Combining Conventional Organic Solvent Extraction, Ultrasound-Assisted Extraction, and Chromatographic Techniques to Obtain Pure Betanin from Beetroot for Clinical Purposes.

Red beetroot extract (E162) is a natural colorant that owes its color to betanin, its major red pigment. Betanin displays remarkable antioxidant, anti-inflammatory, and chemoprotective properties mediated by its structure and influence on gene expression. However, the betanin employed in most preclinical assays is a beetroot extract diluted in dextrin, not pure betanin, as no isolated compound is commercially available. This makes its use inaccurate concerning product content estimates and biological effect assessments. Herein, a combination of conventional extraction under orbital shaking and ultrasound-assisted extraction (UAE) to purify betanin by semi-preparative HPLC was performed. The employed methodology extracts betalains at over a 90% yield, achieving 1.74 ± 0.01 mg of pure betanin/g beetroot, a 41% yield from beetroot contents increasing to 50 %, considering the betalains pool. The purified betanin exhibited an 85% purity degree against 32 or 72% of a commercial standard evaluated by LC-MS or HPLC methods, respectively. The identity of purified betanin was confirmed by UV-Vis, LC-MS, and 1H NMR. The combination of a conventional extraction, UAE, and semi-preparative HPLC allowed for betanin purification with a high yield, superior purity, and almost three times more antioxidant power compared to commercial betanin, being, therefore, more suitable for clinical purposes.

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.

Anticancer effects of root and beet leaf extracts (Beta vulgaris L.) in cervical cancer cells (HeLa).

Cervical cancer is the fourth leading cause of cancer mortality in women worldwide. Beetroot (Beta vulgaris L.) has bioactive compounds that can inhibit the progression of different types of cancer. To analyze the antiproliferative effects of beet leaf and root extracts, we performed MTT, clonogenic survival, cell cycle analysis, Annexin/PI labeling, and western blotting. Here, we report that 10 and 100 µg/ml of root and leaf extracts decreased cell viability and potentiated rapamycin and cisplatin effects while decreased the number of large colonies, especially at 10 µg/ml (293.6 of control vs. 200.0 of leaf extract, p = .0059; 138.6 of root extract, p = .0002). After 48 hr, 100 µg/ml of both extracts led to increased sub-G1 and G0/G1 populations. In accordance, 100 µg/ml of root extract induced early apoptosis (mean = 0.64 control vs. 1.56 root; p = .048) and decreased cell size (p < .0001). Both extracts decreased phosphorylation and expression of mechanistic Target of Rapamycin (mTOR) signaling, especially by inhibiting ribosomal protein S6 (S6) phosphorylation, increasing cleaved poly(ADP-ribose) polysomerase 1 (PARP1) and Bcl-2-like protein 11 (BIM), and decreasing cyclin D1 expression, which regulates cell cycle progression. Here, we demonstrate that beetroot and leaf extracts could be an efficient strategy against cervical cancer.

Beetroot, a Remarkable Vegetable: Its Nitrate and Phytochemical Contents Can be Adjusted in Novel Formulations to Benefit Health and Support Cardiovascular Disease Therapies.

The cardioprotective effects of dietary nitrate from beetroot in healthy and hypertensive individuals are undeniable and irrefutable. Nitrate and nitrate-derived nitrite are precursors for nitric oxide synthesis exhibiting an effect on cardiomyocytes and myocardial ischemia/reperfusion, improving endothelial function, reducing arterial stiffness and stimulating smooth muscle relaxation, decreasing systolic and diastolic blood pressures. Beetroot phytochemicals like betanin, saponins, polyphenols, and organic acids can resist simulated gastrointestinal digestion, raising the hypothesis that the cardioprotective effects of beetroots result from the combination of nitrate/nitrite and bioactive compounds that limit the generation of reactive oxygen species and modulate gene expression. Nitrate and phytochemical concentrations can be adjusted in beet formulations to fulfill requirements for acute or long-term supplementations, enhancing patient adherence to beet intervention. Based on in vitro, in vivo, and clinical trials, beet nitrate and its bioactive phytochemicals are promising as a novel supportive therapy to ameliorate cardiovascular diseases.

Metabolic profile of pitaya (Hylocereus polyrhizus (F.A.C. Weber) Britton & Rose) by UPLC-QTOF-MSE and assessment of its toxicity and anxiolytic-like effect in adult zebrafish.

Pitaya is a Cactacea with potential for economic exploitation, due to its high commercial value and its functional components - such betalains, oligosaccharides and phenolic compounds. Although the biological activities of pitaya have been studied using in vivo and in vitro models (anti-inflammatory and antiproliferative activities, as example), its anxiolytic-like effect is still unexplored. Therefore, the aim of this work was to perform a characterization of pulp and peel of pitaya (Hylocereus polyrhizus (F.A.C. Weber) Britton & Rose) using UPLC-QTOF-MSE, and to assess its toxicity and anxiolytic-like effect in adult zebrafish (Danio rerio). The results showed 16 and 15 compounds (in pulp and peel, respectively), including maltotriose, quercetin-3-O-hexoside, and betalains, putatively identified by UPLC-QTOF-MSE. Thus, pitaya pulp and peel showed no toxicity in both models tested (Vero cell lines and zebrafish model, LC50 ˃ 1 mg/mL); and a significant anxiolytic activity, since the treated fish reduced the permanence in the clear zone (Light & Dark Test) compared to that in the control, exhibiting anxiolytic-simile effect of diazepam. However, these effects were reduced by pre-treatment with the flumazenil suggesting that the pulp and peel of pitaya are anxiolytics agents mediated via the GABAergic system. These findings suggested that H. polyrizhus has the potential of developing an alternative plant-derived anxiolytic therapy. In addition, pitaya peel (which is a waste in the food industry) should be regarded as a valuable product, which has the potential as an economic value-added ingredient for anxiety disorders.

Microencapsulation of betanin in monodisperse W/O/W emulsions.

Betanin is the main pigment of the food color beetroot red (E162). Due to the fair heat and light stability of E162, this pigment is mainly used in minimally processed packaged food products. Encapsulation increases the stability of betanin, but detailing on the effect of different sources of betanin on the properties and stability of multiple emulsions are scarce. Here we describe the encapsulation of E162, spray-dried beetroot juice and betanin in a monodisperse food-grade water-in-oil-in-water (W/O/W) emulsions by using microchannel emulsification. We compare the tinctorial strength of the encapsulated pigments and investigate the effect of temperature, storage period and pigment concentration on emulsion stability and color. Betanin increases the overall stability of the W/O/W emulsion, reduce the oil droplet size and improve size distribution when compared to the negative control without pigment and to emulsions containing betanin from other sources.

Effect of ph on the stability of red beet extract (Beta vulgaris l.) microcapsules produced by spray drying or freeze drying

Abstract Red beets is rich in phenolic acids and has high antioxidant capacity, and can be used to produce a natural dye. This study evaluated the effect of pH (3 to 6) on the stability of red beet extract microcapsules, dried by freeze drying and spray drying and stored at room temperature. The microcapsules were produced using a combination of maltodextrin and xanthan gum as encapsulating agents and stored for 7 days. For all evaluated microcapsules, a degradation of betanin was observed, however, that degradation was independent of pH, with the exception of the sample with maltodextrin and dried by spray drying. The freeze dried products showed lower degradation constants and higher half-life (t1/2) when comparing with the spray dried samples. The microcapsules containing maltodextrin and xanthan gum, dried by spray drying, showed the highest change in the content of phenolic compounds after storage for 7 days. The color parameters showed a reduction for a*, and increase in b* and L*, for all samples during the storage time. In general, the microcapsules produced using maltodextrin and xanthan gum, and dried by freeze dryer, showed higher stability in terms of betanin content, phenolic compounds and color parameters during storage at different pHs.

Betalains and phenolic compounds of leaves and stems of Alternanthera brasiliana and Alternanthera tenella.

Betacyanins and phenolic compounds from acetonitrile:acidified water extracts of Alternanthera brasiliana and Alternanthera tenella were characterized and quantified using a high-performance liquid chromatography system coupled with diode array and electrospray mass spectrometry detection. Four betacyanins (amaranthine, isoamaranthine, betanin and isobetanin) were tentatively identified and quantified. Twenty eight phenolic compounds of four different families (hydroxybenzoic and hydroxycinnamic acids, flavones and flavonols) were separated and characterized on the basis of their accurate MS and MS/MS information out of which ten compounds were confirmed by authentic standards. These plant species could be considered as an especially rich source of natural bioactive compounds and potential food colorants. A. brasiliana showed the highest betacyanin and polyphenols content (89µg/g and 35,243µg/g, respectively). Among polyphenols, flavonols were the more abundant (kaempferol-glucoside, kaempferol-rutinoside and kaempferol-rhamnosyl-rhamnosyl-glycoside). Meanwhile, A. tenella showed a different polyphenols profile with flavones as major compounds (glucopyranosil-vitexin and vitexin). As a novelty, pentosyl-vitexin and pentosyl-isovitexin were detected for the first time in Alternanthera plants. Both A. brasiliana and A. tenella leaves showed high total polyphenol content and in vitro antioxidant activity (FRAP). These results provide an analytical base concerning the phenolic and betalains composition and the antioxidant properties of two members of the promising Alternanthera gender, for subsequent applications, such as functional food ingredients.

Encapsulating betalains from Opuntia ficus-indica fruits by ionic gelation: Pigment chemical stability during storage of beads.

Betalain encapsulation was performed by ionic gelation as a stabilization strategy for these natural pigments. Betalains were extracted from purple cactus fruits and encapsulated in calcium-alginate and in combination of calcium alginate and bovine serum albumin. Beads were characterised by scanning electron microscopy and thermal analysis using differential scanning calorimetry and thermogravimetry. Moisture sorption isotherms were determined. Bead morphology was affected by matrix composition. Pigments storage stability was evaluated at different equilibrium relative humidity and temperatures. Pigment composition of beads was determined by HPLC-MS-MS and degradation products were also analysed after storage; betalamic acid being the major one. Both types of matrices protected the encapsulated pigments, being their storage stability better at low relative humidity than that of the non-encapsulated control material. Antiradical activities of beads were proportional to remaining betalain contents. At high relative humidity, there was no protection and low storage stability was observed in the samples.

Microencapsulation of betalains obtained from cactus fruit (Opuntia ficus-indica) by spray drying using cactus cladode mucilage and maltodextrin as encapsulating agents.

The microencapsulation of betalains from cactus fruit by spray drying was evaluated as a stabilization strategy for these pigments. The betalains used as active agent were extracted from purple fruits of Opuntia ficus-indica (BE) and encapsulated with maltodextrin and cladode mucilage MD-CM and only with MD. The microcapsulates were characterized by scanning electron microscopy (SEM), thermal analysis (TGA-DSC), tristimulus colorimetry, as well as, their humidity, water activity and dietary fiber content were also determined. The active agent content was measured by UV-Vis spectrophotometry and its composition confirmed by HPLC-ESIMS. A pigment storage stability test was performed at 18 °C and different relative humidities. The addition of CM in the formulation increased the encapsulation efficiency, diminished the moisture content, and allowed to obtain more uniform size and spherical particles, with high dietary fiber content. These microencapsulates are promising functional additive to be used as natural colorant in the food industry.

Microencapsulation of pulp and ultrafiltered cactus pear (Opuntia ficus-indica) extracts and betanin stability during storage.

Pulp (CP) and ultrafiltered (UF) cactus pear extracts were encapsulated with Capsul (C) by applying a central composite design (CP-C and UF-C systems) by spray-drying. To evaluate the effect of the extract, microparticles obtained under optimal conditions were characterised and stored at 60 °C. Betacyanin and betaxanthin encapsulation efficiency reached values above 98% for both systems studied. This efficiency was attributed to strong interactions between betalains and the polymer. Betalain degradation in CP-C and UF-C microparticles followed pseudo-first order kinetics. The betacyanin degradation rate constant was significantly higher for CP-C than for UF-C. These results suggested that the mucilage or higher sugar content of CP increased the hygroscopicity of the CP-C microparticles, leading to the degradation of betalain. The hydrolysis pathway was the main mechanism of betanin degradation during microparticle storage. These results demonstrate the potential utility of both CP-C and UF-C microparticles as natural colourants for healthy foods.