Article history:Received 10 April 2013Accepted in revised form 28 May 2013Available online 7 June 2013
Swiss chard (Beta vulgaris cicla, BVc) and beetroot (Beta vulgaris rubra, BVr) are vegetables of theChenopodiaceae family,widely consumed in traditionalwestern cooking. These vegetables representa highly renewable and cheap source of nutrients. They can be cultivated in soils with scarce organicmaterial and little light and water. BVc and BVr have a long history of use in folk medicine. Modernpharmacology shows that BVc extracts possess antihypertensive and hypoglycaemic activity aswell as excellent antioxidant activity. BVc contains apigenin flavonoids, namely vitexin,vitexin-2-O-rhamnoside and vitexin-2-O-xyloside, which show antiproliferative activity on cancercell lines. BVr contains secondary metabolites, called betalains, which are used as natural dyes infood industry and show anticancer activity. In this light, BVc and BVr can be considered functionalfoods. Moreover, the promising results of their phytochemicals in health protection suggestthe opportunity to take advantage of the large availability of this crop for purification ofchemopreventive molecules to be used in functional foods and nutraceutical products.
Red beetroot (Beta vulgaris rubra, BVr) and Swiss chard (Betavulgaris cicla, BVc) are members of the Chenopodiaceae. Thisfamily contains important food crops, such as Spinacia oleracea(spinach), which is the most consumed Chenopodiaceae leafyvegetable in Europe [www.fao.org/economic/ess/en/], Salsolakali, or prickly saltwort, currently used inwestern countries, andChenopodium quinoa, commonly known as quinoa [1].
In the Chenopodiaceae family, there are also twowild ediblerepresentatives: Chenopodium album, known as lambsquarterand in indi as bathua [2], and Chenopodium bonus henricus [3],also called “mountain spinach”, as it grows in the grazing landsof the Alps [4].
BVc and BVr have been used for food since 1000 B.C. by allpopulations of the Mediterranean basin. The Romans utilizedthe BVc and BVr leaves for food, while the roots were used formedicinal applications. BVc became commercially importantin 19th century in Europe, following the development of thesugar beet (Beta vulgaris saccharifera) in Germany [4].
D
A
Fig. 1. Pictures of the main cultivars of Beta vulgaris cicla and rubra. A) Swiss chard, biF1; D) Red Beetroot cv Chioggia; E) Red Beetroot cv. Detroit; F) Red Beetroot cv Rub
In Italy, the two most produced BVc cultivars are Bieta acosta Bianca, for home use, and Bieta erbetta da taglio forindustry use (Fig. 1), whereas there are several varieties ofBVr, 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 wellas on the biological and pharmacological activities of theirphytochemicals.
2. Taxonomy
Beet is classified taxonomically as Dicotyledonae,Caryophyllidae, Chenopodiaceae and Beta [5]. On the basis ofmorphological characters, the genus Betawas sub-divided intotwo groups: cultivated and wild maritime beets. In the lattergroup, the sea beets (Beta vulgaris maritima) is the uniquespecies, which represents the ancestral form of all the species.In the cultivated group, there are sugar beets (Beta vulgarissaccharifera), fodder beets (Beta vulgaris crassa), leaf beets(Beta vulgaris cicla) and garden beets (Beta vulgaris rubra) [6].
E F
B C
eta costa bianca; B) Swiss chard, bieta erbetta da taglio; C) Swiss chard, Hybridy Queen.
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3. Botany
BVc is a biennal plant with strong roots and large ovalleaves, with white veins. BVc is generally seeded in spring forsummer crop and again in summer for the winter and springcrops. BVc is harvested by cutting leaves at the base, while theroots remain in the soil for re-growth. During thewinter, plantsaccumulate nutrients in the roots and begin the newvegetationcycle in the spring. The following summer, the plants produceflowers grouped into glomerula forming spikes in the apicalpart of the stems. After that, the plants produce the seeds andlater die. The seeds of beet normally contain more than oneseed and they are called polygerms. The breeding procedureshave obtained, in recent years, three important results:polyploidy, monogermity and the production of commercialhybrids [7].
BVr or beetroot, or garden beet or table beet, is cultivatedfor its large roots, but the leaves also are utilizable. The shanksmay be red, magenta or white and leaves are small and greenwith thin red veins. BVr is grown for the thick flashy taprootthat forms during the first season. On cooking the color diffusesuniformly through the flesh. In the second season, a tallbranched leafy stem arises to bear clusters of minute greenflowers, that develop into brown corky fruits, commonly calledseed balls.
The taproot varies in shape from flattened oblate to globularand from conical to long tapered. Skin and flash colors areusually dark-purplish red, with some nearly white.
4. Ecology
BVc and BVr grow on friable soils with a good drainingtexture and abundant organic matter. Commercial fertilizers,containing nitrogen, phosphorus and potash may be added tothe soil, or alternative fertilizers, such as green manures, cropresidues, animal manure and compost may be used [5].However, BVc and BVr adapted to environments with elevatedsaline concentrations, such as marine coast, salt marsh andloam [4]. Optimal soil pH ranges between 6.0 and 6.8, butneutral or alkaline pH is well tolerated [5].
BVr must be sown directly in the field, since the sowing ina greenhouse, followed by planting, may lead to bifid roots.BVr is harvested during the summer, when the leaves aredehydrated.
Indeed, BVc and BVr plants are also able to grow in soils withscarce availability of water and organic matter and tolerate
Table 1Fatty acid composition of Beta vulgaris cicla seed oil.
⁎ Average value of three separate analysis on oil extracted withsupercritical CO2.
bb
b
Flavonoids
Fig. 2. Phenolic and flavonoid concentration after planting BVc cultivarsBieta Erbetta, Hybrid F1 and Bieta costa Bianca during 90 days. Phenoliccompounds were assayed by the method of Singleton et al. [109], whereasflavonoid were assayed by the method of Eberhard et al. [110]. Results arethe mean ± SD of three replicates. a, b = same letters indicate statisticallysignificant differences among values of the same curve, for p b 0.05, byANOVA.
conditions of low light [4]. In fact, these vegetables wereproposed for cultivation in space. The Swiss chard varietiesLarge White Ribbed and Lucullus have been demonstrated toprovide 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 whosecomposition in fatty acids has been determined by us throughGC–MS analysis, following the standard protocols reported bynormative ISO 5508:1990. Table 1 shows the fatty acidcomposition of BVc seed oil after extraction with supercriticalCO2. Results show the high content of linoleic acid, followed bya remarkable presence of oleic acid. Among the saturated fattyacids, the dominant component was palmitic acid (17%).
5.2. Polyphenols
BVc and BVr seeds, leaves and roots are rich in phenoliccompounds, whose concentration is dependent on the stage ofplant development [9,10]. For instance, three cultivars of BVcshowed maximum phenolic content at 55–60 days aftertransplantation (Fig. 2). After this time, the phenolic concen-tration dropped progressively with aging; the flavonoidconcentration showed a similar trend (Fig. 2). Therefore, forthe maximum phenolic intake, the leaves should be harvestedat the maturity stage.
The phenolic pool of BVc and BVr is represented by differenttypes of molecules, which include phenolic acids, flavonoidsand phenolic amides, including betalains.
5.2.1. Phenolic acidsPyo et al. [11] measured the concentration of phenolic
acids in Swiss chard leaves. Interestingly, they found syringicacid (44.9 mg/100 g freshweight), caffeic acid (14.8 mg/100 g
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.
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fresh weight) and other phenolic acids in concentrationsranging from 5 to 10 mg/100 g fresh weight.
In our laboratory, we performed the screening of phenoliccompounds through HPLC–MS analysis of BVc seed extractand we found in addition to the above phenolic acids,the presence of vanillic acid, and two aldehydes, namely:2,5-dihydroxybenzaldehyde and2,4,5-trihydroxybenzaldehyde [12].
In BVr, Georgev et al. [13] demonstrated the presence of4-hydroxybenzoic acid, caffeic acid and chlorogenic acid inconcentrations ranging from 0.12 to 0.047 mg/g of dry extract.
5.2.2. FlavonoidsBVc 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), thepurification of which has been performed in our laboratory[12]. Among the BVc flavonoids, Pyo et al. [11] recognized thefollowing: catechin (6.7 mg/100 g fresh weight), myricetin(2.2 mg/100 g fresh weight), quercetin (7.5 mg/100 g freshweight) and kaempferol (9.2 mg/100 g fresh weight).
Geogev et al. [13] revealed in BVr cv. Detroit Dark Red thepresence of catechin-hydrate, epicatechin and rutin, as well asKujala et al. [14] identified in four cultivars of BVr four flavonoids:betagarin, betavulgarin, cochliofilin and dihydroxyisorhamnetin.
5.2.3. Betalains and phenolic amidesBVr contains a large amount of betalains, a group of
numerous water soluble nitrogen containing pigments derivedfrom betalamic acid (Fig. 3A), which the chromophore moietycommon to all molecules. Inside to the betalain family, thereare 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 theranges 0.04–0.21% and 0.02–0.14%, respectively [16]. Themajorbetacyanin pigment in BVr is betanin, which is a betanidin-5-O-β-glucoside. Betanidin, is therefore the aglyconic form of thebetanin. The maior betaxanthin present in BVr is vulgaxanthin I,which is also present, together with betanin, in few pigmentedcultivar of BVc, but their concentration is much lower in BVcthan in BVr [16]. To date, the food colorant extracted from BVr,known as “beetroot red”, is commercially available as E162 inEurope and USA [17] and new BVr crops with increasedpigment quantity are actively searched by experts of breedingand horticultural practices [18].
Beyond to the betalains, Kujala et al. [14] identified in fourcultivars of BVr two additional phenolic amides (N-trans-feruloyltyramine andN-trans-feruloylhomovanillylamine). Thediscovery of the latter compounds stimulated the efforts for thechemical synthesis of this natural molecule to be used as anantioxidant and antitumor agent [19], due to its exceptionalradical-scavenging and photoprotective ability [13].
5.2.4. Organic and inorganic acidsBVc leaves and BVr roots contain oxalates and nitrates
(NO3−), which represent anti-nutritional factors, as theysubtractmicronutrients during the digestive process in humans[20,21]. Another adverse effect is the contribution to theformation of kidney stones [22]. Nitrates are commonly presentin water and vegetables as well as in cured meat [23]. Somenitrates are also endogenously produced principally by theL-arginine–NO pathway [24].The Acceptable Daily Intake (ADI)for nitrate is 3.7 mg/kg b.w./day [25]. NO3− ion shows arelatively low toxicity [26], but one should bear in mind that25% 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 oramides to form N-nitroso compounds, which are well-knowngastric and bladder cancer activators [28]. During the industrialpreparation of BVr juices, the nitrate concentration is consid-erably reduced by fermentation strategies [29].
A positive role of NO3− reduction regards the nitriteconversion of into nitric oxide (NO) in the body, throughnumerous pathways involving deoxyhaemoglobin, myoglobin,ascorbate and polyphenols [30]. In physiological or patholog-ical hypoxia, NO provides several effects, including: vasodila-tion, modulation of cellular respiration, responses to ischaemicstress, lowering blood pressure, vaso-protection and anti-platelet aggregation properties [31,32]. In this light, thebeetroot juice has a potential effect of human blood pressurereduction [33] and therefore it was used in the improvement ofathletic performance [34].
Concerning the oxalates as anti-nutritional factors [20,21],we must infer that the problem addresses mainly BVc oldleaves. In fact, oxalate concentration, which is very low inyoung BVc leaves, increases near to the toxic level in old BVcleaves, during the second year of vegetation [5,35]. From afunctional and nutritional standpoint, therefore, it is better toconsume steam-cooked young BVc leaves, i.e. harvested inthe range of 40–60 days, in order to save the polyphenolcontent and minimize the oxalates [36]. On the contrary, ifone should use old leaves, i.e. those harvested in the secondyear, it is better to boil the leaves in abundant water, in orderto lose most of the oxalates in the boiling water.
5.2.5. MiscellaneousRed beetroot is rich in carbohydrates, fibers, proteins and
minerals, such as sodium, potassium, calcium and iron [3]. Thebeet metabolism also produces geosmin, a bicyclic alcoholwhich provides the characteristic “earthy” flavor to red beetroot[37]. To remove this unpleasant flavor, a distillation processduring juice concentration is applied [16]. Swiss chard leaves area 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 freshBVc and BVr, respectively [38].
6. Biological activity
BVc and BVr have been used for a long time for theirbeneficial health effects, mainly consisting in stimulation ofhaematopoietic and immune systems as well as in theprotection of kidney, liver and gut from toxic compounds.Moreover, they exhibit mineralizing, antiseptic and cholereticactivities as well as they contribute to the reinforcement of thegastric mucosa [39–41].
BVr have been used for therapy of intestinal and genitaltumors, while juices of fresh roots or leaves have beenconsidered effective in the therapy of tumors of the digestivesystem as well as of the lung, liver, breast, prostate and uterus[5].
Inmodern pharmacology, the betalains have been themoststudied BVr health protectivemolecules. They have been linkedto protection against oxidative stress, inflammation andtumors. The stability of betalains in the beetroot juice hasbeen considered crucial for the exhibition of the antitumoreffect. For instance, Patkai et al. [42] studied the retention of
betalains in red beetroot juice, during production andpasteurization.
Modern pharmacologists also addressed the importance ofbioactive molecules from BVc extracts and demonstrated theiranti-diabetic, anti-inflammatory, antioxidant and anticanceractivities.
6.1. Anti-diabetic activity
Experiments performed by Yanardag et al. [43] demon-strated the hypoglycemic effect of BVc extract in diabetic rats.This hypothesis was substantiated by further studies of thesame 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 theextract has been tentatively attributed to saponins, that inhibitgluconeogenesis and glycogenolysis [46]. However, othermolecular pathways potentially involved in hypoglycemiceffects remain to be deeply investigated. In fact, someevidences suggested that the hypoglycemic activity of BVcextract may be due to flavonoids, through the inhibition ofglucose transporters. For instance, quercetin, which is presentin BVc, showed evidence of anti-diabetic effects via inhibitionof the intestinal glucose transporter GLUT2 [47].
Another complementary hypoglycemic mechanism couldbe the flavonoid induced inhibition of the α-amylase andα-glucosidase activities [48]. For instance, two flavonol-glycosides isolated from Salsola kali were demonstrated to beactive inhibitors of α-amylase [49]. The inhibition of thisenzyme could delay the digestion and absorption of carbohy-drates and consequently suppress post-prandial hyperglycemia[50]. SomeC-glycosyl flavones, i.e. vitexin, vitexin-2-O-glycosideand VOR (Fig. 3), contained in BVc leaves and seeds, were foundto strongly inhibit α-glucosidase [51] and could be the mostprobable cause of the hypoglycemic effect earlier evidenced indiabetic rats [44].
6.2. Anti-inflammatory activity
In order to study the pharmacological activity of BVc, someauthors considered the molecules contained in the seeds. Kimet al. [35] extracted phenolic amides from BVc seeds and testedtheir inhibition in nitric oxide (NO) production in murinemacrophages, stimulated by lipopolysaccharides; the authorsfound IC50 values for NO synthase (NOS) inhibition rangingfrom 13 to 18 μM. Therefore, they concluded that BVc seedextract demonstrated in vitro anti-inflammatory activity byinhibiting NOS and suggested to use these phenolic amides forthe development of new anti-inflammatory drugs.
The red pigment betanin has been demonstrated to providea strong anti-inflammatory activity, throughout the inhibitionof cyclooxygenase (COX) activity, which catalyzes the conver-sion of arachidonic acid into chemical mediators of inflamma-tion [52]. Results showed that betanin (100 μg/mL) was moreefficient in the inhibition of COX-2 (97%) than COX-1 (33.5%).The same authors observed that combination of betanin andanthocyanins reduced their respective biological activities thusexhibiting a negative synergistic effect [52].
Atta et al. [53] demonstrated that ethanolic extracts of redbeet roots possess a dose-dependent anti-inflammatoryeffect against both acute and chronic inflammation. However,
Table 2Total phenols, flavonoids and antioxidant capacities in different Chenopodiaceae.
Values are themean ± SD of four independentmeasurements performed in ourlaboratory. Phenolswere assayed as reported by Singleton et al. [109], flavonoidsby Eberhart et al. [110] and ORAC by Ninfali et al. [9]. dw = dry weight.
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these authors did not specify which cultivar they tested andthe origin of their ethanolic extract remains undefined.
6.3. Antioxidant activity
BVc and BVr extracts have been widely studied for theirantioxidant activity both in vitro and in vivo [9,54–57].
In our laboratory, we performed a comparative survey of theantioxidant capacity among edible Chenopodiaceae plants.Table 2 shows the ORAC results, in parallel with the phenolicand flavonoid content in roots, leaves and seeds. Concerning theBeta vulgaris species, it is interesting to note that phenols,flavonoids and ORAC values are lower in roots with respect toleaves. BVr roots, which contain large amounts of carbohydratesand low concentration of secondary metabolites, consequentlyhave lower antioxidant capacity than BVc or BVr leaves.Chenopodiumalbum and Spinacia olearacea leaves showedhigherORAC values than BVc leaves. Among the seeds, Chenopodiumquinoa 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 spinachcontains aminor amount of phenols, but its phenolic compoundsare endowed of powerful antioxidant capacity. The comparisonamong BVc, BVr and spinach is referred to the raw vegetables,but wemust consider that they are eaten boiled or steamed andthe antioxidant capacity as well as the phenolic content mayundergodramatic changes, depending on the thermal processing[58,59]. This aspect addresses also the frozen vegetables as, forthe industrial production, BVc and spinach are mechanicallycollected and immediately transferred to the industry, wherethey are washed, steamed and packaged under frozen condi-tions. Part of the antioxidant capacity is lost during the industrialtreatment, depending on the steaming time. Ninfali et al. [9,60]and Gil et al. [61] measured the phenolic loss after steaming andboiling and showed that nearly 50% of the polyphenols are lost in
the water after boiling, together with 80% of vitamin C content;on the contrary, 15 min steaming depletes only 20% of phenoliccompounds. Kuti et al. [62] also found a similar phenolic andantioxidant capacity decay, measured with the ORACmethod, inspinach leaves steamed in a microwave oven.
From the results of Table 2, it emerges the low antioxidantcapacity of the whole extract of BVr roots. However, whenthe betalains are extracted from BVr roots, their antioxidantcapacity become remarkable. Kujawska et al. [54] investigatedthe protective effect of the beetroot juice in a model ofoxidative stress induced by carbon tetrachloride (CCl4) onmale Wistar rats. Results demonstrated that microsomal lipidperoxidation in the liver, expressed as Thiobarbituric AcidReactive Substances (TBARS) concentration decreased by 38%in rats pretreated with beetroot juices before the administra-tion of CCl4. The pretreatmentwith beetroot juice also caused apartial recovery in the activity of glutathione peroxidase andglutathione reductase by 35% and 66% respectively, after beendepleted by the CCl4 treatment. Beetroot juice was alsodemonstrated to be able to reduce plasma protein carbonyls(~30%) and DNA damage in blood leukocytes (~20%) of ratstreated with juice before xenobiotic administration. Escribanoet al. [39] investigated the anti-radical activity of bothbetaxanthins and betacyanins from Beta vulgaris rubra bymeans the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) (ABTS). The antiradical activity of betacyanins was foundto be greater than that of betaxanthins and increased with thepH of the reaction medium. Georgev et al. [13] compared theantioxidant activity of betalains from hairy root cultures andintact plants of the beetroot Beta vulgaris cv. Detroit dark red.These authors evaluated the antioxidant activity using both theDPPH and the ORAC assays and demonstrated a six-fold higherantioxidant activity of hairy root extracts versus the maturebeetroots.
Tesoriere et al. [63] showed the incorporation of betalainsinto human red blood cells and the consequent protection of thecells from oxidative hemolysis; in parallel, in an in vitro model,Gentile et al. [64] described the ability of betalains to protect theendothelial cells from the oxidation processes, related toinflammatory response. Kanner et al. [65] and Tesoriere et al.[66] demonstrated that betalains may bind LDL and cellularmembranes to inhibit lipid peroxidation in blood. Moreover,Allegra et al. [67] reported the ability of betalains to scavengehypochlorous acid, which is powerful oxidant produced byneutrophils during the inflammatory response. The ability of thebetalains to protect both human LDL and endothelial cellsfrom oxidation due to inflammatory responses, make thesemolecules as functionally important for reducing the risks ofatherosclerotic plaque formation. Finally, Vulic et al. [68]investigated the biological activity of beetroot pomace, a wasteindustrial material mainly constituted by ferulic, vanillic,p-hydroxybenzoic, caffeic and protocatechuic acids andbetalains, including betanin, isobetanin and vulgaxhantin I.The beetroot pomace, showed excellent antiradical activity,measured by ESR spectroscopy, towards DPPH and hydroxylradicals as well as antimicrobial activity against Gram− andGram+ bacteria [68].
Therefore, the finding of BVr cultivars very rich in phenolicacids and betalains as well as to find agronomic practicesappears of nutritional utility, whichmay significantly influencethe total antioxidant concentration. For example, Bavec et al.
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[20] demonstrated that polyphenols and antioxidant capacityof BVr were higher in biodynamic and integrated farmingsystems with respect to conventional farming system.
6.4. Anticancer activity
Earlier investigations of the anticancer properties of thegenus Beta were focused on the BVr. Betalains have been theprimary molecules recognized to provide anticancer effects[15]. The first antitumor mechanism, proposed for betalains,was an interruption in the exchange of metabolites betweentumor cells and surrounding tissues, in such a way that itwould hinder the infiltration capacity of the tumor cells[40,52]. More recently, Sreekanth et al. [69] deeply investi-gated 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 inK-562 cell proliferation [69]. Results also revealed thatbetanin induced apoptosis through the intrinsic pathway,which was mediated by the release of cytochrome-C frommitochondria and Poly ADP-Ribose Polymerase (PARP)cleavage [69]. Cytotoxic activity against Ehrlich carcinomacells, was demonstrated by Vulic et al. [68] with a murinemodel in which Ehrlich carcinoma cells were implanted.These authors observed that the administration of beetrootpomace extract, rich in polyphenols and betalains, was ableto decrease the carcinoma cell numbers and the amount ofthe ascites they produced in respect to the control animalstreated with placebo.
Vitexin,(Fig. 3D), VOX (Fig. 3E), and VOR (Fig. 3F), are theprincipal 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 lastdecade [70,71]. Apigenin has been shown to interfere in thetumor 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 literaturehas led to the initiation of current research concerning theantitumor contribution of vitexin, VOX and VOR. The anticancereffect of vitexin has been studied by Yang et al. [85] in humanoral cancer OC2 cells. These authors demonstrated that vitexindecreased cell viability significantly and up-regulated theexpression of the tumor suppressor p53 mediated signaling,including p53-Bax and p53-PPARγ-caspase 3 pathways, whichled to apoptosis. Meanwhile, vitexin decreased cell migrationvia p53-PAI1-MMP2 cascade [85].
Ninfali et al. [86] observed that a mixture of VOX and VORwas very active in blocking the proliferation of MCF-7 breastcancer cells. Indeed, VOX was purified from BVc seeds andfound to be particularly effective in inhibiting the proliferationof intestinal cancer cells, such as RKO [12], Caco2 and LoVo celllines [87].
Further, VOX is able to synergize with epigallocatechine-3-gallate and glucoraphasatin to inhibit proliferation of LoVo andCaco-2 cell lines [87].
The mechanism by which VOX induces apoptosis inintestinal tumor cell lines has been proposed [87]. The process,described in Fig. 4, beginswith the production of ROS, which inturn leads to glutathione depletion; this cascade increases Baxand concomitantly depletes Bcl-2 proteins, which in turn
changes the mitochondrial membrane potential, releasingcytochrome-C into the cytoplasm. This efflux activates theApoptotic Protease Activating Factor-1 (APAF-1), which in-creases the activity of caspase-9 and caspase-3, ultimatelyresulting in apoptosis. Additionally, ROS uptake caused DNAdamage, possibly via the “clastogenic effect” of the flavonoids[88,89]. This damage increases p53 concentration and conse-quently p21 level, followed by depletion of cyclin E andincrease of Cyclin-dependent Kinase-2 (CdK-2). These latterevents mediated cell cycle arrest in the G1 phase. Therefore,VOX and possibly VOR should be considered as potentialantitumor agents, as they are able to induce apoptosis in tumorcell lines, without significantly affecting normal cell lines [87].
7. Bioavailability of BVc and BVr phytochemicals
The bioavailability of a phytochemical is the percentage ofthe consumed drug that enters the bloodstream [90,91].
The bioavailability of betalains, was documented by severalstudies in animals and humans. Netzel et al. [92] and Frank et al.[93] investigated the pharmacokinetic of betalains in healthyhumans after the ingestion of beet root juice. They observed thatbetacyanins were detected in the urine immediately after theingestion, but the fraction of the unmetabolized betalains,excreted in the urine was very low. As the pigment content inthe urine accounted for 0.5–0.9 % of the ingested dose, theseauthors concluded that the renal clearance scarcely contributesto systemic elimination of betalains [92]. Other pathways ofelimination such as biliary excretion, enterohepatic circulationandmetabolism, including themetabolic contribute of intestinalbacteria have been also hypothesized [93]. Tesoriere et al.[94] simulated in “in vitro” conditions oral, gastric and intestinaldigestion of betalains comparing different fresh foodscontaining the pigments with the purified pigments. Theyfound that the food matrix prevented degradation of betaninand isobetanin at the gastric environment and loss of beta-cyaninswas observed during the digestion in the small intestine,with differences between food containing pigments and purifiedbetalains. In fact, betalamic acid accumulationwas observed afterthe digestive degradations of purified betalains, but not duringdigestion of food containing betalains [94]. The authors conclud-ed that bioavailability of dietary betalains ismainly controlled bytheir chemical stability in the digestive tracts, but other factors,i.e. the type of food matrix, influence the bioaccessibility of thedigestive enzymes [94]. Indeed, intestinal bacteria are activelyinvolved in betalains metabolism, interfering with their absorp-tion and bioavailability [95].
Tesoriere et al. [96] studied the permeability of redbeet indicaxanthin and betanin in Caco-2 monolayers cell.Indicaxanthin showed a better permeability coefficient inrespect to betanin and the key step in the absorption wasattributed to the Multidrug Resistance-associated Protein-2(MRP-2), which controlled the efflux of phytochemicals bymeans of a dose-dependent activity [96].
Overall, these data confirm that betalains availability ishigh in humans with betaxanthins more bioavailable thatbetacyanins, but further research is needed to provide type andconcentration of betalain metabolites in plasma, urine and bile.Due to its better bioavailability and health protective effect, thebetaxanthins have been already used as food supplements inorder to fortify processed food products [97].
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-asparticprotease; PARP: poly ADP-ribose polymerase; CDK-2: cyclin-dependent kinase; PRb: retinoblastoma protein.
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As far as the bioavailability of vitexin flavonoids is con-cerned, few studies are available in the literature on animalmodels. The bioavailability of vitexin, VOR and VOX has beendetermined for the first time using hawthorn leaf extract
(HLE) byMaet al. [98] inmice. By intragastric administration of2 g/kg of HLE, which is particularly rich of these flavonoids, amaximal plasma concentration of VOR was detected within45 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 bythe intestine principally through passive diffusion. Its absorp-tion and secretion are mediated by the efflux transportsystems, such as P-glycoprotein (P-gp). The same authorssuggested that the absorption of VOR can be enhanced if thedrug is administered together with P-gp inhibitors [99].
In our laboratory, we assessed the bioavailability of vitexinflavonoids, purified from Swiss chard, by two methods. First,using an ex vivo system, where a flavonoid is incubated withred blood cells (RBC) in the presence of 2′,7′dichlorofluoresceindiacetate (DCFH-DA), with subsequent measurement of thecellular antioxidant activity (CAA) [100]. During incubation,DCFH-DA enters the RBC and is hydrolysed by internalesterases into DCFH, which becomes fluorescent when oxi-dized to DCF. The flavonoid is able to enter the RBC and it is alsoable to protect the DCFH probe from oxidation, thus allowingmeasurement of its intracellular antioxidant capacity. Fromcomparison of the fluorescence curve areas, with quercetin as astandard, it is possible to assess both the CAA and thepermeability of the flavonoid across the cell membrane. Thismethod was only predictive of the in vivo bioavailability andwe applied it to compare CAAof VOX in respect to apigenin andluteolin (Table 3). VOX was found to be bioavailable at theextent of 10% with respect to its aglycone apigenin, whosebioavailability has been demonstrated in vivo [101,102]. Thelower bioavailability of VOX is most likely due to prohibitedmovement across the RBCmembrane owing to the polarity andto steric hindrance of its sugar moiety.
Second, the bioavailability of vitexin flavonoids wasstudied by means of polyclonal antibodies, produced in ourlaboratory, and ELISA for detection of the flavonoid and itsmetabolites in the blood. As flavonoids are low molecularweight compounds, to obtain an adequate antibody titre, thebiapigenin hinokiflavone was coupled with bovine serumalbumin (BSA) [103] and injected into mice as the immuno-gen. Immunization of Balb/c mice provided an antiserumwith antibody titre of 1:1600. Plasma concentration of vitexinflavonoids was found to be 3.42 ± 0.72 μg/mL in mice fedwith 170 mg/kg VOX [104].
In the study of vitexin flavonoids bioavailability, an im-portant issue is the understanding of the metabolic contributeperformed by the gut microbiota. In fact, flavonoids also serveas substrates for intestinal bacteria, particularly when they arepresent in their glycosylated form. As a result, sugar moietiesare released from flavonoid and, in some cases, also theflavonoid basic structure is further metabolized [105]. Recentstudies [106] showed that isovitexin, but not its isomer vitexin,was cleaved into the aglyconic formby an anaerobic cellulolytic
Table 3Cellular antioxidant capacity of flavonoids from Beta vulgaris cicla.
⁎ μMolQE/g represents the units of the cellular antioxidant activity in RBCreferred to themicromolar equivalents of quercetin, which is used as standardFor details, see reference [100].
.
bacterium, found in sheep and cow rumen as well as in mouseintestine. This study indicates the importance of the selectionand the contribution of the microbiota to the metabolism andabsorption of flavonoid glycosydes.
8. Conclusions
BVc and BVr are commercially important crops, whichrepresent a plentiful and inexpensive source of nutrients. Inour country, the utilization of these vegetables as a food iswide, as they are used in several home-made meals. Industrialprocedures for preparing frozen BVc or pre-cooked BVr providepackaged vegetables of good nutritional quality, which satisfythe exigency of convenience products for families andrestaurants. At the same time, BVc and BVr have revealedinteresting properties in phytomedicine. BVc is a rich source ofvitexin, VOR and VOX, the isolation of which is workable atpresent and results are more convenient than the chemicalsynthesis [12]. In fact, BVc leaves and seeds represent a cheapand highly renewable material, which makes large-scalepurification industrially possible, with little environmentalharm. Starting from the BVc seeds it is possible to isolate VOXat 98% purity. By using the leaves as starting material, thepurification process can provide a mixture of VOR and VOX.
Further studies are needed to scale-up the purificationprotocol to obtain sufficient amount of these flavonoids for invivo studies of their biochemical properties.
VOR and VOX show therapeutic potential owing to theirantioxidant capacity, low toxicity and anti-proliferative activityon tumor cells and merit to be brought to clinical trials, forfurther investigation as cancer chemopreventive agents.
The betalains of BVr are natural colorants for food use[107] but also strong antiradical and antioxidant agents ableto protect against oxidative stress related disorders in vivo.Consumers may benefit from regular consumption of betalain-rich beetroot juice, which is commercially available as lacto-fermented juice with low nitrates content. This juice showsinteresting properties, such as detoxification and cholesterollowering effect. Future research should address the circulatingmetabolites of the betalains and their physiological properties.Moreover, breeding procedures should be investigated in orderto obtain cultivars more rich in betalains, with respect to thoseactually present in market. Moreover, it is necessary to deeplyunderstand how betalains change their properties when addedto foods, as in some cases the food matrix can positively affectthe stability of the pigment, whereas in some cases it workscontrariwise.
BVc and BVr are popular vegetables in the MediterraneanDiet, which has been associated, through epidemiologicalstudies, to a statistically significant protective effect on coloncancer risk [108]. The apigenin flavonoids, vitexin, VOR and VOX,together with the betalains, constitute an important group of theChenopodiacea phytochemicals that can be fittingly enclosed inthe arsenal that nature has given us to protect our health.
Acknowledgments
Authors wish to thank Dr. Whitney N. Ajie, Department ofFood Science and Human Nutrition of the University ofIllinois at Urbana-Champaign, IL, for helpful discussion andsupport 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 providingsamples of fresh crops of beets.
References
[1] Ruales J, Nair BM. Properties of starch and dietary fibre in raw andprocessed quinoa (Chenopodium quinoa, Willd) seeds. Plant FoodsHum Nutr 1994;45:223–46.
[2] Yadav SK, Sehgal S. Effect of home processing and storage on ascorbicacid and beta-carotene content of Bathua (Chenopodium album) andfenugreek (Trigonella foenum graecum) leaves. Plant Foods Hum Nutr1997;50:239–47.
[3] Chiej R. Encyclopaedia of medicinal plants. London: TBS The BookService Ltd; 1984 .
[4] Norton PB, Esposito JJ. The New Encyclopaedia Britannica. Chicago:Encyclopedia Britannica Inc.; 1994 .
[5] Duke JA. The handbook of energy crops. http://www.hort.purdue.edu/newcrop/duke_energy/dukeindex.html; 1983.
[6] Lewellen R, Panella R, Harveson R. Introduction — botany of the beetplant. In: Harveson R, Hanson LE, Hein GO, editors. Compendium ofthe beet diseases and insects. St.Paul, MN: APS Press; 2009. p. 2–3.
[7] Lange W, Brandeburg WA, De Bock TSM. Taxonomy and cultonomy ofbeet (Beta vulgaris L.). Bot J Linn Soc 1999;130:81–96.
[8] Logendra LS, Gilrain MR, Gianfagna TJ, Janes HW. Swiss chard: a saladcrop for the space program. Life Support Biosph Sci 2002;8:173–9.
[9] Ninfali P, Bacchiocca M. Polyphenols and antioxidant capacity ofvegetables under fresh and frozen conditions. J Agric Food Chem2003;51:2222–6.
[10] Vali L, Stefanovits-Banyai E, Szentmihalyi K, Febel H, Sardi E, Lugasi A,et al. Liver-protecting effects of table beet (Beta vulgaris var. rubra)during ischemia–reperfusion. Nutrition 2007;23:172–8.
[12] Gennari L, Felletti M, Blasa M, Angelino D, Celeghini C, Corallini A,et al. Total extract of Beta vulgaris var. cicla seeds versus its purifiedphenolic components: antioxidant activities and antiproliferativeeffects against colon cancer cells. Phytochem Anal 2011;22:272–9.
[13] Georgiev VG, Weber J, Kneschke EM, Denev PN, Bley T, Pavlov AI.Antioxidant activity and phenolic content of betalain extracts fromintact plants and hairy root cultures of the red beetroot Beta vulgariscv. Detroit dark red. Plant Foods Hum Nutr 2010;65:105–11.
[14] Kujala TS, Vienola MS, Klika KD, Loponen JN, Pihlaja K. Betalain andphenolic compositions of four beetroot (Beta vulgaris) cultivars. EurFood Res Technol 2002;214:505–10.
[15] Stintzing FC, Reinhold C. Functional properties of anthocyanins andbetalains in plants, food, and in human nutrition. Trends Food SciTechnol 2004;15:19–38.
[16] Kugler F, Graneis S, Stintzing FC, Carle R. Studies on betaxanthinprofiles of vegetables and fruits from the Chenopodiaceae andCactaceae. Z Naturforsch C 2007;62:311–8.
[17] Castellar MR, Solano F, Obon JM. Betacyanin and other antioxidantsproduction during growth of Opuntia stricta (Haw.) fruits. Plant FoodsHum Nutr 2012;67:337–43.
[18] Feugang JM, Konarski P, Zou D, Stintzing FC, Zou C. Nutritional andmedicinal use of Cactus pear (Opuntia spp.) cladodes and fruits. FrontBiosci 2006;11:2574–89.
[19] Mee SPH, Lee V, Baldwin JE, Cowley A. Total synthesis of 5,5′,6,6′-tetrahydroxy-3,3′-biindolyl, the proposed structure of a potent antiox-idant found in beetroot (Beta vulgaris). Tetrahedron 2004;60:3695–712.
[20] Bavec M, Turinek M, Grobelnik-Mlakar S, Slatnar A, Bavec F. Influence ofindustrial and alternative farming systems on contents of sugars, organicacids, total phenolic content, and the antioxidant activity of red beet (Betavulgaris L. ssp. vulgaris Rote Kugel). J Agric Food Chem 2010;58:11825–31.
[21] Guil JL, Rodriguez-Garcia I, Torija E. Nutritional and toxic factors inselected wild edible plants. Plant Foods Hum Nutr 1997;51:99–107.
[22] Chai W, Liebman M. Effect of different cooking methods on vegetableoxalate content. J Agric Food Chem 2005;53:3027–30.
[23] Santamaria P. Nitrate in vegetables: toxicity, content, intake and ECregulation. J Sci Food Agric 2006;86:10–7.
[24] Leaf CD, Wishnok JS, Tannenbaum SR. L-arginine is a precursor for nitratebiosynthesis in humans. BiochemBiophys Res Commun1989;163:1032–7.
[25] European Food Safety Authority (EFSA), Nitrate in vegetables.Scientific opinion of the panel on contaminants in the food chain.EFSA J 2008;689:1–79.
[26] Mensinga TT, Speijers GJ, Meulenbelt J. Health implications ofexposure to environmental nitrogenous compounds. Toxicol Rev2003;22:41–51.
[27] Lundberg JO, Govoni M. Inorganic nitrate is a possible source forsystemic generation of nitric oxide. Free Radic Biol Med 2004;37:395–400.
[28] Lundberg JO, Weitzberg E, Cole JA, Benjamin N. Nitrate, bacteria andhuman health. Nat Rev Microbiol 2004;2:593–602.
[29] Czapski J, Maksymiuk M, Grajek W. Analysis of biodenitrificationconditions of red beet juice using the response surface method. J AgricFood Chem 1998;46:4702–5.
[30] Lidder S, Webb AJ. Vascular effects of dietary nitrate (as found ingreen leafy vegetables and beetroot) via the nitrate–nitrite–nitricoxide pathway. Br J Clin Pharmacol 2013;75:677–96.
[31] Lundberg JO, Weitzberg E, Gladwin MT. The nitrate–nitrite–nitricoxide pathway in physiology and therapeutics. Nat Rev Drug Discov2008;7:156–67.
[32] Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, et al.Acute blood pressure lowering, vasoprotective, and antiplateletproperties of dietary nitrate via bioconversion to nitrite. Hypertension2008;51:784–90.
[33] Coles LT, Clifton PM. Effect of beetroot juice on lowering bloodpressure in free-living, disease-free adults: a randomized, placebo-controlled trial. Nutr J 2012;11:106.
[34] Lansley KE, Winyard PG, Bailey SJ, Vanhatalo A, Wilkerson DP,Blackwell JR, et al. Acute dietary nitrate supplementation improvescycling time trial performance. Med Sci Sports Exerc 2011;43:1125–31.
[35] Simpson TS, Savage GP, Sherlock R, Vanhanen LP. Oxalate content ofsilver beet leaves (Beta vulgaris var. cicla) at different stages ofmaturation and the effect of cooking with different milk sources. JAgric Food Chem 2009;57:10804–8.
[36] Bacchiocca M, Biagiotti E, Ninfali P. Nutritional and technologicalmotives for evaluating the antioxidant capacity of vegetable products.Ital J Food Sci 2006;18:209–17.
[37] Lu G, Edwards CG, Fellman JK, Mattinson DS, Navazio J. Biosyntheticorigin of geosmin in red beets (Beta vulgaris L.). J Agric Food Chem2003;51:1026–9.
[38] European Institute of Oncology. Food composition database forepidemiological studies in Italy. http://www.ieo.it/bda2008/uk/index.aspx; 2008.
[39] Escribano J, Pedreno MA, Garcìa-Carmona F, Munoz R. Characteriza-tion of the antiradical activity of betalains from Beta vulgaris L. roots.Phytochem Anal 1998;9:124–7.
[40] Kapadia GJ, Azuine MA, Sridhar R, Okuda Y, Tsuruta A, Ichiishi E, et al.Chemoprevention of DMBA-induced UV-B promoted, NOR-1-inducedTPA promoted skin carcinogenesis, and DEN-induced phenobarbitalpromoted liver tumors in mice by extract of beetroot. Pharmacol Res2003;47:141–8.
[41] Winkler C, Wirleitner B, Schroecksnadel K, Schennach H, Fuchs D. Invitro effects of beet root juice on stimulated and unstimulated peripheralblood mononuclear cells. Am J Biochem Biotechnol 2005;1:180–5.
[42] Patkai G, Barta J, Varsanyi I. Decomposition of anticarcinogen factorsof the beetroot during juice and nectar production. Cancer Lett1997;114:105–6.
[43] Yanardag R, Colak H. Effect of chard (Beta vulgaris L. var. cicla) onblood glucose levels in normal and alloxan-induced diabetic rabbits.Pharm Pharmacol Commun 1998;4:309–11.
[44] Bolkent S, Yanardag R, Tabakoglu-Oguz A, Ozsoy-Sacan O. Effects ofchard (Beta vulgaris L. var. Cicla) extract on pancreatic B cells instreptozotocin-diabetic rats: a morphological and biochemical study. JEthnopharmacol 2000;73:251–9.
[45] Ozsoy-Sacan O, Karabulut-Bulan O, Bolkent S, Yanardag R, Ozgey M.Effects of chard (Beta vulgaris L. var cicla) on the liver of the diabeticrats: a morphological and biochemical study. Biosci BiotechnolBiochem 2004;68:1640–8.
[46] Massiot G, Dijoux MG, Lavaud C, le Men-Olivier L, Connolly JD, SheeleyDM. Seco-glycosides of oleanolic acid from Beta vulgaris. Phytochem-istry 1994;37:1667–70.
[47] Song J, Kwon O, Chen S, Daruwala R, Eck P, Park JB, et al. Flavonoidinhibition of sodium-dependent vitamin C transporter 1 (SVCT1) andglucose transporter isoform 2 (GLUT2), intestinal transporters forvitamin C and Glucose. J Biol Chem 2002;277:15252–60.
[48] Yilmazer-Musa M, Griffith AM, Michels AJ, Schneider E, Frei B. Grapeseed and tea extracts and catechin 3-gallates are potent inhibitors ofalpha-amylase and alpha-glucosidase activity. J Agric Food Chem2012;60:8924–9.
[49] Tundis R, Loizzo MR, Statti GA, Menichini F. Inhibitory effects on thedigestive enzymealpha-amylase of three Salsola species (Chenopodiaceae)in vitro. Pharmazie 2007;62:473–5.
198 P. Ninfali, D. Angelino / Fitoterapia 89 (2013) 188–199
[50] Bischoff H. The mechanism of alpha-glucosidase inhibition in themanagement of diabetes. Clin Invest Med 1995;18:303–11.
[51] Li H, Song F, Xing J, Tsao R, Liu Z, Liu S. Screening and structuralcharacterization of alpha-glucosidase inhibitors from hawthorn leafflavonoids extract by ultrafiltration LC–DAD–MS(n) and SORI–CIDFTICR MS. J Am Soc Mass Spectrom 2009;20:1496–503.
[52] Reddy MK, exander-Lindo RL, Nair MG. Relative inhibition of lipidperoxidation, cyclooxygenase enzymes, and human tumor cell prolifer-ation by natural food colors. J Agric Food Chem 2005;53:9268–73.
[53] Atta AH, Alkofahi A. Anti-nociceptive and anti-inflammatory effects ofsome Jordanian medicinal plant extracts. J Ethnopharmacol 1998;60:117–24.
[54] Kujawska M, Ignatowicz E, Murias M, Ewertowska M, Mikolajczyk K,Jodynis-Liebert J. Protective effect of red beetroot against carbontetrachloride- and N-nitrosodiethylamine-induced oxidative stress inrats. J Agric Food Chem 2009;57:2570–5.
[55] Pavlov A, Kovatcheva P, Tuneva D, Ilieva M, Bley T. Radical scavengingactivity and stability of betalains from Beta vulgaris hairy root culturein simulated conditions of human gastrointestinal tract. Plant FoodsHum Nutr 2005;60:43–7.
[56] Vinson JA, Hao Y, Su X, Zubik L. Phenol antioxidant quantity andquality in foods: vegetables. J Agric Food Chem 1998;46:3630–4.
[57] Wettasinghe M, Bolling B, Plhak L, Xiao H, Parkin K. Phase IIenzyme-inducing and antioxidant activities of beetroot (Beta vulgarisL.) extracts from phenotypes of different pigmentation. J Agric FoodChem 2002;50:6704–9.
[58] Onyeike EN, Ihugba ACGC. Influence of heat processing on the nutrientcomposition of vegetable leaves consumed in Nigeria. Plant Foods HumNutr 2003;58:1–11.
[59] Singh G, Kawatra A, Sehgal S. Nutritional composition of selectedgreen leafy vegetables, herbs and carrots. Plant Foods Hum Nutr 2001;56:359–64.
[60] Ninfali P, Mea G, Giorgini S, Rocchi M, Bacchiocca M. Antioxidantcapacity of vegetables, spices and dressings relevant to nutrition. Br JNutr 2005;93:257–66.
[61] Gil MI, Ferreres F, Tomas-Barberan FA. Effect of postharvest storageand processing on the antioxidant constituents (flavonoids and vitaminC) of fresh-cut spinach. J Agric Food Chem 1999;47:2213–7.
[62] Kuti JO, Konuru HB. Antioxidant capacity and phenolic content in leafextracts of tree spinach (Cnidoscolus spp.). J Agric Food Chem2004;52:117–21.
[63] Tesoriere L, Butera D, Allegra M, Fazzari M, Livrea MA. Distribution ofbetalain pigments in red blood cells after consumption of cactus pearfruits and increased resistance of the cells to ex vivo induced oxidativehemolysis in humans. J Agric Food Chem 2005;53:1266–70.
[64] Gentile C, Tesoriere L, Allegra M, Livrea MA, D'Alessio P. Antioxidantbetalains from cactus pear (Opuntia ficus-indica) inhibit endothelialICAM-1 expression. Ann N Y Acad Sci 2004;1028:481–6.
[65] Kanner J, Harel S, Granit R. Betalains–a new class of dietary cationizedantioxidants. J Agric Food Chem 2001;49:5178–85.
[66] Tesoriere L, Butera D, D'Arpa D, Di GF, Allegra M, Gentile C, et al.Increased resistance to oxidation of betalain-enriched human lowdensity lipoproteins. Free Radic Res 2003;37:689–96.
[67] Allegra M, Furtmuller PG, Jantschko W, Zederbauer M, Tesoriere L,Livrea MA, et al. Mechanism of interaction of betanin andindicaxanthin with human myeloperoxidase and hypochlorous acid.Biochem Biophys Res Commun 2005;332:837–44.
[68] Vulic JJ, Cebovic TN, Canadanovic VM, Cetkovic GS, Djilas SM,Canadanovic-Brunet JM, et al. Antiradical, antimicrobial and cyto-toxic activities of commercial beetroot pomace. Food Funct 2013;4:713–21.
[69] Sreekanth D, Arunasree MK, Roy KR, Chandramohan RT, Reddy GV,Reddanna P. Betanin a betacyanin pigment purified from fruits ofOpuntia ficus-indica induces apoptosis in human chronic myeloidleukemia Cell line-K562. Phytomedicine 2007;14:739–46.
[71] Sato F, Matsukawa Y, Matsumoto K, Nishino H, Sakai T. Apigenininduces morphological differentiation and G2-M arrest in rat neuronalcells. Biochem Biophys Res Commun 1994;204:578–84.
[72] Lepley DM, Pelling JC. Induction of p21/WAF1 and G1 cell-cycle arrestby the chemopreventive agent apigenin. Mol Carcinog 1997;19:74–82.
[73] Engelmann C, Blot E, Panis Y, Bauer S, Trochon V, Nagy HJ, et al. Apigenin–strong cytostatic and anti-angiogenic action in vitro contrasted by lack ofefficacy in vivo. Phytomedicine 2002;9:489–95.
[74] Gupta S, Afaq F, Mukhtar H. Selective growth-inhibitory, cell-cyclederegulatory and apoptotic response of apigenin in normal versushuman prostate carcinoma cells. Biochem Biophys Res Commun2001;287:914–20.
[75] McVean M, Xiao H, Isobe K, Pelling JC. Increase in wild-type p53stability and transactivational activity by the chemopreventive agentapigenin in keratinocytes. Carcinogenesis 2000;21:633–9.
[76] Yin F, Giuliano AE, Law RE, Van Herle AJ. Apigenin inhibits growth andinduces G2/M arrest by modulating cyclin-CDK regulators and ERKMAP kinase activation in breast carcinoma cells. Anticancer Res2001;21:413–20.
[77] Zhu F, Liu XG, Liang NC. Effect of emodin and apigenin on invasion ofhuman ovarian carcinoma HO-8910 PM cells in vitro. Ai Zheng2003;22:358–62.
[78] Gupta S, Afaq F, Mukhtar H. Involvement of nuclear factor-kappa B,Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigeninin human prostate carcinoma cells. Oncogene 2002;21:3727–38.
[79] Kobayashi T, Nakata T, Kuzumaki T. Effect of flavonoids on cell cycleprogression in prostate cancer cells. Cancer Lett 2002;176:17–23.
[80] Lindenmeyer F, Li H, Menashi S, Soria C, Lu H. Apigenin acts on thetumor cell invasion process and regulates protease production. NutrCancer 2001;39:139–47.
[81] Segaert S, Courtois S, Garmyn M, Degreef H, Bouillon R. The flavonoidapigenin suppresses vitamin D receptor expression and vitamin Dresponsiveness in normal human keratinocytes. Biochem Biophys ResCommun 2000;268:237–41.
[82] Shukla S, Gupta S. Apigenin-induced prostate cancer cell death isinitiated by reactive oxygen species and p53 activation. Free Radic BiolMed 2008;44:1833–45.
[83] Takagaki N, Sowa Y, Oki T, Nakanishi R, Yogosawa S, Sakai T. Apigenininduces cell cycle arrest and p21/WAF1 expression in a p53-independentpathway. Int J Oncol 2005;26:185–9.
[84] Zheng PW, Chiang LC, Lin CC. Apigenin induced apoptosis throughp53-dependent pathway in human cervical carcinoma cells. Life Sci2005;76:1367–79.
[85] Yang SH, Liao PH, Pan YF, Chen SL, Chou SS, Chou MY. The novelp53-dependent metastatic and apoptotic pathway induced byvitexin in human oral cancer OC2 cells. Phytother Res 2012. http://dx.doi.org/10.1002/ptr.4841.
[86] Ninfali P, Bacchiocca M, Antonelli A, Biagiotti E, Di Gioacchino AM,Piccoli G, et al. Characterization and biological activity of the mainflavonoids from Swiss Chard (Beta vulgaris subspecies cycla).Phytomedicine 2007;14:216–21.
[87] Papi A, Farabegoli F, Iori R, Orlandi M, De Nicola GR, Bagatta M, et al.Vitexin-2-O-xyloside, raphasatin and (−)-epigallocatechin-3-gallatesynergistically affect cell growth and apoptosis of colon cancer cells.Food Chem 2013;138:1521–30.
[88] Horvathova K, Chalupa I, Sebova L, Tothova D, Vachalkova A.Protective effect of quercetin and luteolin in human melanomaHMB-2 cells. Mutat Res 2005;565:105–12.
[89] Vanhees K, de Bock L, Godschalk RW, van Schooten FJ, van Waalwijkvan Doorn-Khosrovani SB. Prenatal exposure to flavonoids: implica-tion for cancer risk. Toxicol Sci 2011;120:59–67.
[90] The European Agency for the Evaluation of Medicinal Products. Notefor guidance on the investigation of bioavailability and bioequiva-lence; 2010 1–18.
[91] Toutain PL, Bousquet-Melou A. Bioavailability and its assessment. J VetPharmacol Ther 2004;27:455–66.
[92] Netzel M, Stintzing FC, Quaas D, Straß G, Carle R, Bitsch R, et al. Renalexcretion of antioxidative constituents from red beet in humans. FoodRes Int 2005;38:1051–8.
[93] Frank T, Stintzing FC, Carle R, Bitsch I, Quaas D, Strass G, et al. Urinarypharmacokinetics of betalains following consumption of red beet juicein healthy humans. Pharmacol Res 2005;52:290–7.
[94] Tesoriere L, Fazzari M, Angileri F, Gentile C, Livrea MA. In vitrodigestion of betalainic foods. Stability and bioaccessibility ofbetaxanthins and betacyanins and antioxidative potential of fooddigesta. J Agric Food Chem 2008;56:10487–92.
[95] Rechner AR, Smith MA, Kuhnle G, Gibson GR, Debnam ES, Srai SK, et al.Colonic metabolism of dietary polyphenols: influence of structure onmicrobial fermentation products. Free Radic Biol Med 2004;36:212–25.
[96] Tesoriere L, Gentile C, Angileri F, Attanzio A, Tutone M, Allegra M, et al.Trans-epithelial transport of the betalain pigments indicaxanthin andbetanin across Caco-2 cell monolayers and influence of food matrix.Eur J Nutr 2013;52:1077–87.
[97] Leathers RR, Davis C, Zrÿd JP. Betalain producing cell cultures of Betavulgaris L. var. bikores monogerm (red beet). In vitro - Plant 1992;28:39–45.
[98] Ma LY, Liu RH, Xu XD, Yu MQ, Zhang Q, Liu HL. The pharmacokineticsof C-glycosyl flavones of Hawthorn leaf flavonoids in rat after singledose oral administration. Phytomedicine 2010;17:640–5.
[99] Xu YA, Fan G, Gao S, Hong Z. Assessment of intestinal absorption ofvitexin-2″-o-rhamnoside in hawthorn leaves flavonoids in rat using insitu and in vitro absorptionmodels. DrugDev IndPharm2008;34:164–70.
199P. Ninfali, D. Angelino / Fitoterapia 89 (2013) 188–199
[100] Blasa M, Angelino D, Gennari L, Ninfali P. The cellular antioxidantactivity in red blood cells (CAA-RBC): a new approach to bioavailabil-ity and synergy of phytochemicals and botanical extracts. Food Chem2011;125:685–91.
[101] Cai H, Boocock DJ, Steward WP, Gescher AJ. Tissue distribution in miceand metabolism in murine and human liver of apigenin and tricin,flavones with putative cancer chemopreventive properties. CancerChemother Pharmacol 2007;60:257–66.
[102] Meyer H, Bolarinwa A,WolframG, Linseisen J. Bioavailability of apigeninfrom apiin-rich parsley in humans. Ann Nutr Metab 2006;50:167–72.
[103] Molyneux RJ, Waiss Jr AC, HaddonWF. Oxidative coupling of apigenin.Tetrahedron 2011;26:1409–16.
[104] Ninfali P, Dominic S, Angelino D, Gennari L, Buondelmonte C,Giorgi L. An ELISA test for the measurement of plasma flavonoidsin apigenin-C-glycoside fed mice. J Sci Food Agric 2013. http://dx.doi.org/10.1002/jsfa.6143.
[105] Braune A, Blaut M. Deglycosylation of puerarin and other aromaticC-glucosides by a newly isolated human intestinal bacterium. EnvironMicrobiol 2011;13:482–94.
[106] Braune A, Blaut M. Intestinal bacterium Eubacterium cellulosolvensdeglycosylates flavonoid C- and O-glucosides. Appl Environ Microbiol2012;78:8151–3.
[107] Jackman RL, Smith JL. Anthocyanins and betalains. In: Hendry GAF,Houghton JD, editors. Natural food colorants. London: Chapman &Hal; 1996. p. 244–309.
[108] Aune D, Lau R, Chan DS, Vieira R, Greenwood DC, Kampman E, et al.Nonlinear reduction in risk for colorectal cancer by fruit and vegetableintake based on meta-analysis of prospective studies. Gastroenterol-ogy 2011;141:106–18.
[109] Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of totalphenols and other antioxidant substrates and antioxidant by mean ofFolin-Ciocalteu reagent. Meth Enzymol 1999;299:152–78.
[110] Eberhardt MV, Lee CY, Liu RH. Antioxidant activity of fresh apples.Nature 2000;405:903–4.
