigment of interest in a process that combined two techniques: ATPS (aqueous two-phase system) and SOES (salting-out extraction system). The results reveal that the studied techniques could work as a novel process for the extraction of betanin from red beet juice employing ionic liquids, which have not been tested for this purpose in other research.

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.

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.

Betacyanins from Gomphrena globosa L. flowers: Incorporation in cookies as natural colouring agents.

A betacyanin rich extract was obtained from the flowers of Gomphrena globosa L. by ultrasound-assisted extraction and dried either by lyophilization or spray-drying, was tested as a natural colourant in cookies and compared to a commercial colourant. The extracts were characterized in terms of betacyanin content and antioxidant potential. The effects of the colourants incorporation in the cookies were assessed through proximate composition, soluble sugars, fatty acids, color, texture and microbial load, over a shelf life of 30 days. Considering all the assays and analyzing the results through a 2-way analysis of variance, the cookies incorporated with spray-dried colourant showed the most intense pink coloration while cookies incorporated with lyophilized extract lost less color intensity over time. Thus, betacyanin extracts have potential as pink natural alternatives to synthetic colourants in the food industry.

Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells.

Microglial activation can release free radicals and various pro-inflammatory cytokines, which implicates the progress of a neurodegenerative disease. Therefore suppression of microglial activation can be an appropriate strategy for combating neurodegenerative diseases. Betanin is a red food dye that acts as free radical scavenger and can be a promising candidate for this purpose. In this study, purification of betanin from red beetroots was carried out by normal phase colum chromatography, yielding 500 mg of betanin from 100 g of red beetroot. The purified betanin was evaluated by TLC, UV-visible, HPLC, ESI-MASS, FT-IR spectroscopy. Investigation on the inhibitory effect of betanin on activated microglia was performed using primary microglial culture. The results showed that betanin significantly inhibited lipopolysaccharide induced microglial function including the production of nitric oxide free radicals, reactive oxygen species, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1ß). Moreover, betanin modulated mitochondrial membrane potential, lysosomal membrane permeabilization and adenosine triphosphate. We further investigated the interaction of betanin with TNF-α, IL-6 and Nitric oxide synthase (iNOS or NOS2) using in silico molecular docking analysis. The docking results demonstrated that betanin have significant negative binding energy against active sites of TNF-α, IL-6 and iNOS.

Betacyanin as Bioindicator Using Time-Temperature Integrator for Smart Packaging of Fresh Goat Milk.

Smart packaging is a packaging system with embedded sensor or indicator technology, which provides information on the quality of the product, especially perishable foods such as goat milk. One application of smart packaging is to use a time-temperature bioindicator. The purpose of this study was to determine the quality of fresh goat milk during storage at freezing temperatures (-20 ± 2°C) for 31 days and room temperature (25 ± 3°C) for 24 hours using a time-temperature indicator by utilizing a natural dye betacyanin. The method used was descriptive analysis, and the data obtained were processed using the correlation regression test. The samples were observed at freezing temperature every 24 hours and room temperature at 0, 1, 2, 3, 4, 5, 6, 8, 10, and 24 hours. The observation criteria consisted of changes in bioindicator color, milk pH, and total microbes. The results showed that color changes of the bioindicator film at room temperature were more noticeable than at freezing temperature. Based on changes in color of the bioindicator at room temperature, the sample was safe for consumption until the 5th hour with pH 6.51, and the biofilm color characteristics were L∗ = 82.49, a∗ = 21.46, and b∗ = -7.33, but the total number of microbes did not fulfil Indonesian National Standard at the 24th hour, i.e., 1.36 × 106 CFU/ml. At freezing temperatures, fresh goat milk was still safe for consumption until the 31st day with pH 6.51 and total microbe of 1.89 × 105 CFU/ml, and the biofilm color characteristics were L∗ = 80.52, a∗ = 24.15, and b∗ = -7.91. The results of this study concluded that the milk expiration limit based on the betacyanin indicator was 5 hours at room temperature and 31 days at freezing temperature.

Ultrasound and Microwave Assisted Extraction of Opuntia Fruit Peels Biocompounds: Optimization and Comparison Using RSM-CCD.

Ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) of bioactive compounds, peels from Opuntia engelmannii cultivar (cv.) Valencia were optimized by response surface methodology. Randomized extraction runs were performed for each of the technologies employed in order to build effective models with maximum (bioactive molecules content and yield) and minimum (antioxidant activity) responses. A 5-level, 4-factor central composite design was used to obtain target responses as a function of extraction time (t), solid to liquid ratio (S/L), methanol concentration (metOH), and temperature (T). Specific response optimization for each technology was analyzed, discussed, and general optimization from all the responses together was also gather. The optimum values for each factor were: t = 2.5 and 1.4 min, S/L = 5 and 5 g/L, metOH = 34.6 and 0% of methanol and T = 30 and 36.6 °C, achieving maximum responses of 201.6 and 132.9 mg of betalains/g, 13.9 and 8.0 mg of phenolic acids/g, 2.4 and 1.5 mg of flavonoids/g, 71.8% and 79.1% of extractable solid and IC50 values for the antioxidant activity of 2.9 and 3.6, for UAE and MAE, respectively. The present study suggested UAE as the best extraction system, in order to maximize recovery of bioactive compounds with a high antioxidant activity.

Betanin assisted synthesis of betanin@silver nanoparticles and their enhanced adsorption and biological activities.

Natural red purple dye, Betanin, was extracted from the beetroot, purified by aqueous two- phase extraction and gel permeation column chromatography, and used as a reducing agent for the synthesis of silver-betanin core-shell triangular nanodisks for the first time. Spectroscopic data show that the nanoparticle structure is core@shell like with Ag as core and betanin as shell. Langmuir monolayer model (qm = 32.4 mg/g, RL = 0.99 and R2 = 0.997) was the best fit adsorption isotherm for the dye removal. Adsorption kinetics is well explained by pseudo-second-order equation. Gibbs free energy (ΔG0 = -2.59 kJ mol-1), enthalpy (ΔH0 = 18.05 kJ mol-1), and entropy (ΔS0 = 68.92 J/K/mol) were calculated and discussed. The antibacterial activity of betanin-AgNPs were determined against Escherichia coli MTCC-450 (E. coli) and Staphylococcus aureus MTCC-3160 (S. aureus) bacteria.

Extraction of red beet extract with ß-cyclodextrin-enhanced ultrasound assisted extraction: A strategy for enhancing the extraction efficacy of bioactive compounds and their stability in food models.

Improving the extraction efficiency and stability of red beet compounds has gained the attention of researchers due to their high nutritional and health benefits. In this study, ß-cyclodextrin (ß-CD) enhanced ultrasound assisted extraction was used for the extraction of red beet extract, and lyophilized extracts were characterized with FTIR and DSC analyses. The samples extracted with aqueous 5% ß-CD solutions revealed the highest content of betanin (2.243 ±â€¯0.04 mg) and total phenolic compounds (20.03 ±â€¯1.28 mg GAE/g DW), and the highest DPPH inhibition activity (59.87 ±â€¯4.94%). Additionally, complexation with ß-CD significantly enhanced the stability of betanin, phenolic compounds and antiradical activity in the stored beverage and gummy candy models at various pH and temperature conditions during 28 days. In conclusion, ß-CD-enhanced ultrasound assisted extraction is a suitable approach to extracting and stabilizing the red beet compounds for application in food, nutraceutical, and medical fields.

Thermoprotective properties of Opuntia ficus-indica f. inermis cladodes and mesocarps on sheep lymphocytes.

This study aims to investigate the thermoprotective properties of Opuntia ficus-indica f. inermis. Extracts were prepared from cladodes (CE) and mesocarps (ME), then subjected to a spectrophotometric and LC-MS analyses. Lymphocytes were isolated from peripheral blood of non-stressed sheep, supplemented with CE, ME, betanin or α-tocopherol, and subjected to two thermal treatments: 40 and 41 °C, for 6 h. Viable lymphocytes and H2O2 production were evaluated. The antioxidant activity of ME was 3.43 folds higher than CE. The LC-MS analysis of CE and ME allowed identifying 11 phenolic acids, 2 flavanones, 6 flavones, 3 flavonols and 1 betanin type betacyanin. Lymphocytes mortality increased linearly as function of the severity and the duration of heat stress. This mortality was correlated with H2O2 production. At 41 °C, only ME allowed maintaining lymphocytes viability. Moreover, ME was more efficient than CE in reducing H2O2 production. This thermoprotection was ensured by betaxanthin and betacyanin pigments. Interestingly, betanin was more efficient than α-tocopherol in preventing hyperthermia-induced lymphocytes' mortality. We report here for the first time the thermoprotective properties of cladodes and mesocarps of Opuntia ficus-indica f. inermis. Betanin was able to maintain lymphocyte viability through reducing H2O2 production, and therefore the oxidative-induced heat stress.

Betanin, a Natural Food Additive: Stability, Bioavailability, Antioxidant and Preservative Ability Assessments.

Betanin is the only betalain approved for use in food and pharmaceutical products as a natural red colorant. However, the antioxidant power and health-promoting properties of this pigment have been disregarded, perhaps due to the difficulty in obtaining a stable chemical compound, which impairs its absorption and metabolism evaluation. Herein, betanin was purified by semi-preparative HPLC-LC/MS and identified by LC-ESI(+)-MS/MS as the pseudomolecular ion m/z 551.16. Betanin showed significant stability up to -30 °C and mild stability at chilling temperature. The stability and antioxidant ability of this compound were assessed during a human digestion simulation and ex vivo colon fermentation. Half of the betanin amount was recovered in the small intestine digestive fluid and no traces were found after colon fermentation. Betanin high antioxidant ability was retained even after simulated small intestine digestion. Betanin, besides displaying an inherent colorant capacity, was equally effective as a natural antioxidant displaying peroxy-radical scavenger ability in pork meat. Betanin should be considered a multi-functional molecule able to confer an attractive color to frozen or refrigerated foods, but with the capacity to avoid lipid oxidation, thereby preserving food quality. Long-term supplementation by beetroot, a rich source of betanin, should be stimulated to protect organisms against oxidative stress.

Biofilm inhibiting activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

AIMS: To investigate the biofilm inhibitory activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. METHODS AND RESULTS: The pulp of red pitahaya and the leaves of red spinach were extracted using methanol followed by subfractionation to obtain betacyanin fraction. The anti-biofilm activity was examined using broth microdilution assay on polystyrene surfaces and expressed as minimum biofilm inhibitory concentration (MBIC). The betacyanin fraction from red spinach showed better anti-biofilm activity (MBIC: 0·313-1·25 mg ml-1 ) against five Staph. aureus strains while the betacyanin fraction from red pitahaya showed better anti-biofilm activity (MBIC: 0·313-0·625 mg ml-1 ) against four P. aeruginosa strains. Both betacyanin fraction significantly reduced hydrophobicity of Staph. aureus and P. aeruginosa strains. Numbers of Staph. aureus and P. aeruginosa attached to polystyrene were also reduced without affecting their cell viability. CONCLUSION: Betacyanins can act as anti-biofilm agents against the initial step of biofilm formation, particularly on a hydrophobic surface like polystyrene. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to investigate the use of betacyanin as a biofilm inhibitory agent. Betacyanin could potentially be used to reduce the risk of biofilm-associated infections.

Enhancing the antimicrobial and antifungal activities of a coloring extract agent rich in betacyanins obtained from Gomphrena globosa L. flowers.

Although less explored than beetroot (Beta vulgaris L.), the flowers of Gomphrena globosa L. are a very suitable source of betacyanins with strong pigmentation features, together with many other desirable bioactive properties. Thus, the aim of this study was to enhance the antimicrobial and antifungal activities of a pigmented extract obtained from G. globosa flowers by ultrasound assisted extraction (UAE). The procedure was supported with the application of the response surface methodology, a robust optimization technique that allows to study jointly the effects of several variables and responses. To enhance the antimicrobial (Bacillus cereus, Listeria monocytogenes, Escherichia coli and Salmonella typhimurium) and antifungal (Aspergillus flavus, Aspergillus niger, Penicillium ochrochloron and Penicillium verrucosum) activities, the responses were evaluated in terms of the concentrations needed to obtain minimum inhibitory (MIC), minimum bactericidal (MBC) and minimum fungicidal (MFC) concentrations. It was found that the optimal UAE conditions were 10.8 min, 410.5 W, 57.8% of ethanol, and 5 g L-1 of the solid-liquid ratio providing the following response values: (1) from the studied species of bacteria, the MIC ranged from ∼0.15 to 0.35 g L-1 and the MBC ranges were ∼0.30 to 0.65 g L-1; and (2) from the studied fungus species, the MIC ranged from ∼0.20 to 0.30 g L-1 and the MFC ranges were ∼0.40 to 0.65 g L-1. The antibacterial activity dose levels were lower than the antifungal ones. In conclusion, the results obtained in this study highlight extracts from G. globosa flowers as natural sources of betacyanins with application as food colorants with important antimicrobial and antifungal activities.

Liquid chromatographic techniques in betacyanin isomers separation from Gomphrena globosa L. flowers for the determination of their antimicrobial activities.

High-speed counter-current chromatography (HSCCC) combined with HPLC were established to purify, for the first time, betacyanin isomers from Gomphrena globosa L. flowers. The mixture of tert-butyl methyl ether - butanol - methanol - water TBME-BuOH-MeOH-H2O (1.0% heptafluorobutyric acid HFBA) 2:2:1:5 (v/v/v/v) in reversed phase mode was used for preliminary purification of gomphrenin derivatives. Then, semiprep-HPLC was used to obtained ten pure betacyanins: gomphrenins II and III with their cis-isomers as well as sinapoyl-gomphrenin I and its corresponding diastereomers. This is the first study on separation of naturally coexisting acylated gomphrenin isomers. The isolated compounds showed a broad antimicrobial spectrum (in the range of 0.19-1.5 mg mL-1) by inhibiting the growth of all of food-borne pathogens tested. The antimicrobial activity of acylated betacyanins was higher than that of non-acylated gomphrenin I/isogomphrenin I. Therefore, the acylation with phenolic acids increases the activity of betacyanins against fungi and bacteria, suggesting a synergetic effect of the acylated moieties and the pigment chromophoric system on the activity, which was evaluated for the first time.

Microencapsulated betacyanin from colored organic quinoa (Chenopodium quinoa Willd.): optimization, physicochemical characterization and accelerated storage stability.

BACKGROUND: Betalains are presently gaining popularity as pigments for use as natural colorants and/or bioactive compounds in functional foods. Quinoa (Chenopodium quinoa Willd.) has been recognized as an extremely nutritious grain and has recently been found to be a novel and good betalain source. Microencapsulation has been studied as a protected-delivery procedure to stabilize betalains. There are no studies about microencapsulation of betacyanins extracted from quinoa using spray-drying technology. RESULTS: Optimal microencapsulation was obtained at a drying temperature of 165 °C, a rotameter air flow rate of 47 mm (940 L h-1 ) and 10% w/w maltodextrin, which produced good encapsulation yield (58.1%) and efficiency (100%). Optimized maltodextrin-betacyanin microcapsules (diameter 4.4 µm) have low moisture (1.64 ± 0.08%) and water activity (0.127 ± 0.006), a betacyanin content of 0.1995 ± 0.0017 g kg-1 and saponin content <0.080 mg kg-1 . The oxygen consumption rate by betacyanin was -4.373 × 10-5 bar min-1 at 80 °C and -6.67 × 10-5 bar min-1 at 90 °C, which was accompanied by fading of the color. CONCLUSION: Microencapsulated betacyanin was optimized by response surface methodology, and its stability was measured under accelerated conditions by oxygen consumption. Microencapsulations contain betacyanin and low saponin concentration, which might confer unique health-promoting properties. © 2018 Society of Chemical Industry.

Aqueous biphasic systems formed by deep eutectic solvent and new-type salts for the high-performance extraction of pigments.

Deep eutectic solvent (DES) composed of polypropylene glycol 400 (PPG 400) and tetrabutylammonium bromide (TBAB) was combined with a series of new-type salts such as quaternary ammonium salts, amino acid and polyols to form Aqueous Biphasic Systems (ABSs). Phase-forming ability of the salts was investigated firstly. The results showed that polyols had a relatively weak power to produce phases within studied scopes. And the shorter of carbon chain length of salts, the easier to obtain phase-splitting. Then partitioning of three pigments in PPG 400/betaine-based ABSs was addressed to investigate the effect of pigments' hydrophobicity on extraction efficiency. It was found that an increase in hydrophobicity contributed to the migration of pigments in the DES-rich phase. On the other hand, with a decline in phase-forming ability of salts, the extraction efficiency of the whole systems started to go down gradually. Based on the results, selective separation experiment was conducted successfully in the PPG 400/betaine-based systems, including more than 93.00% Sudan Ⅲ in the top phase and about 80.00% sunset yellow FCF/amaranth in the bottom phase. Additionally, ABSs constructed by DES/betaine for partitioning amaranth were further utilized to explore the performances of influence factors and back extraction. It can be concluded that after the optimization above 98.00% amaranth was transferred into the top phase. And 67.98% amaranth can be transferred into the bottom phase in back-extraction experiment. At last, dynamic light scattering (DLS) and transmission electron microscope (TEM) were applied to probe into extraction mechanism. The results demonstrated that hydrophobicity played an important role in the separation process of pigments. Through combining with new-type DES, this work was devoted to introducing plentiful salts as novel compositions of ABSs and providing an eco-friendly extraction way for partitioning pigments, which boosted development of ABSs in the monitoring food safety field.

Studies on polar high-speed counter-current chromatographic systems in separation of amaranthine-type betacyanins from Celosia species.

Betacyanins, natural plant pigments exhibiting antioxidant and chemopreventive properties, were extracted from Celosia spicata (Thouars) Spreng. inflorescences and separated by high-speed counter-current chromatography (HSCCC) in two polar solvent systems composed of: TBME - 1-BuOH - ACN - H2O (0.7% HFBA, 2:2:1:5, v/v/v/v) (system I) and EtOH - ACN - 1-PrOH - (NH4)2SO4satd.soln - H2O (0.5:0.5:0.5:1.2:1, v/v/v/v/v) (system II). The systems were used in the head-to-tail (system I) and tail-to-head (system II) 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 73.5% (system I) and 80.0% (system II). For the identification of separated betacyanins in the crude extract as well as in the HSCCC fractions, LC-DAD-ESI-MS/MS analyses were performed. Depending on the target compounds, each of the systems exhibit meaningfully different selectivity and applicability. For the pairs of amaranthines (1/1') and betanins (2/2'), the best choice is the system II, but the acylated amaranthine pairs (3/3' and 4/4') can be resolved only in the ion-pair system I. For the indication of the most suitable solvent system for Celosia plumosa hort., Celosia cristata L. and Celosia spicata (Thouars) Spreng. species, the profiles of betacyanins in different plant parts were studied.

Separation of betacyanins from purple flowers of Gomphrena globosa L. by ion-pair high-speed counter-current chromatography.

Betacyanins, known as antioxidants and chemopreventive natural compounds with colourful properties, were extracted from purple flowers of Gomphrena globosa L. belonging to the Amaranthaceae family and separated for the first time by ion-pair high-speed counter-current chromatography (HSCCC). The pigments were detected by LC-DAD-ESI-MS/MS technique. Separation of betacyanins (300mg) by HSCCC was accomplished in four solvent systems: tert-butyl methyl ether - butanol - acetonitrile - water (0.7% and 1.0% HFBA - heptafluorobutyric acid - system I and III) and tert-butyl methyl ether - butanol - methanol - water (0.7% and 1.0% HFBA - system II and IV) (2:2:1:5, v/v/v/v) in the head-to-tail mode. The mobile phase (aqueous phase) was run at 2.0ml/min and the column rotation speed was 860rpm. The applied systems enabled to study the influence of HFBA concentration as well as systems polarity on betacyanins separation. Comparison of the systems containing 0.7% HFBA (systems I-II) demonstrates that the replacement of acetonitrile by methanol increases the resolution (Rs) between all betacyanins and does not influence the retention of the stationary phase (Sf=76%). Higher concentration of the acid in systems III-IV slightly decreases Sf to 71% in the systems with 1.0% HFBA. Comparison of the resolution values for betacyanins in the systems with 0.7% and 1.0% HFBA demonstrates that higher concentration of the acid improves the separation effectiveness for all betacyanins as a result of increasing of the chemical affinity of the pigments to the organic stationary phase in HSCCC. The systems III-IV with 1% HFBA are the most effective for the separation of all the studied betacyanins.