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.


Introduction
Vegetables are important components of a balanced diet due to their constituents, comprising many bioactive compounds. These compounds, termed functional nutrients, provide benefits for the promotion and maintenance of human health [1,2]. Epidemiological studies have demonstrated that dietary nitrate (NO 3 − ) from certain vegetables can provide a physiological substrate for the production of nitric oxide (NO) which, in turn, supports cardiovascular function, causes vasodilation, and decreases blood pressure [3][4][5][6]. Furthermore, secondary metabolites found in vegetables are involved in protective responses to different abiotic plant stresses [6]. In the last decade, systematic reviews and meta-analyses have demonstrated the potential health benefits of the dietary intake of plant polyphenols, mainly antioxidants, to decrease the risk of chronic and degenerative diseases [7][8][9]. It is estimated that at least 8,000 polyphenols have been described, considering natural, semi-synthetic, or synthetic compounds. Food matrices generally contain a complex mixture of those compounds, at variable concentrations, which may not yet have been well characterized [3].
Red beetroot (Beta vulgaris L. species) is a source of bioactive compounds, including dietary NO 3 − , betanin, antioxidant substances, and phenolic compounds (PCs), as well as a source of dietary fiber, minerals (potassium, sodium, iron, copper, magnesium, calcium, phosphorus, and zinc) and vitamins the proliferation of benefic bacterial flora that, in turn, boosts the immune system and prevents and manages gastrointestinal tract infections. Values are expressed as means ± SD. Different letters within the same line indicate differences between samples at a significance level of p < 0.05. Beetroot-cereal bar and gel values are reproduced from Baião et al. [1] and da Silva et al. [12], respectively.
The physicochemical characteristics of beetroot food interventions were considered to design the new formulations, since high water activity (a w ) may promote undesirable modifications, such as non-enzymatic browning and crispness reduction, sensory attributes inherent to cereal bars and chips. In addition, moisture-rich food matrices can favor the growth of spoilage microorganisms, consequently decreasing product shelf-life [28]. The moisture percentage of beetroot-cereal bar and chips was maintained lower than 15%, except for the beetroot gel and juice, which presented higher moisture, as expected for pasty and liquid food products [29]. Beetroot formulations were processed under satisfactory hygienic conditions, stored at cold temperature, and adequately packed in accordance with Brazilian legislation for human food consumption, taking into account the time intervention period.

Bioactive Compounds in Beetroot Product Interventions
Insufficient clinical evidence concerning the efficacy and safety dosage of bioactive compounds found in fruits and vegetables makes it difficult to recommend the intake of these phytochemicals. If they are consumed in a balanced diet, putative health benefits include decreased risk for chronic, i.e., cardiovascular, diseases, even if the physiological targets and mechanism of action of several of these non-nutrients are still not fully understood [30]. Many bioactive compounds found in fresh vegetables and fruits display antioxidant activity against harmful reactive oxygen species, while others stimulate cellular defense mechanisms, enhancing stress responses, competing for active enzymes and receptor binding sites in subcellular structures, modulating the gene expression of proteins/enzymes capable of acting against oxi-degenerative processes that may occur in molecules and cellular structures [31].

NO 3 − and NO 2 −
Beetroot juice is the most common NO 3 − source used for supplementation, although its NO 3 − concentration is lower when compared to other beetroot formulations [5,32]. Dietary NO 3 − concentrations normalized to 100 g or 100 mL of the product was higher in beetroot-cereal bars (14.0 ± 0.05 mmol) when compared to beetroot gel (6.30 ± 0.01 mmol), chips (6.90 ± 0.02 mmol), and juice (4.10 ± 0.01 mmol). NO 2 − contents ranged in low concentrations, from 0.10 ± 0.02 mmol to 0.20 ± 0.01, with no physiological significance (Table 1). Most beetroot formulations must be offered in large serving portions to reach effective NO 3 − concentrations, taking into account the objectives of each intervention. Thus, a serving portion of 200 mL of beetroot juice, 100 g of beetroot gel, and chips can be used to supplement over 6.3 mmol of dietary NO 3 − /day.
However, some gastrointestinal effects, as well as beeturia, may occur, impacting adherence to long-term supplementation [5,20,31,33]. On the other hand, the beetroot-cereal bar design provides an easy way to administer the beet-intervention product, in a convenient serving portion, as a healthy snack containing effective but higher dietary NO 3 − dosages (≈6.3 mmol in 45 g of product) than beetroot juice and gel, previously used to treat individuals at risk of developing CVD. Considered a snack, beetroot-cereal bars can be administered between meals, facilitating adhesion to NO 3 − nutritional interventions. Due to the mixture of fresh juice and powder, NO 3 − amounts can be adjusted and the beetroot-cereal bar can be used for both acute or chronic NO 3 − supplementations, presenting beneficial cardiovascular system effects for both healthy and patient populations.

Saponins
Few studies report the saponin content of beetroot products, but it is known that saponin content and types may vary according to the plant cultivar and food matrix processing [12]. Saponin contents in beetroot food interventions ranged from 2599 ± 1.27 to 8648 ± 1.85 mg/100 g, and the cereal bar was verified as the richest source (Table 2). Interestingly, in soybean, considered the main dietary saponin source, contents found in germs, cotyledons, and soy molasses varied from 935 ± 50.7 to 6583 ± 250.5 mg/100 g, lower than in beetroot [34]. Beetroot intervention products should be considered adequate for dietary saponin supplementation and may eventually replace soybean. Several beneficial bioactivities are attributed to isoprenoid or terpenoid compounds, where an aglycone is attached by a covalent bond to one or two sugar chains, forming a mono-or di-desmoside. Furthermore, oleanoic acids, betavulgarosides II, III, and IV, found in Beta vulgaris L. roots have been shown to promote hypoglycemic effects in rats. [35].

Organic Acids (OAs)
Beetroots are rich in OAs, similar to most plants, where these acids are used to cope with nutrient deficiencies, metal detoxification, and tolerance, and pathogens, as well as endophytic and symbiotic-microbe interactions operating at the root-soil interface [36]. Humans can also benefit from the ingestion of these compounds.
Beetroot-cereal bars present the highest total OA content (9.19 ± 0.71 mg/g) compared to chips (5.34 ± 0.35 mg/g), gel (4.17 ± 0.35 mg/g), and juice (2.84 ± 0.7 mg/g) ( Table 2). Six distinct OAs including citric, ascorbic, malic, fumaric, succinic, and oxalic acids have been quantified in the beetroot-cereal bar, whereas succinic acid and oxalic acid have been found only in beetroot-cereal bars, both derived from the cereals added during bar formulation, while citric acid, ascorbic acid, malic acid, and fumaric acid are found in beets and present in all beet-derivatives. Malic acid and citric acid are the most abundant in beet formulations [1] ( Table 2). The overall OA content found in some beetroot product interventions is close to those found in the most dense-dietary sources of OAs, such as kefir (≈12.0 mg/mL) and milk (≈5.0 mg/mL) [37].
In roots, OAs are present as partially neutralized potassium (K + ) salts, such as those formed by citrate, malate, and, less efficiently, by oxalate, and their contents can be influenced by soil characteristics, temperature and precipitation regimes, conventional or organic farming systems and post-harvest processing (fresh, cooked, juice, or chips) [38].
Phosphoric acid and citric acid are predominant in beetroot juice, followed by oxalic acid and malic acid. Subsequently, shikimic acid, the precursor for the synthesis of aromatic amino acids such as phenylalanine, tyrosine and tryptophan, and betalains, are detected in high concentrations in organic and conventional farming beets, while citric acid, malic acid, and fumaric acid are also observed, but at lower concentrations [39]. Malic acid is present at the highest concentrations in beetroot formulations, including juice, chips, powder, and cooked vegetables, followed by citric acid and ascorbic acid [18].
Some OAs are involved in the beneficial effect promoted by certain foods against oxidative stress, aiding in chronic and degenerative conditions, including cardiovascular diseases [40].
Malic acid is a putative adjuvant in the conservative treatment of calcium (Ca 2+ ) renal stone disease, due to its potential ability to complex with Ca 2+ in urine, preventing the formation of Ca 2+ oxalate (CaOx), the main kidney stone component. Malic alkalizing effects increase citrate excretion, improving hypocitraturia [41][42][43][44].
Citric acid acts as a synergistic antioxidant alongside other compounds and has been pointed out as a chelating agent, protecting molecules from metal-catalyzed oxidation [45,46]. Like malic acid, the ingestion of foods rich in citric acid can be an alternative for the treatment of hypocitraturia, reducing predisposition to renal stone formation [35,47].
Ascorbic acid, known as vitamin C, is a potent antioxidant also present in beetroot formulations ( Table 2). Ascorbic acid contents found in cereal bars (1.55 ± 0.21 mg/g) and chips (0.53 ± 0.04 mg/g) are higher than in citrus fruits (0.53 mg/g), i.e., orange and lemon, which are considered good sources of vitamin C but present similar amounts to those reported in beetroot chips [48]. Ascorbic acid is a powerful antioxidant, able to donate a hydrogen atom, generating the ascorbyl-free radical to protect biomolecules from damage caused by oxidative compounds generated in cell metabolism or following exposure to xenobiotic compounds [49]. Vitamin C functions as a cofactor for monooxygenase and dioxygenase enzymes involved in the degradation or detoxification of toxins and pollutants [50].
Succinic acid (butanedioic acid) was detected at 0.51 ± 0.01 mg/g in beetroot cereal bars ( Table 2). This acid is involved in angiogenesis via the vascular endothelial growth factor, epidermal growth factor receptor, platelet-derived growth factor, and glucose transporter 1, while also participating in the crossing to other metabolic pathways, such as the tricarboxylic acid cycle and the respiratory chain [67][68][69][70]. Another important succinate role is the activation of succinate-receptor 1 (SUCNR1) signaling, promoting the generation of endothelial NO and prostaglandin E2 (PGE2), and the synthesis and release of renin, supporting blood pressure regulation by the renin-angiotensin system [71,72]. Succinate is also involved in mitochondrial integrity by maintaining the ubiquinone (CoQH2) pool and inhibiting mitochondrial lipid peroxidation [73][74][75]. Therefore, succinic acid could support the vascular effects of beet NO 3 − .
To the best of our knowledge, human intervention trials assessing the direct effect of each beetroot compound, such as betagarin, betavulgarin, flavonoids, vanillic, p-coumaric, and syringic phenolic acids, are not yet widely available. When tested in cell cultures and animals, some of these compounds have shown antibacterial, anti-inflammatory, antioxidant, anti-tumoral, and protective effects against reperfusion ischemia injury [85][86][87][88].
Beetroot is a dietary source of PCs, although their concentrations vary according to the plant part, high in plant skin, and less concentrated in the crown and flesh [89,90]. PCs identified in beetroot juices obtained from organic and conventional cultivars and beet varieties include ferulic, caffeic, gallic, p-coumaric, chlorogenic, p-hydroxybenzoic, syringic and vanillic acids, quercetin, and myricetin [37,91].
Chlorogenic acid is a class of compounds formed by hydroxyl cinnamic esters with quinine acid [140,141]. Beetroot-product interventions have reported chlorogenic acid concentrations ranging from 5.94 ± 0.033 mg/100 g in cereal bars to 2.90 ± 0.003 mg/100 g in juice ( Table 2).

Figure 1. Biosynthesis pathway and general structures of betalains (reproduced from [161]).
Red beetroot is an excellent source of betanin (75-95%) but it also contains lower concentrations of isobetanin, betanidin, and betaxanthin [162,163]. Betanin content in red beet may be affected by farming conditions, including soil fertilization, moisture, post-harvest storage conditions, and, mainly, exposure to light and high temperatures [89,90,164].
Considering the betanin content found in beetroots and prospecting the amount in different beetroot formulations, beet chips would show the highest content (1274 mg/g) followed by juice, gel, and cereal bars [165] (Table 2).
In the food industry, betanin obtained from beetroot is used in sorbets, dairy derivatives like yogurts and ice creams, as well as meats (i.e., sausage), since betanin display good stability in a wide pH range (pH 3-7). The use of betanin as a natural red-violet dye for food is regulated by the Food and Drug Administration (FDA) and European Food Safety Authorities, under E-number E162 [166,167]. Betanin can also be considered a natural food preservative and alternative to synthetic antioxidants (i.e., BHA and BHT), due to its ability to prevent lipid peroxidation [165,168].
The exact mechanisms of betanin absorption, metabolic breakdown, and route excretion in humans have not yet been completely elucidated, and identification of chemical intermediates, such as glucuronides, sulfates, or conjugates of methylated betalain, in plasma and urine is still scarce. It is known that the bioavailability of betanin can be influenced by the source matrix (i.e., different food sources or forms of preparation) and by human interindividual variability such as genetics, sex, age, and health conditions, which alter its absorption and excretion profile [24,27,[169][170][171]. Red beetroot is an excellent source of betanin (75-95%) but it also contains lower concentrations of isobetanin, betanidin, and betaxanthin [162,163]. Betanin content in red beet may be affected by farming conditions, including soil fertilization, moisture, post-harvest storage conditions, and, mainly, exposure to light and high temperatures [89,90,164].
Considering the betanin content found in beetroots and prospecting the amount in different beetroot formulations, beet chips would show the highest content (1274 mg/g) followed by juice, gel, and cereal bars [165] (Table 2).
In the food industry, betanin obtained from beetroot is used in sorbets, dairy derivatives like yogurts and ice creams, as well as meats (i.e., sausage), since betanin display good stability in a wide pH range (pH 3-7). The use of betanin as a natural red-violet dye for food is regulated by the Food and Drug Administration (FDA) and European Food Safety Authorities, under E-number E162 [166,167]. Betanin can also be considered a natural food preservative and alternative to synthetic antioxidants (i.e., BHA and BHT), due to its ability to prevent lipid peroxidation [165,168].
The exact mechanisms of betanin absorption, metabolic breakdown, and route excretion in humans have not yet been completely elucidated, and identification of chemical intermediates, such as glucuronides, sulfates, or conjugates of methylated betalain, in plasma and urine is still scarce. It is known that the bioavailability of betanin can be influenced by the source matrix (i.e., different food sources or forms of preparation) and by human interindividual variability such as genetics, sex, age, and health conditions, which alter its absorption and excretion profile [24,27,[169][170][171].
Betanin stability and antioxidant ability have been evaluated in assays mimicking in vitro human digestion and ex vivo colonic fermentation [165]. Over half of the original betanin content is preserved after oral, gastric, and small intestine digestion, as observed in vitro simulation. No betanin was recovered from the ex vivo colon fermentation assay. The betanin chemical structure was preserved during simulated gastrointestinal digestion, as well as its antioxidant activity, confirmed by different antioxidant assays. The ability of betanin to inhibit the OH-radical within the total antioxidant potential (TAP) and its reductive ability to alter the ferric ion of the tripyridyltriazine complex (Fe 3+ -TPTZ) to the ferrous ion (Fe 2+ -TPTZ) was demonstrated in the ferric reducing ability of plasma (FRAP), as well as in the reduction of the 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS + ) radical in the trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays [165]. In agreement with these findings, betanin absorption through epithelial cell membranes occurred with no chemical transformation in a trans-epithelial transport assessment carried out with Caco-2 cells, [171].
Human betanin bioavailability is low as 2.7% of total oral intake is excreted in urine and feces [165,[172][173][174]. Betanin reaches a maximum plasma concentration after ≈3 h and is no longer detected after 12 h of ingestion [169]. Absorbed betanin is excreted primarily by urine, and some individuals can present reddish urine (beeturia) following oral administration [175], while renal excretion is lower than 3% of the administered dose [173]. A very small part of administered betanin (≤1%) through the ingestion of beet juice was excreted in the urine of volunteers after 2-7.5 h mainly as isobetanin, suggesting the occurrence of betanin isomerization due to the temperature of the human organism [175,176]. In addition, other trials have shown that about 90% of the betanin and isobetanin ingested are rapidly excreted as an unchanged structure from 0 to 4 h after administration, indicating that a part is quickly absorbed, while excretion between 8-24 h occurs predominantly in its aglycone form (betanidin and isobetanidin) [27]. The plasma bioavailability of betanin has shown significant variability in different clinical trials, perhaps due to the aforementioned biological variability of each individual and differences in administered doses, although, the low detection of betanin in plasma is a common finding. In another study, betanin was not detected in plasma at any time point post-ingestion of 250 mL of beetroot juice or 300 g of whole beetroot, containing near 194 and 66 mg of betanin respectively [177]. Minimal amounts of betanin in plasma (< 1 µg), 3, 8, and 24 h after supplementing male patients with extracts containing 16 and 35 mg of betanin and after 2 weeks of supplementation (<3 µg) have been reported [178]. Regular consumption for long periods (between 1 and 6 weeks) of beetroot products seems to be the solution to overcome low betanin levels in biological fluids by promoting stabilization of the systemic levels, where betanin and their deglucosylated, decarboxylated and dehydrogenated metabolites are consistently described [24]. The free radical scavenging activity of betanin, due to its ability to donate electrons and hydrogen, relies on the cyclic amine present in its structure, resembling ethoxyquin, a strong antioxidant, as well as hydroxyl groups (-OH), which are excellent hydrogen donors [176]. Due to its ability to remove reactive oxygen species (ROS), betanin prevents oxidative damage to lipid macromolecules and DNA, reversing tissue damage [179][180][181].
In vascular tissue, betanin antiradical activity maintains endothelial function and reduces the atherogenesis process ( Figure 2). In addition, betanin can modulate redox-mediated signal transduction pathways involved in inflammation responses in endothelial cells by inhibiting the intercellular cell adhesion molecule-1 (ICAM-1), resulting in antiproliferative effects in human tumoral cells [182,183]. Since excessive ROS are removed by betanin, NF-κB activation, and cytokine expression down-regulation are noted [184].
Therefore, the effects of betanin on inflammation, oxidative stress, and diabetes in rodent models are well documented, these findings have not yet been confirmed in humans.

Beetroot Product Interventions Increase Nitric Oxide Production and Promote Health Benefits
Vegetables are important health-promoting foods in a balanced diet, due to the bioactivities of their phytochemicals [189,190]. It is widely recognized that dietary NO 3 − from beetroot and green leafy vegetables may provide a physiological substrate for the generation of NO and other bioactive nitrogen oxides, leading to vasodilation and consequent improvement in cardiovascular function [191].
Dietary NO 3 − is well absorbed in the upper gastrointestinal tract. About 25% of dietary NO 3 − is captured by the salivary glands, where it is reduced to NO 2 − by commensal bacteria that express and secret NO 3 − -reductase enzyme in saliva [6,191]. The metabolic activity of the hundreds of commensal bacteria species belonging to the Granulicatella, Actinomyces, Veillonella, Prevotella, Neisseria, Haemophilus, and Rothia genera that live on the tongue can directly influence the NO 3 − to NO metabolism.
Individuals with a higher abundance of NO 3 − -reducing bacteria were able to generate more salivary NO 2 − and, consequently, NO at a faster rate following the ingestion of dietary NO 3 − [192]. In contrast, the enzymatic activity of bacteria in the mouth and conversion of NO 3 − to NO 2 − may be disrupted by antibiotic use or mouth rinsing with an anti-bacterial mouthwash. Oral nitrate-reducing microbiota are beneficial to the host and participate in the control of cardiovascular NO homeostasis [6,192,193].
After the conversion of dietary NO 3 − to NO 2 − in the oral cavity, the NO 2 − in the saliva is swallowed and reaches the stomach, where NO 2 − is non-enzymatically decomposed into NO and other bioactive nitrogen oxides in this acidic environment, by vitamin C or polyphenols. In addition to dietary sources, NO 3 − and NO 2 − can be endogenously originated from NO synthetized by the three isoforms of the nitric oxide synthase (NOS), family from the amino acid L-arginine and O 2 , namely the neuronal (nNOS or NOS-I) and endothelial (eNOS or NOS-III) isoforms, both constitutive and dependent on Ca 2+ -calmodulin, and the inducible isoform (iNOS or NOS-II). In addition, L-arginine is metabolized by arginase to L-ornithine and urea to eliminate excess nitrogenous compounds [193]. NO is a low molecular weight compound (30.01 g/mol) with a short-life (from 5 to 10 s) produced in gas form, containing 11 electrons in its valence shell with an unpaired electron. This radical character confers high reactivity to this compound, since it rapidly oxidizes to NO 2 − and NO 3 − . NO displays an affinity for lipophilic environments and accumulates in the lipid milieu, such as cell membranes and lipoproteins [193]. In human physiology, NO can exert antioxidant functions and is considered a secondary messenger, acting on the vascular endothelium, central and peripheral neurons, and immune system, inhibiting platelet activation, adhesion, and aggregation, modulating vascular tone, and improving human skeletal muscle function [5,27,194,195]. Multiple pathways are used by NO to promote these actions, which depend on the cell tissue and the amount of produced NO ( Figure 3). As mentioned previously, NO's free-radical scavenging ability reduces ROS, promoting cardioprotective effects on the atherosclerotic process by preventing LDL cholesterol oxidation, and reducing RNO production rates [196].
In immune cells, NO is produced as part of the inflammatory response by macrophages and other immune system cells, which express the inducible isoform type II NO synthase. The formed NO reacts with the superoxide anion (O2 •− ), generating peroxynitrite (ONOO − ), which, in turn, causes lethal damage to pathogens or tumoral cells by attacking copper and iron-metalloproteins [5,27,192].
NO formed by the neuronal NO synthase (nNOS) acts as a neurotransmitter in the central and peripheral nervous systems, mediating synapse plasticity in nerve impulse transmission and favoring the secretion of neurotransmitters or hormones in neuronal junctions. The nervous impulse transmission occurs when glutamate, the main excitatory neurotransmitter, diffuses from the presynaptic terminal to bind to the N-methyl-D-aspartate type (NMDA) receptors at the postsynaptic terminal. NMDA receptors are coupled to Ca 2+ ion channels and their activation by glutamate allows the flow of Ca 2+ into the postsynaptic terminal. Ca 2+ associates with calmodulin and activates nNOS, promoting the formation of NO. NO may diffuse to the presynaptic terminal and stimulating the generation of cyclic guanosine monophosphate (cGMP) from guanosine-5 -triphosphate (GTP) catalyzed by the soluble guanylate cyclase (sGC), cGMP then activates protein kinases triggering phosphorylation of target enzymes, activating or inhibiting them [194]. However, the predominant mechanism that mediates the effects of NO signaling in the nervous system involves post-translational modification of thiol nitrosylation of Cys residues, termed S-nitrosylation, Tyr nitration, termed 3-nitrotyrosination (NO 2 − Tyr via ONOO − formation), and PKG-dependent phosphorylation of Ser residues of the target proteins [197].   Both endothelium-and platelet-derived NO prevent platelet aggregation and fibrin formation, inhibiting the spread of thrombi generation [196]. NO exerts its inhibitory action by reducing cytoplasmic Ca 2+ through increasing Ca 2+ extrusion rates and sarcoplasmic reticulum Ca 2+ -ATPase and decreased Ca 2+ input from the extracellular medium. NO promotes phosphorylation of thromboxane-2 receptor and down-regulates P-selectin expression, preventing platelet activation and adhesion [198]. In addition, NO modulates fibrinogen binding via the glycoprotein IIb and IIIa (GPIIb/IIIa) receptor, increasing the dissociation constant of this receptor by fibrinogen, reducing the total number of GPIIb/IIIa receptors on the platelet surface, resulting in unfavorable conditions for platelet aggregation. Furthermore, NO stimulates tyrosine nitrosylation in the ONOO − pathway, thereby inhibiting thromboxane-2 synthesis [199]. NO regulates vascular tone by diffusing across endothelial cells, reaching vascular smooth muscle cells and, through sGC, activates the sarcoplasmic Ca 2+ pump, decreasing intracellular Ca 2+ and promoting vasodilation as a result of diminished vascular tone [200].
Under low O 2 levels and pH, any member of the NO 2 − reductase class enzymes, including xanthine, aldehyde oxidases, aldehyde dehydrogenase type 2, carbonic anhydrase, or deoxyhemoglobin, can reduce NO 2 − to NO [201]. The NO generated alongside NO 2 − from the dietary-NO 3 − conversion improves oxidative phosphorylation efficiency, evidenced by an increased P/O ratio, indicating no uncoupling mechanisms, such as proton leaks towards ATP synthesis and turnover, improving ATP supply to skeletal muscle [27]. Several studies report beneficial effects of dietary NO 3 − in the stimulation of NO production and biochemical, hemodynamic, and vascular parameters following the intake of doses ranging from 6.3 to 22.0 mmol. Different beetroot product interventions have been formulated with distinct nutritional compositions and tested to achieve their claimed health effects (Table 3) [12,19,33,202].  [203] Beetroot juice (140 mL ↓ platelet-monocyte aggregates ↓ stimulated P-selectin expression ↑ FMD ↓ AIx ↓ aPWV ↓ SBP but not DBP and heart rate da Silva et al. [12] Beetroot Gel (100 g)   ↓ arterial stiffness through AIx, aoPP, and PWV ↓ arterial blood pressures ↓ endothelial dysfunction by improvements in cutaneous microvascular conductance peak No changes in endothelial dysfunction, arterial stiffness, and arterial blood pressure after placebo cereal bar ingestion Bezerra et al. [209] Beetroot juice (  Single-blind Randomized Placebo-controlled 1-week ingestion and 1-week washout -↓ cold symptom severity and global sickness during and after final exams Healthy vs asthma group interaction was significant for cold symptom severity and global sickness, indicating that the advantage of the beetroot juice group was greater for participants with asthma than for healthy volunteers. Rokkedal-Lausch et al. [215] Beetroot juice (140 mL) NO3 --depleted beetroot juice (140 mL) Twenty healthy male cyclists Randomized Double-blinded Counter balanced-crossover Placebo-controlled 1-week ingestion and 1 week washout ↑ plasmatic NO3 − and NO2 − concentrations prior to time trial tests in normoxia and hypoxia conditions. ↑ TT performance with no difference between normoxia and hypoxia.
↑ VO2 and VE during TT, with no difference between normoxia and hypoxia. No changes in heart rate, oxygen saturation, or muscle oxygenation during TT.
Jones et al. [216] Beetroot juice (70 mL Smith et al. [217] Beetroot juice (70 mL) Nitrate-depleted placebo (70 mL) Twelve recreational trained male university students Randomized Double-blind Crossover Placebo-controlled Acute ingestion (1-week washout) -No changes on sprint performance and total work done in either temperate or hot, humid conditions.
However, to obtain the maximum cardioprotective effect of NO 3 − intake, the dosage, supplementation regimen and the health status of the assessed individuals must be considered. Minimal or no hemodynamic and vascular beneficial effects in healthy individuals have been observed following acute NO 3 − administration from 1 to 7 days. An intake of 7.0 mmol of NO 3 − in 140 mL of beetroot juice by 27 treated-hypertensive volunteers for 7 days resulted in increased NO synthesis, as assessed by plasmatic, urinary, and salivary NO 3 − and NO 2 − , but no differences in home blood pressure (BP) and 24 h ambulatory systolic (SBP) and diastolic blood pressure (DBP) [203]. A supply of 9.92 mmol of NO 3 − in 100 g of beetroot gel to 25 healthy and physically active runners for 1 week promoted increases in urinary NO 3 − , creatinine, and NO 2 − after 90 min of beetroot ingestion and after exercise. However, urinary levels of nitrous compounds were not related to changes in oxygen volume (VO 2peak ), time to fatigue during treadmill running, respiratory quotient, SBP, and DBP [19]. , while no changes in 24 h ambulatory SBP and DBP were detected [209]. Minimal effects were observed in 15 healthy volunteers treated by 7.3 mmol of NO 3 − on brachial SBP, not sustained over 24 h, and carotid to femoral pulse wave velocity ( cf PWV) [213]. Regardless of the use of higher doses of dietary NO 3 − and a proved improvement in NO synthesis, these aforementioned results indicate a critical role of vascular impairment caused by some chronic non-communicable diseases such as hypertension, dyslipidemia, obesity, and aging, impairing NO effects in target tissues [218]. Furthermore, NO 3 − supplementation benefits on physical performance have been suggested as more meaningful in healthy, but non physically active, individuals, rather than active ones. Physiological adaptations of endurance training may stimulate the expression and activity of the NOS enzyme through the endogenous pathway (via L-arginine/NO), increasing NO bioavailability. Due to the activation of the NO endogenous biosynthesis, the dependency of NO bioavailability derived from dietary NO 3 − supplementation seems to be reduced [19].
However, in individuals presenting one or more risk factors for the development of cardiovascular diseases, the reversal of endothelial dysfunction evaluated by decreased large-artery stiffness and BP is achieved following the intake of up to 6.0 mmol of NO 3 − if long-term supplementation is performed [5,204,218]. Three weeks administration of 6.45 mmol of NO 3 − in 70 mL beetroot juice to 24 older and overweight volunteers promoted an increase in NO synthesis, estimated by urinary and salivary NO 3 − and NO 2 − , resulting in SBP decreases of up to 7.3 mm Hg [202]. The intake of 250 mL of beetroot juice containing 6.4 mmol of NO 3 − by 34 drug-naive hypertensive patients for 4 weeks increased NO synthesis and cGMP levels, accompanied by decreases in arterial stiffness and a ≈20% improvement in endothelial function proven by decreases in 24 h ambulatory and home BPs [29]. The intake of 60 g of beetroot-cereal bars containing 9.57 mmol of NO 3 − for 3 weeks by five patients presenting at least three risk factors for the development of CVD promoted increases in the NO synthesis and improvements in cutaneous microvascular conductance peak decreases in arterial stiffness (through assessments concerning the augmentation index-AIx, aortic pulse pressureao PP, and PWV index) and decreases in SBP and DBP [208]. for long periods would be necessary to result in beneficial effects on blood pressure and endothelial function and should be recommended to populations with compromised vascular responsiveness.

Bioactive Beetroot Compounds-NO 3 − and Betanin-Modulate the Transcription of Genes Responsible for Regulating Redox Imbalance in a Rodent Model
The cellular and systemic improvements observed after dietary NO 3 − intervention may be due to up-and down-gene expression in endothelial function regulation and platelet and macrophage recruitment and vasodilation, while also reducing imbalances in the redox state of the cardiovascular system, associated with mRNA inhibition of endogenous ROS generators, as well as NADPH oxidases. Meanwhile, activations of GPx, CAT, and SOD gene expressions are also noted, increasing the availability of scavenging enzymatic effectors [188]. Transcriptional patterns in aged mice whole thoracic aortas after chronic NaNO 3 − supplementation highlight changes in the expression of genes encoding the calcium-signaling pathway, as well as in detoxification and antioxidant defenses. As a long-term effector, NO 3 − promoted up-regulation of genes encoding Ca 2+ -signaling proteins, including those able to increase Ca 2+ in the cytosol, such as the sarcoplasmic Ca 2+ channel, the ryanodine receptor 2 (Ryr2), the inositol triphosphate receptor (Itpr2, Itpr3, Itpka); and L-type calcium channel (Cacna1d and Ppapdc2), and also the broad spectrum protein regulators, like Ca 2+ /calmodulin-dependent protein kinase II (Calm2, Camk2, Camk4) which, together, can cause smooth muscle cell relaxation [219][220][221][222][223].
A transcriptome analysis of ischemic stress responses following NO 3 − intake indicates the up-regulation of genes enrolled in the lipid and carbohydrate metabolisms and the intracellular transport of molecules, as well as genes related to protein synthesis, turnover, and repair, including those encoding glucokinase, pyruvate dehydrogenase kinase, acetyl coenzyme A acetyltransferase 2, acyl CoA synthetase short-chain,17-dehydrocholesterol reductase, retinol dehydrogenase 11, farnesyl diphosphate synthase, nucleoside transporter, sodium/bile acid co-transporter family member, carbonic anhydrase 3, G2 cyclin, Rho GTPase, activating protein 9, glutamyl aminopeptidase and beta-lactamase 2 [224]. Betanin promotes healthy benefits to the cardiovascular system due to its anti-radical scavenger effect, reducing the reactivity of these molecules, protecting from endothelial tissue from damage. Simultaneously, betanin down-regulates the mRNA of pro-inflammatory mediators while reinforcing endogenous antioxidant defenses. Furthermore, several lines of evidence implicate betanin in the transcriptional regulation of metabolic and antioxidant/detoxification genes [184]. In human hepatic cells, betanin induced translocation of Nrf2 from the cytosol to the nuclear compartment, where it can bind to the antioxidant response element, and, in turn, control mRNA expression and protein levels of several detoxifying/antioxidant enzymes, including glutathione S-transferases, quinone dehydrogenase 1 NAD(P)H dependent and heme oxygenase-1 [186,188,225].
Betanin may, therefore, be a supportive therapeutic alternative to attenuate the main mechanisms involved in CVD without any harmful effects. Although the exact mechanisms by which betanin exerts its cardioprotective role have not been yet fully elucidated, its ability to act directly on ROS/RNS species alongside the induction of the antioxidant and cytoprotective Nrf2-ARE pathway and suppression of the inflammatory NFk-B pathway in CVD can account for all betanin health-promoting benefits [184,226]. Furthermore, betanin is bioaccessible, bioavailable, approved for use in foods in quantium satis, and has not shown any harmful or deleterious effects in animals. Thus, clinical trials should be conducted to determine the effective dose and supplementation regimen to achieve the desired health outcomes in human beings.

Conclusions
Interventions with dietary NO 3 − from beetroot are reported as affecting cardiovascular and metabolic functions by regulating the gene expression patterns or modulating the activity of proteins and enzymes involved in these cellular processes. The cytoprotective effects of NO-derived from NO 3 − -NO 2 − /NO pathway may be collectively reinforced by certain bioactive compounds naturally found in beetroot. PCs and OAs identified at high concentrations in beetroot should also be considered antioxidant defense adjuvants in health promotion and chronic disease prevention. However, the most remarkable compound found in beetroot seems to be betanin. Thus, betanin could be a putative candidate to attenuate the oxidative stress status in humans.
If previously described betanin effects in rodent models are confirmed in humans, it can be expected that short-term betanin intake will be able to attenuate the redox state of human cells by cytoprotective effects, regulating glucose and lipid metabolisms, controlling insulin resistance and lipid peroxidation, and, thus, protecting the cardiovascular system, liver, and kidneys from damage.