Antimicrobial betalains.

Betalains are nitrogen-containing plant pigments that can be red-violet (betacyanins) or yellow-orange (betaxanthins), currently employed as natural colourants in the food and cosmetic sectors. Betalains exhibit antimicrobial activity against a broad spectrum of microbes including multidrug-resistant bacteria, as well as single-species and dual-species biofilm-producing bacteria, which is highly significant given the current antimicrobial resistance issue reported by The World Health Organization. Research demonstrating antiviral activity against dengue virus, in silico studies including SARS-CoV-2, and anti-fungal effects of betalains highlight the diversity of their antimicrobial properties. Though limited in vivo studies have been conducted, antimalarial and anti-infective activities of betacyanin have been observed in living infection models. Cellular mechanisms of antimicrobial activity of betalains are yet unknown; however existing research has laid the framework for a potentially novel antimicrobial agent. This review covers an overview of betalains as antimicrobial agents and discussions to fully exploit their potential as therapeutic agents to treat infectious diseases.

Exploration of Betalains and Determination of the Antioxidant and Cytotoxicity Profile of Orange and Purple Opuntia spp. Cultivars in Greece.

Replacing synthetic dyes with natural pigments has gained great attention over the past years in the food industry, due to the increased alertness of consumers for nontoxic and natural additives. Betalains are water-soluble nitrogenous natural pigments that are used as natural colorants in food industries, due to their applicability and their rich pharmacological profile including antioxidant, antimicrobial, and anticancer properties. Therefore, there is a need for a detailed exploration of betalains to fully exploit their properties. Opuntia spp. plants are one of the primary sources of betalains. The objective of this study was to identify betalain phytochemical content in prickly pear cactus of two different Opuntia species from Greece (an Opuntia ficus-indica (L.) Mill (OFI) orange prickly pear cultivar and an Opuntia spp. purple prickly pear cultivar) using modern analytical techniques as also to evaluate their antioxidant and cytotoxicity profile. To achieve this we used an array of analytical techniques, including ultra-violet-vis (UV-Vis) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) as also cell based in vitro assays. These enabled us to establish a rapid approach that can distinguish the different Opuntia spp. cultivars based on their phytochemical constituents through untargeted metabolomics analysis using ultra-high performance liquid chromatography-mass spectrometry - quadrupole time-of-flight (UPLC/MS Q-TOF). These findings could allow a further exploitation of Opuntia species and especially their enriched betalain phytochemical profile as viable source of natural food colorants.

Red-Beet Betalain Pigments Inhibit Amyloid-ß Aggregation and Toxicity in Amyloid-ß Expressing Caenorhabditis elegans.

Betalain pigments are mainly produced by plants belonging to the order of Caryophyllales. Betalains exhibit strong antioxidant activity and responds to environmental stimuli and stress in plants. Recent reports of antioxidant, anti-inflammatory and anti-cancer properties of betalain pigments have piqued interest in understanding their biological functions. We investigated the effects of betalain pigments (betanin and isobetanin) derived from red-beet on amyloid-ß (Aß) aggregation, which causes Alzheimer's disease. Non-specific inhibition of Aß aggregation against Aß40 and Aß42 by red-beet betalain pigments, in vitro was demonstrated using the thioflavin t fluorescence assay, circular dichroism spectroscopy analysis, transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, we examined the ability of red-beet betalain pigments to interfere with Aß toxicity by using the transgenic Caenorhabditis elegans model, which expresses the human Aß42 protein intracellularly within the body wall muscle. It responds to Aß-toxicity with paralysis and treatment with 50 µM red-beet betalain pigments significantly delayed the paralysis of C. elegans. These results suggest that betalain pigments reduce Aß-induced toxicity.

Antiradical Activity of Beetroot (Beta vulgaris L.) Betalains.

Flavonoids, phenolic acids, and anthocyanidins are widely studied polyphenolics owing to their antiradical activity. Recently, beetroot dyes have drawn an attention as possible radical scavengers, but scant information can be found on this topic. In this study selected compounds were investigated using computational chemistry methods. Implicit water at physiological pH was chosen as the environment of interest. Betalains' dissociation process and electronic structure were examined, as well as the reactivity in six pathways against some common radicals, such as hydroxyl, hydroperoxide, superoxide, and nitric oxide. The study showed that all carboxyl groups are dissociated in the given conditions. The dissociation process impacts the electronic structure, which has consequences for the overall activity. Highly stabilized conjugated structures favor the electron-accepting type of scavenging reactions, primarily by a radical adduct formation mechanism. Betanidin and indicaxanthin were found to be the most promising of the compounds studied. Nevertheless, the study established the role of betalains as powerful antiradical dietary agents.

Biological Properties and Applications of Betalains.

Betalains are water-soluble pigments present in vacuoles of plants of the order Caryophyllales and in mushrooms of the genera Amanita, Hygrocybe and Hygrophorus. Betalamic acid is a constituent of all betalains. The type of betalamic acid substituent determines the class of betalains. The betacyanins (reddish to violet) contain a cyclo-3,4-dihydroxyphenylalanine (cyclo-DOPA) residue while the betaxanthins (yellow to orange) contain different amino acid or amine residues. The most common betacyanin is betanin (Beetroot Red), present in red beets Beta vulgaris, which is a glucoside of betanidin. The structure of this comprehensive review is as follows: Occurrence of Betalains; Structure of Betalains; Spectroscopic and Fluorescent Properties; Stability; Antioxidant Activity; Bioavailability, Health Benefits; Betalains as Food Colorants; Food Safety of Betalains; Other Applications of Betalains; and Environmental Role and Fate of Betalains.

Potential Therapeutic Effects of Natural Plant Compounds in Kidney Disease.

BACKGROUND: The blockade of the progression or onset of pathological events is essential for the homeostasis of an organism. Some common pathological mechanisms involving a wide range of diseases are the uncontrolled inflammatory reactions that promote fibrosis, oxidative reactions, and other alterations. Natural plant compounds (NPCs) are bioactive elements obtained from natural sources that can regulate physiological processes. Inflammation is recognized as an important factor in the development and evolution of chronic renal damage. Consequently, any compound able to modulate inflammation or inflammation-related processes can be thought of as a renal protective agent and/or a potential treatment tool for controlling renal damage. The objective of this research was to review the beneficial effects of bioactive natural compounds on kidney damage to reveal their efficacy as demonstrated in clinical studies. METHODS: This systematic review is based on relevant studies focused on the impact of NPCs with therapeutic potential for kidney disease treatment in humans. RESULTS: Clinical studies have evaluated NPCs as a different way to treat or prevent renal damage and appear to show some benefits in improving OS, inflammation, and antioxidant capacity, therefore making them promising therapeutic tools to reduce or prevent the onset and progression of KD pathogenesis. CONCLUSIONS: This review shows the promising clinical properties of NPC in KD therapy. However, more robust clinical trials are needed to establish their safety and therapeutic effects in the area of renal damage.

Biological effects of red beetroot and betalains: A review.

Over the past few years, the use of natural substances as protective or therapeutic agents has gained much attention worldwide. Recent modern studies have shown a variety of health benefits for red beetroot and its active compounds betalains (also betanin) such as antioxidative, anti-inflammation, anticancer, blood pressure and lipid lowering, also antidiabetic and anti-obesity effects. Betanin, the main component of the red beetroot, is a betalain glycosidic pigment, which is used as a food additive. This review summarizes findings in the literature and shows the therapeutic potential of red beetroot and its active compounds (betalains) as promising alternatives for supplemental therapies in multiple diseases.

Encapsulation Preserves Antioxidant and Antidiabetic Activities of Cactus Acid Fruit Bioactive Compounds under Simulated Digestion Conditions.

Cactus acid fruit (Xoconostle) has been studied due its content of bioactive compounds. Traditional Mexican medicine attributes hypoglycemic, hypocholesterolemic, anti-inflammatory, antiulcerogenic and immunostimulant properties among others. The bioactive compounds contained in xoconostle have shown their ability to inhibit digestive enzymes such as α-amylase and α-glucosidase. Unfortunately, polyphenols and antioxidants in general are molecules susceptible to degradation due to storage conditions, (temperature, oxygen and light) or the gastrointestinal tract, which limits its activity and compromises its potential beneficial effect on health. The objectives of this work were to evaluate the stability, antioxidant and antidiabetic activity of encapsulated extract of xoconostle within double emulsions (water-in-oil-in-water) during storage conditions and simulated digestion. Total phenols, flavonoids, betalains, antioxidant activity, α-amylase and α-glucosidase inhibition were measured before and after the preparation of double emulsions and during the simulation of digestion. The ED40% (treatment with 40% of xoconostle extract) treatment showed the highest percentage of inhibition of α-glucosidase in all phases of digestion. The inhibitory activity of α-amylase and α-glucosidase related to antidiabetic activity was higher in microencapsulated extracts than the non-encapsulated extracts. These results confirm the viability of encapsulation systems based on double emulsions to encapsulate and protect natural antidiabetic compounds.

Analgesic activity and mechanism of action of a Beta vulgaris dye enriched in betalains in inflammatory models in mice.

Evidence demonstrates the pronounced anti-inflammatory activity of a beetroot (Beta vulgaris) dye enriched in betalains obtained using precipitation with ethanol. Herein, we expand upon our previous observations and demonstrate the analgesic and antioxidant effect of betalains. Betalains [10-1000 mg/kg; intraperitoneal route (i.p.)] diminished acetic acid- and PBQ-induced abdominal contortions, and the overt pain-like behaviour induced by complete Freund`s adjuvant (CFA) and formalin (intraplantar; i.pl.) injection. Moreover, betalains (100 mg/kg) administered by various routes [i.p. or subcutaneous (s.c.)] or as a post-treatment reduced carrageenin- or CFA-induced hyperalgesia. Mechanistically, betalains mitigated carrageenin-induced tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, superoxide anion levels, and lipid peroxidation. Betalains also stopped the depletion of reduced glutathione (GSH) levels and ferric reducing ability produced by carrageenin, as well as upregulated Nrf2 and Ho1 transcript expression in the plantar tissue of mice. Furthermore, betalains showed hydroxyl radical, 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS+), and 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH•) scavenging ability and iron-chelating activity (bathophenantroline assay), and inhibited iron-independent and iron-dependent lipid peroxidation (LPO) in vitro. Finally, betalains-treated bone marrow-derived macrophages exhibited lower levels of cytokines (TNF-α and IL-1ß), and superoxide anion levels and nuclear factor kappa B (NF-κB) activation following lipopolysaccharide (LPS) stimulation. Therefore, this betalain-rich dye extracted using a novel precipitation approach presents prominent analgesic effect in varied models of pain by mechanisms targeting cytokines and oxidative stress.

Red Beetroot and Betalains as Cancer Chemopreventative Agents.

Carcinogenesis is the process whereby a normal cell is transformed into a neoplastic cell. This action involves several steps starting with initiation and followed by promotion and progression. Driving these stages are oxidative stress and inflammation, which in turn encompasses a myriad of aberrant gene expressions, both within the transforming cell population and the cells within the surrounding lesion. Chemoprevention of cancer with bioreactive foods or their extracted/purified components occurs via normalizing these inappropriate gene activities. Various foods/agents have been shown to affect different gene expressions. In this review, we discuss whereby the chemoprevention activities of the red beetroot itself may disrupt carcinogenesis and the activities of the water-soluble betalains extracted from the plant.

C-Glycosyl Flavonoids from Beta vulgaris Cicla and Betalains from Beta vulgaris rubra: Antioxidant, Anticancer and Antiinflammatory Activities-A Review.

The green beet (Beta vulgaris var. cicla L.) and red beetroot (B. vulgaris var. rubra L.) contain phytochemicals that have beneficial effects on human health. Specifically, the green beet contains apigenin, vitexin, vitexin-2-O-xyloside and vitexin-2-O-rhamnoside, while the red beetroot is a source of betaxanthins and betacyanins. These phytochemicals show considerable antioxidant activity, as well as antiinflammatory and antiproliferative activities. Vitexin-2-O-xyloside, in combination with betaxanthins and betacyanins, exerts antiproliferative activity in breast, liver, colon and bladder cancer cell lines, through the induction of both intrinsic and extrinsic apoptotic pathways. A significant body of evidence also points to the role of these phytochemicals in the downregulation of the pro-survival genes, baculoviral inhibitor of apoptosis repeat-containing 5 and catenin beta-1, as well as the genes controlling angiogenesis, hypoxia inducible factor 1A and vascular endothelial growth factor A. The multi-target action of these phytochemicals enhances their anticancer activity. Vitexin-2-O-xyloside, betaxanthins and betacyanins can be used in combination with conventional anticancer drugs to reduce their toxicity and overcome the multidrug resistance of cancer cells. In this review, we describe the molecular mechanisms that enable these dietary phytochemicals to block the proliferation of tumor cells and inhibit their pro-survival pathways. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Health-promoting bioactivities of betalains from red dragon fruit (Hylocereus polyrhizus (Weber) Britton and Rose) peels as affected by carbohydrate encapsulation.

BACKGROUND: Betalains, which are red-purple and yellow pigments, are ideal alternatives to synthetic colorants as they possess strong coloring potential and excellent health-contributing properties. However, the instability of betalains toward normal storage and biological conditions, in addition to the limited number of betalain sources, impedes their food application and diminishes their bioactivities. This study aimed to evaluate the health-promoting bioactivities of betalains from red dragon fruit (Hylocereus polyrhizus (Weber) Britton and Rose) peels as affected by encapsulation in maltodextrin-gum Arabic and maltodextrin-pectin matrices. RESULTS: Encapsulation in maltodextrin-gum Arabic and maltodextrin-pectin matrices afforded dry betalain powders after lyophilization. Optical microscopy imaging showed that the betalain powders consisted of matrix-type and shard-like microparticles. ABTS antioxidant assay revealed that maltodextrin-gum Arabic-betalain (MGB) and maltodextrin-pectin-betalain (MPB) microparticles possessed higher antioxidant capacities (195.39 ± 8.63 and 201.76 ± 4.06 µmol Trolox g-1 microparticles respectively) than the non-encapsulated betalain extract (151.07 ± 2.57 µmol Trolox g-1 extract). Duck embryo chorioallantoic membrane (CAM) vascular irritation assay showed that the anti-inflammatory activity of encapsulated betalains was five- to six-fold higher than that of non-encapsulated betalains (P ≤ 0.05). Antiangiogenic activity, as evaluated by duck embryo CAM assay, was enhanced two- to four-fold by carbohydrate encapsulation. Glutathione S-transferase (GST)-inducing activity of betalains was likewise improved four- to five-fold. CONCLUSION: The study showed that the antioxidant, anti-inflammatory, antiangiogenic and GST-inducing activities of betalains from red dragon fruit peels were enhanced through carbohydrate encapsulation. © 2016 Society of Chemical Industry.

Basella alba L. (Malabar Spinach) as an Abundant Source of Betacyanins: Identification, Stability, and Bioactivity Studies on Natural and Processed Fruit Pigments.

The antioxidant and anti-inflammatory activities of acylated and decarboxylated gomphrenins, as well as Basella alba L. fruit extract, were investigated in relation to gomphrenin, known for its high biological potential. The most abundant natural acylated gomphrenins, namely, 6'-O-E-caffeoyl-gomphrenin (malabarin) and 6'-O-E-4-coumaroyl-gomphrenin (globosin), were isolated from B. alba extract for the studies. In addition, controlled thermal decarboxylation of gomphrenin in the purified B. alba extract at 65-75 °C resulted in the formation of the most prevalent decarboxylated products, including 17-decarboxy-gomphrenin and 2,17-bidecarboxy-gomphrenin, along with their isoforms. The structures of the decarboxylated pigments were confirmed by NMR analyses. Exploring the matrix effect on pigment reactivity revealed a tremendous increase in the stability of all betacyanins after the initial stage of extract purification using a cation exchanger under various conditions. This indicates the removal of a substantial portion of the unfavorable matrix from the extract, which presumably contains reactive species that could otherwise degrade the pigments. Furthermore, the high concentration of citrates played a significant role in favoring the formation of 2-decarboxy-gomphrenin to a considerable extent. In vitro screening experiments revealed that the tested compounds demonstrated strong anti-inflammatory properties in lipopolysaccharide (LPS)-activated human macrophages. This effect encompassed the selective inhibition of cytokine and chemokine release from activated macrophages, modulation of the chemotactic activity of immune cells, and the regulation of tissue remodeling mediators' release.

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.

Characterization of betalain-loaded liposomes and its bioactive potential in vivo after ingestion.

Betalains are plant pigments characterized by showing a wide range of beneficial properties for health. Its bioactive potential has been studied for the first time after its encapsulation in liposomes and subsequent administration to the animal model Caenorhabditis elegans. Phenylalanine-betaxanthin and indoline carboxylic acid-betacyanin encapsulated at concentrations of 25 and 500 µM managed to reduce lipid accumulation and oxidative stress in the nematodes. Highly antioxidant betalains dopaxanthin and betanidin were also included in the survival analyses. The results showed that phenylalanine-betaxanthin was the most effective betalain by increasing the lifespan of C. elegans by 21.8%. In addition, the administration of encapsulated natural betanidin increased the nematodes' survival rate by up to 13.8%. The preservation of the bioactive properties of betalains manifested in this study means that the stabilization of the plant pigments through encapsulation in liposomes can be postulated as a new way for administration in pharmacological and food applications.

Betalains isolated from underexploited wild plant Atriplex hortensis var. rubra L. exert antioxidant and cardioprotective activity against H9c2 cells.

Atriplex hortensis var. rubra L. extracts prepared from leaves, seeds with sheaths, and stems were characterized for betalainic profiles by spectrophotometry, LC-DAD-ESI-MS/MS and LC-Orbitrap-MS techniques. The presence of 12 betacyanins in the extracts was strongly correlated with high antioxidant activity measured by ABTS, FRAP, and ORAC assays. Comparative assessment between samples indicated the highest potential for celosianin and amaranthin (IC50 21.5 and 32.2 µg/ml, respectively). The chemical structure of celosianin was elucidated for the first time by complete 1D and 2D NMR analysis. Our findings also demonstrate that betalain-rich A. hortensis extracts and purified pigments (amaranthin and celosianin) do not induce cytotoxicity in a wide concentration range in rat cardiomyocytes model (up to 100 µg/ml for extracts and 1 mg/ml for pigments). Furthermore, tested samples effectively protect H9c2 cells from H2O2-induced cell death and prevent from apoptosis induced by Paclitaxel. The effects were observed at sample concentrations between 0.1 and 10 µg/ml.

Differential Effects of Betacyanin and Betaxanthin Pigments on Oxidative Stress and Inflammatory Response in Murine Macrophages.

SCOPE: Betalain pigments are increasingly highlighted for their bioactive and anti-inflammatory properties, although research is lacking to demonstrate contributions of individual betalains. The work herein aimed to compare effects of four main betalains on inflammatory and cell-protective markers and to highlight potential structure-related relationships of the two main subgroups: betacyanins vs betaxanthins. METHODS AND RESULTS: Murine RAW 264.7 macrophages were stimulated with bacterial lipopolysaccharide following incubation with betacyanins (betanin, neobetanin) and betaxanthins (indicaxanthin, vulgaxanthin I) in concentrations from 1 to 100 µM. All betalains suppressed expression of pro-inflammatory markers IL-6, IL-1ß, iNOS, and COX-2 with tendency for stronger effects of betacyanins compared to betaxanthins. In contrast, HO-1 and gGCS showed mixed and only moderate induction, while more emphasized effects were observed for betacyanins. While all betalains suppressed mRNA levels of NADPH oxidase 2 (NOX-2), a superoxide generating enzyme, only betacyanins were able to counteract hydrogen peroxide induced reactive oxygen species (ROS) generation, in alignment with their radical scavenging potential. Furthermore, betaxanthins exerted pro-oxidant properties, elevating ROS production beyond hydrogen peroxide stimulation. CONCLUSION: In summary, all betalains display anti-inflammatory properties, although only betacyanins demonstrate radical scavenging capacities, indicating potential differing responses under oxidative stress conditions, which requires further research.

Betalains Alleviate Exercise-Induced Oxidative Stress, Inflammation, and Fatigue and Improve Sports Performance: an Update on Recent Advancement.

PURPOSE OF REVIEW: Beetroot juice is a popular natural food supplement commonly consumed for its health and ergogenic benefits. It contains an abundance of phytochemical compounds, which have been shown to enhance sports endurance and recovery. Among them, nitrate is well-studied and known for improving performance during exercise. On the other hand, betalains, the bioactive pigment, have shown various biological activities including antioxidant, anti-inflammatory, and anti-hypertensive, which may improve exercise performance and post-exercise recovery. Additionally, free radical scavenging activities of betalains could increase nitric oxide availability in the blood, thereby improving blood flow and oxygen supply during strenuous exercise. This review article provides a critical discussion of the non-pathological conditions induced by prolonged or strenuous exercise and betalains' potential in reducing such conditions including muscle damage, inflammation, and fatigue. Additionally, the real-time application of betalains as an ergogenic compound in competitive athletes has been discussed. Finally, future directions and conclusions on the potential of betalains as a natural ergogenic aid in sport endurance are outlined. RECENT FINDINGS: Betalains in beetroot are the major water-soluble nitrogen-containing pigment possessing high antioxidant, anti-inflammatory, and anti-fatigue activities. Betalain supplementation could alleviate exercise-induced oxidative stress, inflammation, and fatigue in competitive athletes. Betalains have the potential to become a natural ergogenic aid or nutraceutical compound for sports people during exercise and competitive performance.

Betalains: colours for human health.

In the last years, the use of natural phytochemical compounds as protective agents in the prevention and treatment of obesity and the related-metabolic syndrome has gained much attention worldwide. Different studies have shown health benefits for many vegetables such Opuntia ficus-indica and Beta vulgaris and their pigments collectively referred as betalains. Betalains exert antioxidative, anti-inflammation, lipid lowering, antidiabetic and anti-obesity effects. This review summarizes findings in the literature and highlights the therapeutic potential of betalains and their natural source as valid alternative for supplementation in obesity-related disorders treatment. Further research is needed to establish the mechanisms through which these natural pigments exert their beneficial effects and to translate the promising findings from animal models to humans.