Bioactive compounds and antioxidant activity in garambullo fruit (Myrtillocactus geometrizans) at different ripening stages.

Garambullo (Myrtillocactus geometrizans) is endemic in México, and although popularly consumed locally, its nutritional characteristics and value have not been studied in details. The objective of this work was to investigate the bioactive compounds and antioxidant activity in garambullo fruit from different sites at three ripening stages. Fruit from the three ripening stages (red, purple, and dark purple) were investigated for their physicochemical characteristics, hydrophilic (phenolic compounds, betalains, and ascorbic acid), and lipophilic (carotenoids, tocopherols, and fatty acids) bioactive compounds, using spectrophotometry, gas chromatography (GC-FID), and high-pressure liquid chromatography coupled to mass spectrometry (HPLC/DAD-ESI-MS). The antioxidant capacity was measured with the 2,2'-diphenyl-1-picrylhydrazyl and the ferric-ion-reducing antioxidant power assays. The color components of the fruit, chroma and a* values increased, whereas lightness (L*) and b* significantly decreased during ripening. Five betacyanins and four betaxanthins were tentatively identified with HPLC/DAD-ESI-MS, and betacyanins were more abundant than betaxanthins. Betalains content and antioxidant capacity of hydrophilic extracts significantly increased during ripening. Ten phenolic compounds were identified, with ferulic acid being the most abundant. Tocopherols were low (0.023-0.033 mg/100 g fw). Five fatty acids were abundant, and linoleic acid was the most important. Phenolic compounds, ascorbic acid, total carotenoids, and fatty acids decreased during fruit ripening. Garambullo fruit is rich in phytochemical compounds of importance for human nutrition and health. PRACTICAL APPLICATION: The physicochemical and bioactive compounds characterization in garambullo fruit is important to establish maturation and harvesting indices, postharvest strategies to preserve fruit quality and prolong postharvest life, promote the consumption and utilization of the fruit, and the designing of proper functional foods. In addition, the knowledge on the bioactive components might be useful to include this fruit in personalized nutritional approaches for patients with risks of certain chronic diseases. The methodology used in this study could be useful for the study of other fruits, especially those from the Cactaceae family.

Influence of High-Pressure Homogenization on the Physicochemical Properties and Betalain Pigments of Red Beetroot (Beta vulgaris L.) Juice.

High-pressure homogenization (HPH) is considered an innovative and modern method of processing and preserving liquid and semi-liquid foods. The aim of this research was to examine the impact of HPH processing on the content of betalain pigments and physicochemical properties of beetroot juice. Combinations of the following HPH parameters were tested: the pressure used (50, 100, 140 MPa), the number of cycles (1 and 3) and the applied cooling or no cooling. The physicochemical analysis of the obtained beetroot juices was based on the determination of the extract, acidity, turbidity, viscosity and color values. Use of higher pressures and a greater number of cycles reduces the turbidity (NTU) of the juice. Moreover, in order to maintain the highest possible extract content and a slight color change of the beetroot juice, it was crucial to perform sample cooling after the HPH process. The quantitative and qualitative profiles of betalains have been also determined in the juices. In terms of the content of betacyanins and betaxanthins, the highest values were found in untreated juice at 75.3 mg and 24.8 mg per 100 mL, respectively. The high-pressure homogenization process resulted in a decrease in the content of betacyanins in the range of 8.5-20.2% and of betaxanthins in the range of 6.5-15.0%, depending on the parameters used. Studies have shown that that the number of cycles was irrelevant, but an increase in pressure from 50 MPa to 100 or 140 MPa had a negative effect on pigment content. Additionally, juice cooling significantly limits the degradation of betalains in beetroot juice.

Chemical characterization of yellow-orange and purple varieties of Opuntia ficus-indica fruits and thermal stability of their betalains.

Betalains are plant pigments with biological properties and can be used instead of synthetic colorants to confer color and functional properties to foods. The objective of this work was to carry out the chemical characterization of two varieties of prickly pear of Opuntia ficus-indica, one of yellow-orange coloration (Mandarina) and the other of purple coloration (Vigor), through measurements of chemical parameters and color in pulp, antioxidant activity, total phenolic compounds, and betalain content. Considering the thermolability of betalains and their potential applications in food, the thermal stability and activation energy of betacyanins from Vigor variety and betaxanthins from the Mandarina variety were also evaluated and compared with those from beetroot, the main source of betalains. Results for chemical characterization agreed with previous prickly pear reports of other regions, while the thermal degradation kinetics of betalains showed a first-order degradation pattern with respect to time and temperature treatment. Betacyanins from Vigor prickly pear showed similar thermal stability to those from beetroot, which was reflected in similar values of activation energy, while betaxanthins from Mandarina prickly pear showed a higher stability, and therefore a higher activation energy, than those from beetroot. Based on the results, the prickly pear varieties used in this study can be considered as a good source of betalains with potential applications in food and, in addition, the methodology for the evaluation of thermostability can be used to compare the stability of betalains from different sources in a temperature range of 50-90°C. PRACTICAL APPLICATION: The varieties of prickly pear used in this study can be considered a good source of red-purple and yellow-orange easily extractable pigments. In addition, we report a methodology that can be used for the evaluation of the thermal stability of these pigments and to compare this stability between different plant sources. Gaining knowledge on betalain thermal stability will make it possible to propose specific applications, for example, in processed foods requiring different pigment stabilities.

Bioaccessibility of Betalains in Beetroot (Beta vulgaris L.) Juice under Different High-Pressure Techniques.

The influence of high hydrostatic pressure (HHP) and supercritical carbon dioxide (SCCD) on the bioaccessibility of betalains in beetroot (Beta vulgaris L.) juice was investigated. Freshly squeezed juice (FJ) was treated at a mild temperature of 45 °C for 10 min (T45), pasteurization at 85 °C for 10 min (T85), HHP at 200, 400, and 500 MPa at 20 °C for 5 min (HHP200, HHP400, HHP500) and SCCD at 10, 30 and 60 MPa at 45 °C for 10 min (SCCD10, SCCD30, SCCD60). The juice was subjected to an in vitro digestion system equipped with dialysis. The content of betalains was measured with the aid of a High-Performance Liquid Chromatography (HPLC), the antioxidant capacity (AC) (ABTS•+, DPPH•) was analyzed during each digestion step, and the bioaccessibility of betacyanins and betaxanthins was assessed. The SCCD at 30 and 60 MPa significantly increased pigments' bioaccessibility compared with other samples. The 30 MPa proved particularly advantageous, as it increased the bioaccessibility of the total betacyanins and the betaxanthins by 58% and 64%, respectively, compared to the T85 samples. Additionally, higher bioaccessibility of betacyanins was noted in HHP200 and HHP400, by 35% and 32%, respectively, compared to FJ, T45, and T85 samples. AC measured by ABTS•+ and DPPH• assays were not unequivocal. However, both assays showed significantly higher AC in SCCD60 compared to T85 (21% and 31%, respectively). This research contributed to the extended use of the HHP and/or SCCD to design food with higher health-promoting potentials.

Betalains nanodispersions: Effects on betalains stability and on rheological properties of Greek yogurt.

Red beetroot (Beta vulgaris L.) is a great source of betalains. The main betalains are the betacyanins, responsible for the purple color, and betaxanthins, which present a brownish color. These pigments can present antioxidant activity and are very unstable under certain conditions, such as temperature, extreme ranges of pH, and exposure to light. The aim of this work was to obtain beetroot extract (BE) via ultrasound and transform it into nanoparticles by using polyethylene glycol (PBE) and polyethylene glycol with low molecular weight chitosan (PCBE) as dispersants. The stability of the main betalains in the nanodispersions and the effects of the nanodispersions on the color and rheological properties of commercial Greek yogurt were evaluated. Compared to pristine BE, PCBE nanoparticles presented increased stability for the main betalains in acidic conditions (pH 3.0 and 5.0) of 56% and 22%, respectively. Both PBE and PCBE showed enhanced relative thermal stability compared to pristine BE. Furthermore, PCBE improved commercial Greek yogurt's rheological properties and color parameters. PCBE nanodispersions can be successfully applied as a color additive to commercial Greek yogurt.

Betacyanins and Betaxanthins in Cultivated Varieties of Beta vulgaris L. Compared to Weed Beets.

There are 11 different varieties of Beta vulgaris L. that are used in the food industry, including sugar beets, beetroots, Swiss chard, and fodder beets. The typical red coloration of their tissues is caused by the indole-derived glycosides known as betalains that were analyzed in hypocotyl extracts by UV/Vis spectrophotometry to determine the content of betacyanins (betanin) and of betaxanthins (vulgaxanthin I) as constituents of the total betalain content. Fields of beet crops use to be also infested by wild beets, hybrids related to B. vulgaris subsp. maritima or B. macrocarpa Guss., which significantly decrease the quality and quantity of sugar beet yield; additionally, these plants produce betalains at an early stage. All tested B. vulgaris varieties could be distinguished from weed beets according to betacyanins, betaxanthins or total betalain content. The highest values of betacyanins were found in beetroots 'Monorubra' (9.69 mg/100 mL) and 'Libero' (8.42 mg/100 mL). Other beet varieties contained less betacyanins: Sugar beet 'Labonita' 0.11 mg/100 mL; Swiss chard 'Lucullus,' 0.09 mg/100 mL; fodder beet 'Monro' 0.15 mg/100 mL. In contrast with weed beets and beetroots, these varieties have a ratio of betacyanins to betaxanthins under 1.0, but the betaxanthin content was higher in beetcrops than in wild beet and can be used as an alternative to non-red varieties. Stability tests of selected varieties showed that storage at 22 °C for 6 h, or at 7 °C for 24 h, did not significantly reduce the betalain content in the samples.

Comparative UHPLC-HRMS Profiling, Toxicological Assessment, and Protection Against H2O2-Induced Genotoxicity of Different Parts of Opuntia ficus indica.

Opuntia ficus indica has been an important dietary source and a traditionally used medicinal plant. Given the promising health-promoting properties of this plant, a comparative toxicological assessment and antioxidant bioevaluation of extracts from different parts of the plant were carried out in relation to their chemical profile. Toxicity was examined at multiple endpoints using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Comet and the γH2AX In-Cell Western Assay, while hyphenated ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis was carried out to identify main constituents. None of the extracts showed any cytotoxic and genotoxic effect on cell lines used, apart from the flower extract in HepG2 cells at the highest concentration tested (2.5 mg/mL). Both fruit flesh and seed extracts demonstrated a prominent protective effect against H2O2-induced genotoxicity in almost all concentrations tested, while extracts originated from flowers and cladodes were effective only at the low non-cytotoxic (0.312 and 0.625 mg/mL) and high (1.25 and 2.5 mg/mL) concentrations, respectively. In total, 2 phenolic acids, 12 flavonoids, along with 3 feruloyl derivatives and the plant pigment indicaxanthin, were tentatively identified by UHPLC-HRMS analysis. Phenolic acids (compounds 1 and 2) were mainly distributed in cladodes (64.6%), while flavonoids (3-14) in the flowers (81.8%). Overall, the highest amount of total flavonoids (22.76 ± 0.015 mg of quercetin equivalent [QE]/g) and total phenolics (62.80 ± 0.009 mg gallic acid equivalents [GAE]/g) was found in the flower extract. Flavonoid glycosides have not been detected in the seeds and the flesh, while the fruit seed extract contained mainly feruloyl derivatives. Our data provide convincing evidences for the lack of cytotoxic and genotoxic effects of O. ficus indica aqueous extracts and, in parallel, support the potential for further exploitation of this plant in the food supplement or functional food sector.

Implications of the Red Beet Ripening on the Colour and Betalain Composition Relationships.

The evolution during ripening of Beta vulgaris (var. Pablo) on colour and betalain composition, not previously conducted in conjunction in red beets, has been examined. According to the results, it could be asserted that the ripening stage significantly (p < 0.05) influenced on all the studied parameters. On the basis of the betalain content, the optimum ripening stage corresponded to a medium weigh-to-calibre ratio, in the light of the significantly (p < 0.05) higher content of betalains, especially betanin and vulgaxanthin I. Moreover, colour attributes also differed during ripening, having the medium-ripened beetroots a significantly (p < 0.05) more reddish hue (hab) and lower lightness (L*), probably due to the higher content of betaxanthins in this stage. The colour differences among red beets in the stage II and the rest of stages were visually appreciable (ΔE*ab > 3) and mainly qualitative. A new range of opportunities for diversification of colorant market, from a nutritional and colorimetric point of view, could be possible by employing red beets with different stages of ripening.

Encapsulation of cactus (Opuntia megacantha) betaxanthins by ionic gelation and spray drying: A comparative study.

The encapsulation of betaxanthins from orange Opuntia megacantha fruits was performed as a stabilisation strategy for these natural pigments. Betaxanthin-rich extract (BE) was encapsulated by two methodologies, spray drying (SD) and ionic gelation (IG). Encapsulates were obtained by using two encapsulating agents (EA), a mixture of maltodextrin-cactus cladode mucilage and sodium alginate (SA) for SD and IG, respectively. Their properties were compared with the freeze-dried cactus fruit pulp (CP) as control. Total betaxanthin content, moisture content, water activity (aw), particle size, morphology by SEM, colour parameters (CIELab), and thermogravimetric properties were analysed in the encapsulates. Additionally, the antioxidant activity, total dietary fibre content, and pigment stability of encapsulates under two relative humidity conditions were also evaluated. The results showed that the encapsulation technique, EA concentration, and the BE/EA ratio affected the particle size and morphology, the glass transition temperature, and the mass loss of encapsulated materials. The best pigment stabilisation was obtained for the SD capsules, although both encapsulation techniques allowed improving the pigment stability in comparison with the CP taken as control. These results provided evidence that betaxanthins from Opuntia megacantha have the potential to be used as natural pigments, soluble in water, with functional properties for the food industry.

Contribution to the characterization of Opuntia spp. juices by LC-DAD-ESI-MS/MS.

Opuntia spp. fruits are considered as health promoting foods due to the diversity of bioactive molecules found in these fruits. The composition in organic acids, flavonols and betalains in the Opuntia ficus-indica juice from a region of Portugal was accomplished for the first time by liquid chromatography and tandem mass spectrometry using an electrospray ionization source operating in negative and positive mode. The methodology used allowed the detection of 44 compounds, from which 32 were identified. Isorhamnetin derivatives were the dominant flavonol glycosides. A total of 9 betalains including 6 betaxanthins and 3 betacyanin were also detected in the fruit juice samples and indicaxanthin, betanin and isobetanin were the major pigments. Phenolic acid and phenylpyruvic acid derivatives were also identified. To our knowledge, it is the first time derivative compounds from piscidic acid, phenolic compounds and betalains are characterized in cactus pear juice using a single LC-DAD-ESI-MS/MS method.

The effect of thermal treatment on antioxidant capacity and pigment contents in separated betalain fractions.

BACKGROUND: Increased consumption of fruits and vegetables significantly reduces the risk of cardio-vascular disease. This beneficial effect on the human organism is ascribed to the antioxidant compounds these foods contain. Unfortunately, many products, particularly vegetables, need to be subjected to thermal processing before consumption. The aim of this study was to determine the effect of such thermal treatment on the antioxidant capacity and pigment contents in separated fractions of violet pigments (betacyanins) and yellow pigments (betaxanthins and betacyanins). METHODS: Fractions of violet and yellow pigments were obtained by separation of betalain pigments from fresh roots of 3 red beet cultivars using column chromatography and solid phase extraction (SPE). The betalain pigment content was determined in all samples before and after thermal treatment (90°C/30 min) by spectrophotometry, according to Nilsson's method [1970] and antioxidant capacity was assessed based on ABTS. Betalain pigments in the separated fractions were identified using HPLC-MS. RESULTS: After thermal treatment of betacyanin fractions a slight, but statistically significant degradation of pigments was observed, while the antioxidant capacity of these fractions did not change markedly. Losses of betacyanin content amounted to 13-15% depending on the cultivar, while losses of antioxidant capacity were approx. 7%. HPLC/MS analyses showed that before heating, betanin was the dominant pigment in the betacyanin fraction, while after heating it was additionally 15-decarboxy-betanin. Isolated fractions of yellow pigments in red beets are three times less heat-resistant than betacyanin fractions. At losses of yellow pigment contents in the course of thermal treatment reaching 47%, antioxidant capacity did not change markedly (a decrease by approx. 5%). In the yellow pigment fractions neobetanin was the dominant peak in the HPLC chromatogram, while vulgaxanthin was found in a much smaller area, whereas after heating additionally 2-decarboxy-2,3-dehydro-neobetanin was detected. CONCLUSIONS: Both groups of betalain pigments (betacyanins and betaxanthins) exhibit antioxidant capacity before and after heating. Violet beatacyjanins are 3 times more stable when heated than yellow betaxanthins.

HPLC-MS(n) Identification of Betalain Profile of Different Beetroot (Beta vulgaris L. ssp. vulgaris) Parts and Cultivars.

The distribution of betalains in peel, flesh, and petioles of yellow and red beetroot cultivars has been investigated using an High-performance liquid chromatography (HPLC) system with electrospray mass spectrometry. Differences in the levels of betacyanins and betaxanthins between different colored cultivars were individually determined for 3 plant parts. The content of almost all analyzed compounds decreased in the following order: peel > flesh > petiole. Betanin/isobetanin pigments comprised a major portion of the relative peak area measured in red beetroot peel. Isobetanin relative peak areas were also high in leaf petioles (68.94% to 74.16%) of red colored cultivars. However, betacyanins were completely absent from the extracts of all analyzed parts of yellow beet. Glutamine-bx represented a very high relative peak area (59.54% to 64.18%) in flesh of red-colored cultivars analyzed in the study. Our results indicate that red beet cultivars can be utilized as a potential source of red and yellow natural colorants. However, differences in pigment composition among different beetroot parts must be considered and in order to maximize the pigment yields petioles can also be used as a source rich in specific betalain compounds.

Transposon-mediated mutation of CYP76AD3 affects betalain synthesis and produces variegated flowers in four o'clock (Mirabilis jalapa).

The variegated flower colors of many plant species have been shown to result from the insertion or excision of transposable elements into genes that encode enzymes involved in anthocyanin synthesis. To date, however, it has not been established whether this phenomenon is responsible for the variegation produced by other pigments such as betalains. During betalain synthesis in red beet, the enzyme CYP76AD1 catalyzes the conversion of L-dihydroxyphenylalanine (DOPA) to cyclo-DOPA. RNA sequencing (RNA-seq) analysis indicated that the homologous gene in four o'clock (Mirabilis jalapa) is CYP76AD3. Here, we show that in four o'clock with red perianths, the CYP76AD3 gene consists of one intron and two exons; however, in a mutant with a perianth showing red variegation on a yellow background, a transposable element, dTmj1, had been excised from the intron. This is the first report that a transposition event affecting a gene encoding an enzyme for betalain synthesis can result in a variegated flower phenotype.

Physical, chemical, and antioxidant activity characterization of pitaya (Stenocereus pruinosus) fruits.

Fruits with red and orange flesh of the columnar cactus pitaya (Stenocereus pruinosus) were studied to evaluate physical characteristics, total soluble solids, betalains and soluble phenols content, and antioxidant activity. Fruits had, in average, weight of 179.0 g, 9.8 °Brix, 9.4 % carbohydrates, 1.25 % protein, 0.11 % ethereal extract, 0.60 % crude fiber, and 0.62 % ash. Also, fruits resulted rich in Fe (22.8-27.8 mg/kg). Hue angle and contents of betacyanins, betaxanthins (µg/g dry sample), and total soluble phenols (mg GAE/g fresh sample) were 19.8°, 2860.0, 3210.0, and 1.6 in the red material, and 28.9°, 470.0, 2670.0, and 1.2, respectively, in the orange fruit. The antioxidant capacity was higher in the red material, since the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) technique reported 1090.6 and 735.4 µmol of Trolox equivalents/g fresh flesh in red and orange fruits, respectively, while the oxygen radical absorbance capacity (ORAC) assay reported 7.84 and 5.16 µmol of Trolox equivalents/g fresh flesh, respectively. The chromatographic profile showed five betalains in red fruits, but only four of them were observed in those orange fleshed.

Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography.

Polar betacyanin pigments together with betaxanthins from ripe cactus fruits of Hylocereus polyrhizus (Cactaceae) were fractionated by means of preparative ion-pair high-speed countercurrent chromatography (IP-HSCCC) also using the elution-extrusion (EE) approach for a complete pigment recovery. HSCCC separations were operated in the classical 'head-to-tail' mode with an aqueous mobile phase. Different CCC solvent systems were evaluated in respect of influence and effectiveness of fractionation capabilities to separate the occurring pigment profile of H. polyrhizus. For that reason, the additions of two different volatile ion-pair forming perfluorinated carboxylic acids (PFCA) were investigated. For a direct comparison, five samples of Hylocereus pigment extract were run on preparative scale (900 mg) in 1-butanol-acetonitrile-aqueous TFA 0.7% (5:1:6, v/v/v) and the modified systems tert.-butyl methyl ether-1-butanol-acetonitrile-aqueous PFCA (2:2:1:5, v/v/v/v) using 0.7% and 1.0% trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) in the aqueous phase, respectively. The chemical affinity to the organic stationary CCC solvent phases and in consequence the retention of these highly polar betalain pigments was significantly increased by the use of the more lipophilic fluorinated ion-pair reagent HFBA instead of TFA. The HFBA additions separated more effectively the typical cacti pigments phyllocactin and hylocerenin from betanin as well as their iso-forms. Unfortunately, similar K(D) ratios and selectivity factors alpha around 1.0-1.1 in all tested solvent systems proved that the corresponding diastereomers, 15S-type pigments cannot be resolved from the 15R-epimers (iso-forms). Surprisingly, additions of the stronger ion-pair reagent (HFBA) resulted in a partial separation of hylocerenin from phyllocactin which were not resolved in the other solvent systems. The pigments were detected by means of HPLC-DAD and HPLC-electrospray ionization-MS using also authentic reference materials.

Betalains in red and yellow varieties of the Andean tuber crop ulluco (Ullucus tuberosus).

The betalain pigments in ulluco (Ullucus tuberosus), a tuberous crop native to the Andes, have been investigated for the first time using LC-DAD-ESI-MS-MS(2) analyses. Five red, yellow, and red-spotted accessions introduced into New Zealand as a new food crop plus two red tetraploid lines were investigated. Thirty-two different betalains were identified. Both the yellow and red tubers were rich in yellow betaxanthins, and the most prominent among the 20 identified were histidine-betaxanthin, arginine-betaxanthin and glutamine-betaxanthin. Arginine-betaxanthin has been reported to occur naturally only once before and was found in yellow ulluco but not in the red tubers. Twelve betacyanins were found in red tubers, with roughly 50% of this content being betanin/isobetanin. Betacyanin levels were up to 70 microg/g fresh weight in red tubers, but were below quantifiable levels in yellow tubers. Betaxanthin levels were up to 50 microg/g fresh weight in yellow tubers. Interference by betacyanins in measuring levels of betaxanthins by visible spectrophotometry is discussed. Low concentrations of betalains were detected in leaves, whereas stems contained total levels similar to the tubers, with dopamine-betaxanthin and betanin being the major pigments. This is the first report describing both the betacyanin and betaxanthin patterns in a plant from the Basellaceae family.