Nutritional and functional potential of Beta vulgaris cicla and rubra

Fitoterapia 89 (2013) 188–199

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Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Review

Nutritional and functional potential of Beta vulgaris cicla and rubra Paolino Ninfali ⁎, Donato Angelino Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, Via Saffi 2 – 61029 Urbino (PU) Italy

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Article history: Received 10 April 2013 Accepted in revised form 28 May 2013 Available online 7 June 2013 Keywords: Beta vulgaris cicla Beta vulgaris rubra Chenopodiaceae Antitumoral activity Antioxidant capacity Functional foods

a b s t r a c t Swiss chard (Beta vulgaris cicla, BVc) and beetroot (Beta vulgaris rubra, BVr) are vegetables of the Chenopodiaceae family, widely consumed in traditional western cooking. These vegetables represent a highly renewable and cheap source of nutrients. They can be cultivated in soils with scarce organic material and little light and water. BVc and BVr have a long history of use in folk medicine. Modern pharmacology shows that BVc extracts possess antihypertensive and hypoglycaemic activity as well as excellent antioxidant activity. BVc contains apigenin flavonoids, namely vitexin, vitexin-2-O-rhamnoside and vitexin-2-O-xyloside, which show antiproliferative activity on cancer cell lines. BVr contains secondary metabolites, called betalains, which are used as natural dyes in food industry and show anticancer activity. In this light, BVc and BVr can be considered functional foods. Moreover, the promising results of their phytochemicals in health protection suggest the opportunity to take advantage of the large availability of this crop for purification of chemopreventive molecules to be used in functional foods and nutraceutical products. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . Taxonomy . . . . . . . . . . . . . . . . . Botany . . . . . . . . . . . . . . . . . . Ecology . . . . . . . . . . . . . . . . . . Chemical composition . . . . . . . . . . . 5.1. Essential oil . . . . . . . . . . . . . 5.2. Polyphenols . . . . . . . . . . . . 5.2.1. Phenolic acids . . . . . . . 5.2.2. Flavonoids . . . . . . . . . 5.2.3. Betalains and phenolic amides 5.2.4. Organic and inorganic acids . 5.2.5. Miscellaneous . . . . . . .

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Abbreviations: ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); APAF-1, Apoptotic Protease Activating Factor-1; Apo1, Apoptosis Antigen 1; BAX, Bcl2-Associated X protein; Bcl2, B-cell lymphoma 2; BSA, Bovine Serum Albumin; BVc, Beta vulgaris cicla; BVr, Beta vulgaris rubra; CAA, Cellular Antioxidant Activity; Caspase, Cysteine-Aspartic Proteases; CdK, Cyclin-dependent Kinase; COX, Cyclooxygenase; DCFH-DA, Dichlorofluorescein-Diacetate; DNA, Deoxyribonucleic Acid; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ELISA, Enzyme Linked Immunosorbent Assay; GC-MS, Gas chromatography–mass spectrometry; GLUT2, Glucose Transporter 2; HLE, Hawthorn Leaf Extract; HPLC–MS, High Performance Liquid Chromatography–Mass Spectrometry; IC50, Half maximal Inhibitory Concentration; LDL, Low Density Lipoprotein; MM2, Paraneoplastic Ma antigen 2; MRP-2, Multidrug Resistance-associated Protein-2; NO, Nitric Oxide; NOS, Nitric Oxide Synthase; ORAC, Oxygen Radical Absorbance Capacity; PAI1, Plasminogen Activator Inhibitor-1; PARP, Poly-ADP Ribose Polymerase; P-gp, P-glycoprotein; PPARγ, Peroxisome Proliferator-Activated Receptor gamma; RBC, Red Blood Cell; ROS, Reactive Oxygen Species; TBARS, Thiobarbituric Acid Reactive Substances; VOR, Vitexin-2-O-rhamnoside; VOX, Vitexin-2-O-xyloside; WAF, Cicline-Dependent Kinase-interacting protein 1 ⁎ Corresponding author. Tel.: +39 722 305288; fax: +39 722 305324. E-mail address: [email protected] (P. Ninfali).URL: http://www.uniurb.it/orac/ (P. Ninfali). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.06.004

P. Ninfali, D. Angelino / Fitoterapia 89 (2013) 188–199

6.

Biological activity . . . . . . . . . . . . . 6.1. Anti-diabetic activity . . . . . . . . 6.2. Anti-inflammatory activity . . . . . 6.3. Antioxidant activity . . . . . . . . 6.4. Anticancer activity . . . . . . . . . 7. Bioavailability of BVc and BVr phytochemicals 8. Conclusions . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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1. Introduction Red beetroot (Beta vulgaris rubra, BVr) and Swiss chard (Beta vulgaris cicla, BVc) are members of the Chenopodiaceae. This family contains important food crops, such as Spinacia oleracea (spinach), which is the most consumed Chenopodiaceae leafy vegetable in Europe [www.fao.org/economic/ess/en/], Salsola kali, or prickly saltwort, currently used in western countries, and Chenopodium quinoa, commonly known as quinoa [1]. In the Chenopodiaceae family, there are also two wild edible representatives: Chenopodium album, known as lambsquarter and in indi as bathua [2], and Chenopodium bonus henricus [3], also called “mountain spinach”, as it grows in the grazing lands of the Alps [4]. BVc and BVr have been used for food since 1000 B.C. by all populations of the Mediterranean basin. The Romans utilized the BVc and BVr leaves for food, while the roots were used for medicinal applications. BVc became commercially important in 19th century in Europe, following the development of the sugar beet (Beta vulgaris saccharifera) in Germany [4].

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In Italy, the two most produced BVc cultivars are Bieta a costa Bianca, for home use, and Bieta erbetta da taglio for industry use (Fig. 1), whereas there are several varieties of BVr, such as the Chioggia, Detroit and Ruby Queen (Fig. 1). This review focuses on botany, chemical composition, biological activity and nutritional value of BVc and BVr as well as on the biological and pharmacological activities of their phytochemicals. 2. Taxonomy Beet is classified taxonomically as Dicotyledonae, Caryophyllidae, Chenopodiaceae and Beta [5]. On the basis of morphological characters, the genus Beta was sub-divided into two groups: cultivated and wild maritime beets. In the latter group, the sea beets (Beta vulgaris maritima) is the unique species, which represents the ancestral form of all the species. In the cultivated group, there are sugar beets (Beta vulgaris saccharifera), fodder beets (Beta vulgaris crassa), leaf beets (Beta vulgaris cicla) and garden beets (Beta vulgaris rubra) [6].

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189

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E

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F

Fig. 1. Pictures of the main cultivars of Beta vulgaris cicla and rubra. A) Swiss chard, bieta costa bianca; B) Swiss chard, bieta erbetta da taglio; C) Swiss chard, Hybrid F1; D) Red Beetroot cv Chioggia; E) Red Beetroot cv. Detroit; F) Red Beetroot cv Ruby Queen.

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3. Botany BVc is a biennal plant with strong roots and large oval leaves, with white veins. BVc is generally seeded in spring for summer crop and again in summer for the winter and spring crops. BVc is harvested by cutting leaves at the base, while the roots remain in the soil for re-growth. During the winter, plants accumulate nutrients in the roots and begin the new vegetation cycle in the spring. The following summer, the plants produce flowers grouped into glomerula forming spikes in the apical part of the stems. After that, the plants produce the seeds and later die. The seeds of beet normally contain more than one seed and they are called polygerms. The breeding procedures have obtained, in recent years, three important results: polyploidy, monogermity and the production of commercial hybrids [7]. BVr or beetroot, or garden beet or table beet, is cultivated for its large roots, but the leaves also are utilizable. The shanks may be red, magenta or white and leaves are small and green with thin red veins. BVr is grown for the thick flashy taproot that forms during the first season. On cooking the color diffuses uniformly through the flesh. In the second season, a tall branched leafy stem arises to bear clusters of minute green flowers, that develop into brown corky fruits, commonly called seed balls. The taproot varies in shape from flattened oblate to globular and from conical to long tapered. Skin and flash colors are usually dark-purplish red, with some nearly white. 4. Ecology BVc and BVr grow on friable soils with a good draining texture and abundant organic matter. Commercial fertilizers, containing nitrogen, phosphorus and potash may be added to the soil, or alternative fertilizers, such as green manures, crop residues, animal manure and compost may be used [5]. However, BVc and BVr adapted to environments with elevated saline concentrations, such as marine coast, salt marsh and loam [4]. Optimal soil pH ranges between 6.0 and 6.8, but neutral or alkaline pH is well tolerated [5]. BVr must be sown directly in the field, since the sowing in a greenhouse, followed by planting, may lead to bifid roots. BVr is harvested during the summer, when the leaves are dehydrated. Indeed, BVc and BVr plants are also able to grow in soils with scarce availability of water and organic matter and tolerate

conditions of low light [4]. In fact, these vegetables were proposed for cultivation in space. The Swiss chard varieties Large White Ribbed and Lucullus have been demonstrated to provide maximal yield in low light in a space station [8]. 5. Chemical composition 5.1. Essential oil The seeds of BVc and BVr contain an essential oil whose composition in fatty acids has been determined by us through GC–MS analysis, following the standard protocols reported by normative ISO 5508:1990. Table 1 shows the fatty acid composition of BVc seed oil after extraction with supercritical CO2. Results show the high content of linoleic acid, followed by a remarkable presence of oleic acid. Among the saturated fatty acids, the dominant component was palmitic acid (17%). 5.2. Polyphenols BVc and BVr seeds, leaves and roots are rich in phenolic compounds, whose concentration is dependent on the stage of plant development [9,10]. For instance, three cultivars of BVc showed maximum phenolic content at 55–60 days after transplantation (Fig. 2). After this time, the phenolic concentration dropped progressively with aging; the flavonoid concentration showed a similar trend (Fig. 2). Therefore, for the maximum phenolic intake, the leaves should be harvested at the maturity stage. The phenolic pool of BVc and BVr is represented by different types of molecules, which include phenolic acids, flavonoids and phenolic amides, including betalains. 5.2.1. Phenolic acids Pyo et al. [11] measured the concentration of phenolic acids in Swiss chard leaves. Interestingly, they found syringic acid (44.9 mg/100 g fresh weight), caffeic acid (14.8 mg/100 g

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b

b

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Table 1 Fatty acid composition of Beta vulgaris cicla seed oil.

Flavonoids

Fatty acid

g/100 g total fatty acids⁎

Palmitic acid Stearic acid Oleic acid Linoleic acid Eicosenoic acid cis-11-Eicosenoic acid Arachidonic acid Others

17.16 1.40 31.54 44.08 0.49 0.67 0.35 4.32

± ± ± ± ± ± ± ±

1.68 0.14 2.87 4.01 0.05 0.07 0.04 0.52

⁎ Average value of three separate analysis on oil extracted with supercritical CO2.

Fig. 2. Phenolic and flavonoid concentration after planting BVc cultivars Bieta Erbetta, Hybrid F1 and Bieta costa Bianca during 90 days. Phenolic compounds were assayed by the method of Singleton et al. [109], whereas flavonoid were assayed by the method of Eberhard et al. [110]. Results are the mean ± SD of three replicates. a, b = same letters indicate statistically significant differences among values of the same curve, for p b 0.05, by ANOVA.

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Fig. 3. Chemical structures of betalains and vitexin flavonoids present in Beta vulgaris cicla and rubra. A, betalamic acid; B, betaxanthin; C, betacyanin; D, vitexin; E, vitexin-2-O-rhamnoside; F, vitexin-2-O-xyloside. R1 = tyrosine, tyrosine-betaxanthin. R2 = H, betanidin; R2 = glucose, betanin.

fresh weight) and other phenolic acids in concentrations ranging from 5 to 10 mg/100 g fresh weight. In our laboratory, we performed the screening of phenolic compounds through HPLC–MS analysis of BVc seed extract and we found in addition to the above phenolic acids, the presence of vanillic acid, and two aldehydes, namely: 2,5-dihydroxybenzaldehyde and 2,4,5-trihydroxybenzaldehyde [12]. In BVr, Georgev et al. [13] demonstrated the presence of 4-hydroxybenzoic acid, caffeic acid and chlorogenic acid in concentrations ranging from 0.12 to 0.047 mg/g of dry extract. 5.2.2. Flavonoids BVc is a rich source of flavonoid glycosides derived from apigenin, namely vitexin (Fig. 3D), vitexin-2-O-rhamnoside (VOR) (Fig. 3E), vitexin-2-O-xyloside (VOX) (Fig. 3F), the purification of which has been performed in our laboratory [12]. Among the BVc flavonoids, Pyo et al. [11] recognized the following: catechin (6.7 mg/100 g fresh weight), myricetin (2.2 mg/100 g fresh weight), quercetin (7.5 mg/100 g fresh weight) and kaempferol (9.2 mg/100 g fresh weight). Geogev et al. [13] revealed in BVr cv. Detroit Dark Red the presence of catechin-hydrate, epicatechin and rutin, as well as Kujala et al. [14] identified in four cultivars of BVr four flavonoids: betagarin, betavulgarin, cochliofilin and dihydroxyisorhamnetin. 5.2.3. Betalains and phenolic amides BVr contains a large amount of betalains, a group of numerous water soluble nitrogen containing pigments derived from betalamic acid (Fig. 3A), which the chromophore moiety common to all molecules. Inside to the betalain family, there are two classes of compounds: the yellow-orange betaxanthins (Fig. 3B) and the red-violet betacyanins (Fig. 3C) [15]. The

betacyanin and betaxanthin concentration in BVr varies in the ranges 0.04–0.21% and 0.02–0.14%, respectively [16]. The major betacyanin pigment in BVr is betanin, which is a betanidin-5-Oβ-glucoside. Betanidin, is therefore the aglyconic form of the betanin. The maior betaxanthin present in BVr is vulgaxanthin I, which is also present, together with betanin, in few pigmented cultivar of BVc, but their concentration is much lower in BVc than in BVr [16]. To date, the food colorant extracted from BVr, known as “beetroot red”, is commercially available as E162 in Europe and USA [17] and new BVr crops with increased pigment quantity are actively searched by experts of breeding and horticultural practices [18]. Beyond to the betalains, Kujala et al. [14] identified in four cultivars of BVr two additional phenolic amides (N-transferuloyltyramine and N-trans-feruloylhomovanillylamine). The discovery of the latter compounds stimulated the efforts for the chemical synthesis of this natural molecule to be used as an antioxidant and antitumor agent [19], due to its exceptional radical-scavenging and photoprotective ability [13].

5.2.4. Organic and inorganic acids BVc leaves and BVr roots contain oxalates and nitrates (NO3−), which represent anti-nutritional factors, as they subtract micronutrients during the digestive process in humans [20,21]. Another adverse effect is the contribution to the formation of kidney stones [22]. Nitrates are commonly present in water and vegetables as well as in cured meat [23]. Some nitrates are also endogenously produced principally by the L-arginine–NO pathway [24].The Acceptable Daily Intake (ADI) for nitrate is 3.7 mg/kg b.w./day [25]. NO3− ion shows a relatively low toxicity [26], but one should bear in mind that 25% of all nitrates ingested with diet is converted into nitrite (NO2−) in the saliva and in the upper gastrointestinal tract

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[27]. NO2− is a more toxic form, as it reacts with amines or amides to form N-nitroso compounds, which are well-known gastric and bladder cancer activators [28]. During the industrial preparation of BVr juices, the nitrate concentration is considerably reduced by fermentation strategies [29]. A positive role of NO3− reduction regards the nitrite conversion of into nitric oxide (NO) in the body, through numerous pathways involving deoxyhaemoglobin, myoglobin, ascorbate and polyphenols [30]. In physiological or pathological hypoxia, NO provides several effects, including: vasodilation, modulation of cellular respiration, responses to ischaemic stress, lowering blood pressure, vaso-protection and antiplatelet aggregation properties [31,32]. In this light, the beetroot juice has a potential effect of human blood pressure reduction [33] and therefore it was used in the improvement of athletic performance [34]. Concerning the oxalates as anti-nutritional factors [20,21], we must infer that the problem addresses mainly BVc old leaves. In fact, oxalate concentration, which is very low in young BVc leaves, increases near to the toxic level in old BVc leaves, during the second year of vegetation [5,35]. From a functional and nutritional standpoint, therefore, it is better to consume steam-cooked young BVc leaves, i.e. harvested in the range of 40–60 days, in order to save the polyphenol content and minimize the oxalates [36]. On the contrary, if one should use old leaves, i.e. those harvested in the second year, it is better to boil the leaves in abundant water, in order to lose most of the oxalates in the boiling water. 5.2.5. Miscellaneous Red beetroot is rich in carbohydrates, fibers, proteins and minerals, such as sodium, potassium, calcium and iron [3]. The beet metabolism also produces geosmin, a bicyclic alcohol which provides the characteristic “earthy” flavor to red beetroot [37]. To remove this unpleasant flavor, a distillation process during juice concentration is applied [16]. Swiss chard leaves are a good source of vitamins A, E, B3, B5, B9 and minerals as iron, potassium, calcium, phosphorus and magnesium [5]. The caloric contribution is 17 and 20 kcal/100 g for fresh BVc and BVr, respectively [38]. 6. Biological activity BVc and BVr have been used for a long time for their beneficial health effects, mainly consisting in stimulation of haematopoietic and immune systems as well as in the protection of kidney, liver and gut from toxic compounds. Moreover, they exhibit mineralizing, antiseptic and choleretic activities as well as they contribute to the reinforcement of the gastric mucosa [39–41]. BVr have been used for therapy of intestinal and genital tumors, while juices of fresh roots or leaves have been considered effective in the therapy of tumors of the digestive system as well as of the lung, liver, breast, prostate and uterus [5]. In modern pharmacology, the betalains have been the most studied BVr health protective molecules. They have been linked to protection against oxidative stress, inflammation and tumors. The stability of betalains in the beetroot juice has been considered crucial for the exhibition of the antitumor effect. For instance, Patkai et al. [42] studied the retention of

betalains in red beetroot juice, during production and pasteurization. Modern pharmacologists also addressed the importance of bioactive molecules from BVc extracts and demonstrated their anti-diabetic, anti-inflammatory, antioxidant and anticancer activities. 6.1. Anti-diabetic activity Experiments performed by Yanardag et al. [43] demonstrated the hypoglycemic effect of BVc extract in diabetic rats. This hypothesis was substantiated by further studies of the same group, who demonstrated a 40% reduction of glycaemia, without any loss of weight or impairment of liver functions [44,45]. The mechanism for the hypoglycemic action of the extract has been tentatively attributed to saponins, that inhibit gluconeogenesis and glycogenolysis [46]. However, other molecular pathways potentially involved in hypoglycemic effects remain to be deeply investigated. In fact, some evidences suggested that the hypoglycemic activity of BVc extract may be due to flavonoids, through the inhibition of glucose transporters. For instance, quercetin, which is present in BVc, showed evidence of anti-diabetic effects via inhibition of the intestinal glucose transporter GLUT2 [47]. Another complementary hypoglycemic mechanism could be the flavonoid induced inhibition of the α-amylase and α-glucosidase activities [48]. For instance, two flavonolglycosides isolated from Salsola kali were demonstrated to be active inhibitors of α-amylase [49]. The inhibition of this enzyme could delay the digestion and absorption of carbohydrates and consequently suppress post-prandial hyperglycemia [50]. Some C-glycosyl flavones, i.e. vitexin, vitexin-2-O-glycoside and VOR (Fig. 3), contained in BVc leaves and seeds, were found to strongly inhibit α-glucosidase [51] and could be the most probable cause of the hypoglycemic effect earlier evidenced in diabetic rats [44]. 6.2. Anti-inflammatory activity In order to study the pharmacological activity of BVc, some authors considered the molecules contained in the seeds. Kim et al. [35] extracted phenolic amides from BVc seeds and tested their inhibition in nitric oxide (NO) production in murine macrophages, stimulated by lipopolysaccharides; the authors found IC50 values for NO synthase (NOS) inhibition ranging from 13 to 18 μM. Therefore, they concluded that BVc seed extract demonstrated in vitro anti-inflammatory activity by inhibiting NOS and suggested to use these phenolic amides for the development of new anti-inflammatory drugs. The red pigment betanin has been demonstrated to provide a strong anti-inflammatory activity, throughout the inhibition of cyclooxygenase (COX) activity, which catalyzes the conversion of arachidonic acid into chemical mediators of inflammation [52]. Results showed that betanin (100 μg/mL) was more efficient in the inhibition of COX-2 (97%) than COX-1 (33.5%). The same authors observed that combination of betanin and anthocyanins reduced their respective biological activities thus exhibiting a negative synergistic effect [52]. Atta et al. [53] demonstrated that ethanolic extracts of red beet roots possess a dose-dependent anti-inflammatory effect against both acute and chronic inflammation. However,

P. Ninfali, D. Angelino / Fitoterapia 89 (2013) 188–199 Table 2 Total phenols, flavonoids and antioxidant capacities in different Chenopodiaceae. Name

Total phenols (mg/g dw)

Total flavonoids (mg/g dw)

ORAC (μmolTE/g dw)

Beta vulgaris cicla leaves 11.12 ± 0.56 7.92 ± 0.39 192.8 ± 9.6 Beta vulgaris cicla roots 0.72 ± 0.04 0.88 ± 0.05 8.54 ± 0.43 Beta vulgaris cicla seeds 1.88 ± 0.07 1.57 ± 0.08 49.10 ± 2.76 Beta vulgaris rubra 12.76 ± 0.76 11.64 ± 0.81 200.3 ± 11.2 leaves Beta vulgaris rubra roots 1.77 ± 0.08 1.44 ± 0.15 18.21 ± 0.86 Chenopodium album 10.68 ± 0.49 8.15 ± 0.41 341.4 ± 19.4 leaves Chenopodium album 4.98 ± 0.27 5.90 ± 0.29 192.9 ± 12.4 seeds Chenopodium Bonus 8.52 ± 0.39 3.69 ± 0.19 198.5 ± 12.5 Enricus leaves Liscari sativa leaves 4.25 ± 0.19 5.08 ± 0.23 108.3 ± 7.4 Spinacia olearacea 8.93 ± 0.25 6.15 ± 0.32 287.1 ± 12.5 leaves Chenopodium quinoa 1.23 ± 0.04 0.98 ± 0.04 29.61 ± 1.87 seeds Values are the mean ± SD of four independent measurements performed in our laboratory. Phenols were assayed as reported by Singleton et al. [109], flavonoids by Eberhart et al. [110] and ORAC by Ninfali et al. [9]. dw = dry weight.

these authors did not specify which cultivar they tested and the origin of their ethanolic extract remains undefined.

6.3. Antioxidant activity BVc and BVr extracts have been widely studied for their antioxidant activity both in vitro and in vivo [9,54–57]. In our laboratory, we performed a comparative survey of the antioxidant capacity among edible Chenopodiaceae plants. Table 2 shows the ORAC results, in parallel with the phenolic and flavonoid content in roots, leaves and seeds. Concerning the Beta vulgaris species, it is interesting to note that phenols, flavonoids and ORAC values are lower in roots with respect to leaves. BVr roots, which contain large amounts of carbohydrates and low concentration of secondary metabolites, consequently have lower antioxidant capacity than BVc or BVr leaves. Chenopodium album and Spinacia olearacea leaves showed higher ORAC values than BVc leaves. Among the seeds, Chenopodium quinoa showed the lowest phenols, flavonoids and ORAC values. The ORAC value of spinach is higher than that of BVc and BVr, whereas the phenolic content is lower. This means that spinach contains a minor amount of phenols, but its phenolic compounds are endowed of powerful antioxidant capacity. The comparison among BVc, BVr and spinach is referred to the raw vegetables, but we must consider that they are eaten boiled or steamed and the antioxidant capacity as well as the phenolic content may undergo dramatic changes, depending on the thermal processing [58,59]. This aspect addresses also the frozen vegetables as, for the industrial production, BVc and spinach are mechanically collected and immediately transferred to the industry, where they are washed, steamed and packaged under frozen conditions. Part of the antioxidant capacity is lost during the industrial treatment, depending on the steaming time. Ninfali et al. [9,60] and Gil et al. [61] measured the phenolic loss after steaming and boiling and showed that nearly 50% of the polyphenols are lost in

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the water after boiling, together with 80% of vitamin C content; on the contrary, 15 min steaming depletes only 20% of phenolic compounds. Kuti et al. [62] also found a similar phenolic and antioxidant capacity decay, measured with the ORAC method, in spinach leaves steamed in a microwave oven. From the results of Table 2, it emerges the low antioxidant capacity of the whole extract of BVr roots. However, when the betalains are extracted from BVr roots, their antioxidant capacity become remarkable. Kujawska et al. [54] investigated the protective effect of the beetroot juice in a model of oxidative stress induced by carbon tetrachloride (CCl4) on male Wistar rats. Results demonstrated that microsomal lipid peroxidation in the liver, expressed as Thiobarbituric Acid Reactive Substances (TBARS) concentration decreased by 38% in rats pretreated with beetroot juices before the administration of CCl4. The pretreatment with beetroot juice also caused a partial recovery in the activity of glutathione peroxidase and glutathione reductase by 35% and 66% respectively, after been depleted by the CCl4 treatment. Beetroot juice was also demonstrated to be able to reduce plasma protein carbonyls (~30%) and DNA damage in blood leukocytes (~20%) of rats treated with juice before xenobiotic administration. Escribano et al. [39] investigated the anti-radical activity of both betaxanthins and betacyanins from Beta vulgaris rubra by means the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The antiradical activity of betacyanins was found to be greater than that of betaxanthins and increased with the pH of the reaction medium. Georgev et al. [13] compared the antioxidant activity of betalains from hairy root cultures and intact plants of the beetroot Beta vulgaris cv. Detroit dark red. These authors evaluated the antioxidant activity using both the DPPH and the ORAC assays and demonstrated a six-fold higher antioxidant activity of hairy root extracts versus the mature beetroots. Tesoriere et al. [63] showed the incorporation of betalains into human red blood cells and the consequent protection of the cells from oxidative hemolysis; in parallel, in an in vitro model, Gentile et al. [64] described the ability of betalains to protect the endothelial cells from the oxidation processes, related to inflammatory response. Kanner et al. [65] and Tesoriere et al. [66] demonstrated that betalains may bind LDL and cellular membranes to inhibit lipid peroxidation in blood. Moreover, Allegra et al. [67] reported the ability of betalains to scavenge hypochlorous acid, which is powerful oxidant produced by neutrophils during the inflammatory response. The ability of the betalains to protect both human LDL and endothelial cells from oxidation due to inflammatory responses, make these molecules as functionally important for reducing the risks of atherosclerotic plaque formation. Finally, Vulic et al. [68] investigated the biological activity of beetroot pomace, a waste industrial material mainly constituted by ferulic, vanillic, p-hydroxybenzoic, caffeic and protocatechuic acids and betalains, including betanin, isobetanin and vulgaxhantin I. The beetroot pomace, showed excellent antiradical activity, measured by ESR spectroscopy, towards DPPH and hydroxyl radicals as well as antimicrobial activity against Gram − and Gram + bacteria [68]. Therefore, the finding of BVr cultivars very rich in phenolic acids and betalains as well as to find agronomic practices appears of nutritional utility, which may significantly influence the total antioxidant concentration. For example, Bavec et al.

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[20] demonstrated that polyphenols and antioxidant capacity of BVr were higher in biodynamic and integrated farming systems with respect to conventional farming system. 6.4. Anticancer activity Earlier investigations of the anticancer properties of the genus Beta were focused on the BVr. Betalains have been the primary molecules recognized to provide anticancer effects [15]. The first antitumor mechanism, proposed for betalains, was an interruption in the exchange of metabolites between tumor cells and surrounding tissues, in such a way that it would hinder the infiltration capacity of the tumor cells [40,52]. More recently, Sreekanth et al. [69] deeply investigated the antiproliferative effect of beetroot red, or betanin (E162), on human chronic myeloid leukemia cell line K-562. The authors showed a dose and time dependent decrease in K-562 cell proliferation [69]. Results also revealed that betanin induced apoptosis through the intrinsic pathway, which was mediated by the release of cytochrome-C from mitochondria and Poly ADP-Ribose Polymerase (PARP) cleavage [69]. Cytotoxic activity against Ehrlich carcinoma cells, was demonstrated by Vulic et al. [68] with a murine model in which Ehrlich carcinoma cells were implanted. These authors observed that the administration of beetroot pomace extract, rich in polyphenols and betalains, was able to decrease the carcinoma cell numbers and the amount of the ascites they produced in respect to the control animals treated with placebo. Vitexin,(Fig. 3D), VOX (Fig. 3E), and VOR (Fig. 3F), are the principal antitumor molecules found in BVc leaves and seeds [12]. These flavonoids are glycosides of the flavone apigenin, whose antitumor effects have been widely studied in the last decade [70,71]. Apigenin has been shown to interfere in the tumor cell signaling network [72], induce apoptosis [73–77] and regulate the expression of the following proteins: p53, APO-1, Bcl-2, and p21/CDKN1 [78–84]. The primary focus on apigenin in anticancer literature has led to the initiation of current research concerning the antitumor contribution of vitexin, VOX and VOR. The anticancer effect of vitexin has been studied by Yang et al. [85] in human oral cancer OC2 cells. These authors demonstrated that vitexin decreased cell viability significantly and up-regulated the expression of the tumor suppressor p53 mediated signaling, including p53-Bax and p53-PPARγ-caspase 3 pathways, which led to apoptosis. Meanwhile, vitexin decreased cell migration via p53-PAI1-MMP2 cascade [85]. Ninfali et al. [86] observed that a mixture of VOX and VOR was very active in blocking the proliferation of MCF-7 breast cancer cells. Indeed, VOX was purified from BVc seeds and found to be particularly effective in inhibiting the proliferation of intestinal cancer cells, such as RKO [12], Caco2 and LoVo cell lines [87]. Further, VOX is able to synergize with epigallocatechine3-gallate and glucoraphasatin to inhibit proliferation of LoVo and Caco-2 cell lines [87]. The mechanism by which VOX induces apoptosis in intestinal tumor cell lines has been proposed [87]. The process, described in Fig. 4, begins with the production of ROS, which in turn leads to glutathione depletion; this cascade increases Bax and concomitantly depletes Bcl-2 proteins, which in turn

changes the mitochondrial membrane potential, releasing cytochrome-C into the cytoplasm. This efflux activates the Apoptotic Protease Activating Factor-1 (APAF-1), which increases the activity of caspase-9 and caspase-3, ultimately resulting in apoptosis. Additionally, ROS uptake caused DNA damage, possibly via the “clastogenic effect” of the flavonoids [88,89]. This damage increases p53 concentration and consequently p21 level, followed by depletion of cyclin E and increase of Cyclin-dependent Kinase-2 (CdK-2). These latter events mediated cell cycle arrest in the G1 phase. Therefore, VOX and possibly VOR should be considered as potential antitumor agents, as they are able to induce apoptosis in tumor cell lines, without significantly affecting normal cell lines [87]. 7. Bioavailability of BVc and BVr phytochemicals The bioavailability of a phytochemical is the percentage of the consumed drug that enters the bloodstream [90,91]. The bioavailability of betalains, was documented by several studies in animals and humans. Netzel et al. [92] and Frank et al. [93] investigated the pharmacokinetic of betalains in healthy humans after the ingestion of beet root juice. They observed that betacyanins were detected in the urine immediately after the ingestion, but the fraction of the unmetabolized betalains, excreted in the urine was very low. As the pigment content in the urine accounted for 0.5–0.9 % of the ingested dose, these authors concluded that the renal clearance scarcely contributes to systemic elimination of betalains [92]. Other pathways of elimination such as biliary excretion, enterohepatic circulation and metabolism, including the metabolic contribute of intestinal bacteria have been also hypothesized [93]. Tesoriere et al. [94] simulated in “in vitro” conditions oral, gastric and intestinal digestion of betalains comparing different fresh foods containing the pigments with the purified pigments. They found that the food matrix prevented degradation of betanin and isobetanin at the gastric environment and loss of betacyanins was observed during the digestion in the small intestine, with differences between food containing pigments and purified betalains. In fact, betalamic acid accumulation was observed after the digestive degradations of purified betalains, but not during digestion of food containing betalains [94]. The authors concluded that bioavailability of dietary betalains is mainly controlled by their chemical stability in the digestive tracts, but other factors, i.e. the type of food matrix, influence the bioaccessibility of the digestive enzymes [94]. Indeed, intestinal bacteria are actively involved in betalains metabolism, interfering with their absorption and bioavailability [95]. Tesoriere et al. [96] studied the permeability of red beet indicaxanthin and betanin in Caco-2 monolayers cell. Indicaxanthin showed a better permeability coefficient in respect to betanin and the key step in the absorption was attributed to the Multidrug Resistance-associated Protein-2 (MRP-2), which controlled the efflux of phytochemicals by means of a dose-dependent activity [96]. Overall, these data confirm that betalains availability is high in humans with betaxanthins more bioavailable that betacyanins, but further research is needed to provide type and concentration of betalain metabolites in plasma, urine and bile. Due to its better bioavailability and health protective effect, the betaxanthins have been already used as food supplements in order to fortify processed food products [97].

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Fig. 4. Schematic summary of the main pathways involved in the cell cycle arrest and apoptosis promoted by vitexin-2-O-xyloside on colon cancer cell. ROS: reactive oxygen species; Bcl-2: B-cell lymphoma-2; Bax: Bcl-2 associated X protein; APAF-1: apoptotic protease activating factor-1; caspase: cysteine-aspartic protease; PARP: poly ADP-ribose polymerase; CDK-2: cyclin-dependent kinase; PRb: retinoblastoma protein.

As far as the bioavailability of vitexin flavonoids is concerned, few studies are available in the literature on animal models. The bioavailability of vitexin, VOR and VOX has been determined for the first time using hawthorn leaf extract

(HLE) by Ma et al. [98] in mice. By intragastric administration of 2 g/kg of HLE, which is particularly rich of these flavonoids, a maximal plasma concentration of VOR was detected within 45 minutes and high levels of VOR were observed in the liver

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and kidney, while no detectable level was found in the brain. Xu et al. [99], using HLE, demonstrated that VOR is taken up by the intestine principally through passive diffusion. Its absorption and secretion are mediated by the efflux transport systems, such as P-glycoprotein (P-gp). The same authors suggested that the absorption of VOR can be enhanced if the drug is administered together with P-gp inhibitors [99]. In our laboratory, we assessed the bioavailability of vitexin flavonoids, purified from Swiss chard, by two methods. First, using an ex vivo system, where a flavonoid is incubated with red blood cells (RBC) in the presence of 2′,7′dichlorofluorescein diacetate (DCFH-DA), with subsequent measurement of the cellular antioxidant activity (CAA) [100]. During incubation, DCFH-DA enters the RBC and is hydrolysed by internal esterases into DCFH, which becomes fluorescent when oxidized to DCF. The flavonoid is able to enter the RBC and it is also able to protect the DCFH probe from oxidation, thus allowing measurement of its intracellular antioxidant capacity. From comparison of the fluorescence curve areas, with quercetin as a standard, it is possible to assess both the CAA and the permeability of the flavonoid across the cell membrane. This method was only predictive of the in vivo bioavailability and we applied it to compare CAA of VOX in respect to apigenin and luteolin (Table 3). VOX was found to be bioavailable at the extent of 10% with respect to its aglycone apigenin, whose bioavailability has been demonstrated in vivo [101,102]. The lower bioavailability of VOX is most likely due to prohibited movement across the RBC membrane owing to the polarity and to steric hindrance of its sugar moiety. Second, the bioavailability of vitexin flavonoids was studied by means of polyclonal antibodies, produced in our laboratory, and ELISA for detection of the flavonoid and its metabolites in the blood. As flavonoids are low molecular weight compounds, to obtain an adequate antibody titre, the biapigenin hinokiflavone was coupled with bovine serum albumin (BSA) [103] and injected into mice as the immunogen. Immunization of Balb/c mice provided an antiserum with antibody titre of 1:1600. Plasma concentration of vitexin flavonoids was found to be 3.42 ± 0.72 μg/mL in mice fed with 170 mg/kg VOX [104]. In the study of vitexin flavonoids bioavailability, an important issue is the understanding of the metabolic contribute performed by the gut microbiota. In fact, flavonoids also serve as substrates for intestinal bacteria, particularly when they are present in their glycosylated form. As a result, sugar moieties are released from flavonoid and, in some cases, also the flavonoid basic structure is further metabolized [105]. Recent studies [106] showed that isovitexin, but not its isomer vitexin, was cleaved into the aglyconic form by an anaerobic cellulolytic

Table 3 Cellular antioxidant capacity of flavonoids from Beta vulgaris cicla. Compound

CAA-RBC (μMolQE/g⁎)

Luteolin Apigenin Vitexin-2-O-xyloside

4.32 ± 0.26 4.30 ± 0.34 0.435 ± 0.026

⁎ μMolQE/g represents the units of the cellular antioxidant activity in RBC referred to the micromolar equivalents of quercetin, which is used as standard. For details, see reference [100].

bacterium, found in sheep and cow rumen as well as in mouse intestine. This study indicates the importance of the selection and the contribution of the microbiota to the metabolism and absorption of flavonoid glycosydes. 8. Conclusions BVc and BVr are commercially important crops, which represent a plentiful and inexpensive source of nutrients. In our country, the utilization of these vegetables as a food is wide, as they are used in several home-made meals. Industrial procedures for preparing frozen BVc or pre-cooked BVr provide packaged vegetables of good nutritional quality, which satisfy the exigency of convenience products for families and restaurants. At the same time, BVc and BVr have revealed interesting properties in phytomedicine. BVc is a rich source of vitexin, VOR and VOX, the isolation of which is workable at present and results are more convenient than the chemical synthesis [12]. In fact, BVc leaves and seeds represent a cheap and highly renewable material, which makes large-scale purification industrially possible, with little environmental harm. Starting from the BVc seeds it is possible to isolate VOX at 98% purity. By using the leaves as starting material, the purification process can provide a mixture of VOR and VOX. Further studies are needed to scale-up the purification protocol to obtain sufficient amount of these flavonoids for in vivo studies of their biochemical properties. VOR and VOX show therapeutic potential owing to their antioxidant capacity, low toxicity and anti-proliferative activity on tumor cells and merit to be brought to clinical trials, for further investigation as cancer chemopreventive agents. The betalains of BVr are natural colorants for food use [107] but also strong antiradical and antioxidant agents able to protect against oxidative stress related disorders in vivo. Consumers may benefit from regular consumption of betalainrich beetroot juice, which is commercially available as lactofermented juice with low nitrates content. This juice shows interesting properties, such as detoxification and cholesterol lowering effect. Future research should address the circulating metabolites of the betalains and their physiological properties. Moreover, breeding procedures should be investigated in order to obtain cultivars more rich in betalains, with respect to those actually present in market. Moreover, it is necessary to deeply understand how betalains change their properties when added to foods, as in some cases the food matrix can positively affect the stability of the pigment, whereas in some cases it works contrariwise. BVc and BVr are popular vegetables in the Mediterranean Diet, which has been associated, through epidemiological studies, to a statistically significant protective effect on colon cancer risk [108]. The apigenin flavonoids, vitexin, VOR and VOX, together with the betalains, constitute an important group of the Chenopodiacea phytochemicals that can be fittingly enclosed in the arsenal that nature has given us to protect our health. Acknowledgments Authors wish to thank Dr. Whitney N. Ajie, Department of Food Science and Human Nutrition of the University of Illinois at Urbana-Champaign, IL, for helpful discussion and support during the preparation of the English manuscript.

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Authors wish also to thank SUBA SEEDS COMPANY, Longiano (FO, Italy) for providing Beta vulgaris cicla seeds and Mr. Franco Balducci, Urbino (PU, Italy) for his help in providing samples of fresh crops of beets.

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