Leaves of White Beetroot As a New Source of Antioxidant and Anti-Inﬂammatory Compounds

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Article Leaves of White Beetroot As a New Source of Antioxidant and Anti-Inﬂammatory Compounds

Urszula Gawlik-Dziki 1,* , Laura Dziki 1, Jakub Anisiewicz 1, Ewa Habza-Kowalska 1, Małgorzata Sikora 1 and Dariusz Dziki 2

1 Department of Biochemistry and Food Chemistry, University of Life Sciences, 8 Skromna Str., 20-704 Lublin, Poland; [email protected] (L.D.); [email protected] (J.A.); [email protected] (E.H.-K.); [email protected] (M.S.) 2 Department of Thermal Technology and Food Process Engineering, University of Life Sciences in Lublin, 31 Gł˛ebokaSt., 20-612 Lublin, Poland; [email protected] * Correspondence: [email protected]

Received: 18 June 2020; Accepted: 23 July 2020; Published: 26 July 2020

Abstract: The white beetroot cv. Snie˙znaKula´ is the first betanin-free beetroot registered in the European Union. The aim of this study was to compare the phenolic acids profile and antioxidant capacity of leaves of white (SK) and red (CC) beetroots and red (LC) and white (BL) Swiss chard growing in Poland. LC leaves were the richest source of total phenolics (16.55 mg GAE/g FW) and phenolic acids (1.81 mg/g FW), while the highest content of flavonoids was determined in CC leaves (1.6 mg QE/g FW). The highest antiradical activity was observed for LC, whereas CC extract exhibited the highest chelating power. BL and CC leaf extracts demonstrated high LOX inhibitory potential (EC50 = 53.23 and 56.97 mg FW/mL, respectively). An uncompetitive type of LOX inhibition was obtained for all extracts. SK extracts demonstrated the highest XO inhibitory activity (EC50 = 81.04 mg FW/mL). A noncompetitive type of XO inhibition was obtained in both extracts from red leaves (CC and LC), whereas an uncompetitive mode of inhibition was observed in the case of white leaf (SK and LC) extracts. Thus, it can be assumed that the presence of betanin influences the XO inhibition mechanism.

Keywords: beetroot leaves; phenolic compounds; antioxidant activity; lipoxygenase inhibition; xanthine oxidase inhibition

1. Introduction The red beetroot (Beta vulgaris L.) is commonly cultivated and consumed in Poland and other countries of the East. Recently, a new variety of the red beetroot called “Snie˙znaKula”´ (Eng. “snow ball”) has appeared in the world market. The plant is a result of multiyear work conducted by researchers and produced by a Polish company—TORSEED. Snie˙znaKula´ is the ﬁrst betanin—free beetroot registered in the European Union (Figure1). The vegetable has retained the taste and aromatic properties of the red beetroot as well as agrotechnical requirements. The white beetroot was created mainly for Italian, French, and English markets, where betanin was withdrawn from diets for babies and children due to its allergenic properties.

Plants 2020, 9, 944; doi:10.3390/plants9080944 www.mdpi.com/journal/plants Plants 2020, 9, 944 2 of 14 Plants 2020, 9, x FOR PEER REVIEW 2 of 14

Figure 1. Beetroot “Śnieżna Kula”. Ownership: Torseed S.A. Figure 1. Beetroot “Snie˙znaKula”.´ Ownership: Torseed S.A.

The rootvegetable is the traditionallyhas retained eaten the parttaste ofand these aromatic plants. Whilstproperties most of studies the red assessing beetroot the as beneficial well as eagrotechnicalffect of the red requirements. beetroot have The been wh limitedite beetroot to the was root, created the nutritional mainly for and Italian, nutraceutical French, and potential English of leavesmarkets, is underestimated.where betanin was Red withdr beet leavesawn arefrom consumed diets for inbabies salads and with children other vegetables due to its worldwide. allergenic Inproperties. Korea, leafy vegetables, including red beet leaf, are commonly used for wrapping cooked rice or meatThe [1]. Inroot turn, is athe cultivar traditionally of Beta vulgaris eaten—i.e., part theof th Swissese chardplants. is commonlyWhilst most cultivated studies andassessing consumed the inbeneficial Mediterranean effect of countries. the red beetroot It constitutes have onebeen of lim theited basic to componentsthe root, the ofnutritional the Mediterranean and nutraceutical diet, but ispotential also becoming of leaves part is underestimated. of other diets. Given Red thebeet increasing leaves are awareness consumed of in the salads potential with benefits other vegetables provided byworldwide. plant foods, In Korea, the Swiss leafy chard vegetables, has recently includin gainedg red popularity beet leaf, [2 ].are commonly used for wrapping cookedFood rice is theor mainmeatsource [1]. In of turn, principal a cultivar antioxidants of Beta represented vulgaris— byi.e., phenolic the Swiss compounds. chard is They commonly exhibit anti-inflammatorycultivated and consumed and anticancer in Mediterranean properties, countries. which has It been constitutes well documented one of the [basic3]. One components of the plants of withthe Mediterranean a high level of phenolicdiet, but compoundsis also becoming are beetroots part of andother Swiss diets. chards, Given which the increasing have been awareness undervalued of inthe terms potential of their benefits pro-health provided influence by plant to date foods, [4]. Nevertheless,the Swiss chard the has prominent recently propertiesgained popularity of beetroot [2]. as a promisingFood preventiveis the main or source therapeutic of principal agent in antioxidants oxidative stress represented and inflammatory by phenolic conditions compounds. are reported. They Inexhibit particular, anti-inflammatory there is ample and evidence anticancer now properties, that the contribution which has ofbeen beetroot well documented in cardiovascular [3]. One diseases of the wasplants likely with due a high to its level ability of tophenolic diminish compounds NO bioavailability are beetroots and improve and Swiss endothelial chards, which function have [4]. been undervaluedGiven the in high terms level of their of phenolic pro-health compounds influence and to antioxidantdate [4]. Nevertheless, potential of the beetroot prominent leaves, properties inclusion thereofof beetroot in the as dieta promising may bring preventive natural protective or therap eeuticffects agent against in so-calledoxidative diseasesstress and of ainflammatoryffluence, e.g., cancerconditions or cardiovascular are reported. In disease particular, and other there free is ampl radical-relatede evidence diseasesnow that [2 the]. contribution of beetroot in cardiovascularDisproportion diseases between was reactive likely oxygen due to species its ability (ROS) to diminish and antioxidants NO bioavailability is defined as and an oxidativeimprove stressendothelial (OS). Oxygen function radicals [4]. such as hydroxyl radical, OH, and nonradical substances, such as hydrogen Given the high level of phenolic compounds• and antioxidant potential of beetroot leaves, peroxide H2O2, are examples of ROS. [5]. Overproduction of reactive species leads to OS, which causes damageinclusion to thereof basic biomolecules, in the diet may e.g., proteins,bring natural DNA, pr andotective lipids. effects Additionally, against ROS so-called can interfere diseases with of intracellularaffluence, e.g., signal cancer transduction or cardiovascular processes, disease which and have other been free implicated radical-related in the developmentdiseases [2]. of insulin resistance,Disproportion inflammation, between and variousreactive lifestyleoxygen diseases,species for(ROS) example, and antioxidants cancer, cardiovascular is defined diseases as an includingoxidative atherosclerosis,stress (OS). Oxygen hypertension radicals heartsuch as failure, hydroxyl etc. [ 6radical,,7]. •OH, and nonradical substances, such However,as hydrogen ROS peroxide play anH2O important2, are examples role of in ROS. immune [5]. Overproduction reactions, for example,of reactive in species combating leads pathogensto OS, which [8 causes]. ROS damage can also to be basic generated biomolecules, in the e.g., cells proteins, via enzymatic DNA, and reactions. lipids. Additionally, Free radicals ROS are generatedcan interfere in processeswith intracellular related to signal the respiratory transduction chain, processes, phagocytosis, which prostaglandin have been implicated synthesis, andin the in development of insulin resistance, inflammation, and various lifestyle diseases, for example, cancer, cardiovascular diseases including atherosclerosis, hypertension heart failure, etc. [6,7].

Plants 2020, 9, 944 3 of 14 the cytochrome P450 system [9]. The lipoxygenase (LOX) and xanthine oxidase (XO) enzymes are involved in the production of free radicals. LOX catalyzes incorporation of oxygen to the double bond in polyunsaturated fatty acids, e.g., linoleic acid, oleic acid, and arachidonic acid. This reaction results in the production of hydroperoxides and epoxide. Overexpression of LOX is detected during inflammation and tumorigenesis primarily in immune, epithelial, and tumor cells [10]. XO catalyzes the process of oxidation of hypoxanthine to xanthine and oxidation of xanthine to uric acid. Hydrogen peroxide (H2O2) exhibiting an affinity for iron is generated as a side effect of these reactions. In the reaction with iron, H O is converted into hydroxyl radical ( OH). OH initiates the 2 2 • • − generation of subsequent free radicals in reactions with lipids, proteins, and nucleic acids [11]. To the best of our knowledge, there is no study of the antioxidant activity of the white beetroot in the context of the absence of betanin. Moreover, there are limited studies concerning the antioxidant activity of Beta vulgaris subspecies cycla. Beetroot leaves (especially young ones) are consumed as an ingredient in salads and used for the preparation of soups. White beetroot is a new and little known plant. In turn, Swiss chard is a leafy vegetable gaining popularity in Poland, but it is still occasionally harvested. A comparison of these plants seems relevant since, despite the differences, they are representatives of the same species. Therefore, the aim of this study was to investigate and compare the nutraceutical potential of leaves of two cultivars of beetroots (Beta vulgaris L.): White (Snie˙znaKula´ Eng. “white ball”) and red (Czerwona Kula, Eng. “red ball”) beetroot and two cultivars of Beta vulgaris subsp. vulgaris, Cicla-Group and Flavescens-Group; red—Rhubarb Chard and white—Lukullus growing in Poland.

2. Results

2.1. Analysis of Phenolic Compounds Plants containing a high amount of phenolic compounds are a good source of antioxidants [6]. This fact prompted the determination of the phenolic acids (PA) profile as well as the content of total phenolics (TPC) and flavonoids (TFC) in the samples. As presented in Table1, the red chard leaves were the richest sources of phenolic acids (PA) (180.90 mg/100 g FW). Surprisingly, the lowest content of total PA was determined in red beetroot leaves (116.89 mg/100 g FW). The total contents of PA in the case of the white chard and SK leaves were quite similar (152.35 and 141.64 mg/100g FW, respectively). Interestingly, the PA profile of the SK and LC leaves was the same, but the amounts of phenolic acids differed significantly. The difference is mainly related to the salicylic acid content (Table1).

Table 1. Phenolic acids content in leaves of white beetroot (SK), beetroot (CC), white chard (BL), and red chard (LC) [mg/100 g FW].

Phenolic Acid Red Chard (LC) White Beetroot (SK) Red Beetroot (CC) White Chard (BL) Protocatechuic 2.83 0.03 a * 1.20 0.00 c * 1.72 0.05 b * nd ± ± ± p-OH benzoic 2.26 0.06 a 1.21 0.01 b 0.96 0.00 c 1.37 0.01 b * ± ± ± ± Caffeic 2.11 0.01 a 1.79 0.01 b nd nd ± ± Syryngic 1.45 0.01 b 1.36 0.01 c 1.74 0.06 a 1.34 0.01 c ± ± ± ± Vanilic 1.43 0.02 a 1.36 0.00 a nd nd ± ± p-coumaric 1.40 0.00 a 1.32 0.00 a nd nd ± ± Ferulic 5.32 0.17 a 2.73 0.05 c 4.72 0.21 b 2.81 0.04 c ± ± ± ± Synapic 7.92 0.45 c 23.23 1.29 a 14.90 1.16 b 4.59 0.09 d ± ± ± ± Salicylic 159.01 9.98 a 108.64 6.23 c 94.56 7.60 d 142.24 9.91 b ± ± ± ± Sum 180.90 a 141.64 c 116.89 d 152.35 b * The values are expressed as the mean SD; means with different letter superscripts (a–d) in the rows are significantly different (α = 0.05). ±

Unexpectedly, the highest TPC was determined in the LC leaves (16.55 mg GAE/g FW). The lowest TPC value was obtained for the SK and BL leaf extract (8.15 and 8.94 mg GAE/g FW, respectively) PlantsPlants 2020 2020, ,9 9, ,x x FOR FOR PEER PEER REVIEW REVIEW 44 of of 14 14 Plants 2020, 9, 944 4 of 14 Unexpectedly,Unexpectedly, the the highest highest TPC TPC was was determined determined in in the the LC LC leaves leaves (16.55 (16.55 mg mg GAE/g GAE/g FW). FW). The The lowestlowest TPCTPC valuevalue waswas obtainedobtained forfor thethe SKSK andand BLBL leafleaf extractextract (8.15(8.15 andand 8.948.94 mgmg GAE/gGAE/g FW,FW, (Figure2B). The highest content of TF was determined in the case of CC (1.6 mg QE /g FW) and respectively)respectively) (Figure (Figure 2B). 2B). The The highest highest content content of of TF TF was was determined determined in in the the case case of of CC CC (1.6 (1.6 mg mg QE/g QE/g both chard samples (over 1.5 mg QE/g FW). The lowest ﬂavonoid content (1.31 mg QE/g FW) was FW)FW) and and both both chard chard samples samples (over (over 1.5 1.5 mg mg QE/g QE/g FW). FW). The The lowest lowest flavonoid flavonoid content content (1.31 (1.31 mg mg QE/g QE/g determined in the SK samples (Figure2B). FW)FW) was was determined determined in in the the SK SK samples samples (Figure (Figure 2B). 2B).

20 20 1,8 18 a 1,8 a a 18 a A a B a a A B a 16 1,6 16 1,6 b 14 1,4 b 14 1,4 12 1,2 12 1,2 b bc 10 b bc FW QE/g mg 1 10 c FW QE/g mg 1 c 0,8 8 0,8

mg GAE/g FW GAE/g mg 8 mg GAE/g FW GAE/g mg 6 0,6 6 0,6 4 0,4 4 0,4 2 0,2 2 0,2 0 0 0 0 LC SK CC BL LC SKsample CC BL LC SK CC BL LC SKsample CC BL sample sample FigureFigureFigure 2. 2. Phenolics PhenolicsPhenolics ( A(A)) ) andand and totaltotal total flavonoids flavonoids flavonoids content content content (B ( )B( inB) )in leavesin leaves leaves of redof of red chardred chard chard (LC), (LC), (LC), white white white beetroot beetroot beetroot (SK), (SK),red(SK), beetroot red red beetroot beetroot (CC), and(CC), (CC), white and and chardwhite white (BL). chard chard Results (BL). (BL). areResu Resu presentedltslts are are presented presented as the mean as as the theSD; mean mean the values± ± SD; SD; the designatedthe values values ± designatedbydesignated the different by by the the small different different letters small small (a, b, letters c,letters d) are (a, (a, significantlyb, b, c, c, d) d) are are significantly significantly different (α different= different0.05). ( α(α = = 0.05). 0.05). 2.2. Total Betalain Content 2.2.2.2. Total Total Betalain Betalain Content Content The betalain content in red beetroots has been widely studied; however, there are only sparse TheThe betalain betalain content content in in red red beetroots beetroots has has been been wi widelydely studied; studied; however, however, there there are are only only sparse sparse data on their content in leaves. As expected, the highest content of betalains expressed as a betanin datadata on on their their content content in in leaves. leaves. As As expected, expected, the the highest highest content content of of betalains betalains expressed expressed as as a a betanin betanin equivalent was found in the CC leaves (46.42 mg/kg FW). A significantly lower amount of this pigment equivalentequivalent waswas foundfound inin thethe CCCC leavesleaves (46.42(46.42 mg/kgmg/kg FW).FW). AA significantlysignificantly lowerlower amountamount ofof thisthis was determined in the LC sample (33.22 mg/kg FW). The lowest betalains content (2.55 mg/kg FM) was pigmentpigment waswas determineddetermined inin thethe LCLC samplesample (33.22(33.22 mg/kgmg/kg FW).FW). TheThe lowestlowest betalainsbetalains contentcontent (2.55(2.55 detected in the SK leaves (Figure3). mg/kgmg/kg FM) FM) was was detected detected in in the the SK SK leaves leaves (Figure (Figure 3). 3).

60 60

50 a 50 a

40 40 b b

30 30 mg/kg FW mg/kg FW 20 20

c 10 c 10 d d

0 0 LC SKsample CC BL LC SKsample CC BL Figure 3. Betalains content in leaves of red chard (LC), white beetroot (SK), red beetroot (CC), and FigureFigure 3. Betalains contentcontent inin leaves leaves of of red red chard chard (LC), (LC), white white beetroot beetroot (SK), (SK), red beetrootred beetroot (CC), (CC), and white and white chard (BL). Results are presented as the mean ± SD; the values designated by the different small whitechard chard (BL). Results(BL). Results are presented are presented as the as mean the meanSD; ± the SD; values the values designated designated by the by di fftheerent different small letterssmall letters (a, b, c, d) are significantly different (α± = 0.05). letters(a, b, c, (a, d) b, are c, significantlyd) are significantly different different (α = 0.05). (α = 0.05).

2.3.2.3.2.3. Antioxidant Antioxidant Capacity CapacityCapacity InInIn terms termsterms of ofof the thethe ability abilityability to toto neutralize neutralizeneutralize ABTS ABTSABTS free freefree radicals, radicals,radicals, the thethe highest highesthighest activity activityactivity was waswas noted notednoted for forfor LC LCLC 50 (EC(EC(EC5050 = = 41.65 41.6541.65 mg mg mg FW/mL). FW/mL). FW/mL). A AA slightly slightly slightly lower lower lower but but but statisti statisti statisticallycallycally significant significant significant activity activity was was noticed noticednoticed for forfor the thethe CC CCCC 50 50 samplesamplesample (EC (EC(EC5050 = = 50.00 50.0050.00 mg mg FW/mL). FW/mL). FW/mL). Extracts Extracts from fromfrom SK SKSK and andand BL BLBL showed showedshowed a a similar similar low low activity activity (EC(EC (EC5050 = = 65.20 65.2065.20 andandand 69.97 69.9769.97 mg mgmg FW/mL, FWFW/mL,/mL, respectively) respectively)respectively) (Figure (Figure(Figure 44A). 4A).A). In In turn, turn, the the highest highest ability ability to to neutralize neutralize OH• OH OH• radicals radicalsradicals • 50 waswaswas observed observedobserved for forfor the the LC LC extracts extracts (EC (EC50 = == 50.00 50.00 mg mg FW/mL), FWFW/mL),/mL), whereas whereas SK SK showed showedshowed the thethe lowest lowestlowest activity activityactivity 50 50 (EC(EC(EC5050 = = 29.78 29.7829.78 mg mg FW/mL). FW/mL). FW/mL). LC LC and and BL BL did diddid not notnot exhibi exhibitexhibit tstatistically statisticallystatistically significant significantsignificant differencesdi differencesfferences (EC(EC (EC5050 = = 26.32 26.3226.32

Plants 2020, 9, 944 5 of 14 Plants 2020, 9, x FOR PEER REVIEW 5 of 14 and 26.56 mg FW/mL, respectively) (Figure 4B). Our investigation showed that the CC extract and 26.56 mg FW/mL, respectively) (Figure4B). Our investigation showed that the CC extract exhibited exhibited the highest chelating power (EC50 = 79.98 mg FW/mL), whereas lower values were observed the highest chelating power (EC50 = 79.98 mg FW/mL), whereas lower values were observed for SK for SK and LC (EC50 = 91.80 and 111.05 mg FW/mL, respectively). The lowest activity was noticed for and LC (EC50 = 91.80 and 111.05 mg FW/mL, respectively). The lowest activity was noticed for BL BL (EC50 = 143.46 mg FW/mL) (Figure 4C). The positive control values (expressed as EC50) were 0.103 (EC50 = 143.46 mg FW/mL) (Figure4C). The positive control values (expressed as EC 50) were 0.103 and and 0.036 mg TA/mL for ABTS and OH assays, respectively, and 0.372 mg EDTA/mL for the CHEL 0.036 mg TA/mL for ABTS and OH assays, respectively, and 0.372 mg EDTA/mL for the CHEL assay. assay.

80 35 a a B 70 b A A 30 b b 60 c c 25 50 d 20 40 15

EC50 [mgEC50 FW/ml] 30 EC50 [mg EC50 FW/ml] 10 20

10 5

0 0 LC SKsample CC BL LC SKsample CC BL 160 a 140 C 120 b

100 c d 80

60 EC50 [mgFW/ml] EC50

0 LC SKsample CC BL

Figure 4. ABTS free radicals scavenging ability. ( (AA)) Hydroxyl Hydroxyl radicals radicals scavenging scavenging ability, ability, ( B) and chelating power ( C) of extracts from red chard (LC), white beetroot (SK), red beetroot leaves (CC), and white chard (BL). Results are presented as the meanmean ± SD; the values designated by the different different ± small letters (a, b, c, d) are significantlysignificantly didifferentfferent ((αα == 0.05).

2.4. Lipoxygenase Inhibition As presented in Table2 2,, thethe highesthighest LOXLOX inhibitoryinhibitory potential potential waswas foundfound forfor thethe BLBL andand CCCC leafleaf extracts (EC50 == 53.2353.23 and and 56.97 56.97 mg mg FW/mL, FW/mL, respectively), respectively), whereas whereas the lowest value was determined in the case of the LC extract (78.29 mg FW/mL). FW/mL). As shown in Table 2 2 and and FigureFigure5 ,5, an an uncompetitive uncompetitive type of LOX inhibition was determineddetermined inin allall extracts.extracts.

Table 2.2. ECEC5050 value,value, Ki Ki value, value, Vmax Vmax value, value, and and mode mode of lipoxygenase of lipoxygenase inhibition inhibition by extracts by extracts from leaves from ofleaves white of beetrootwhite beetroot (SK), beetroot (SK), beetroot (CC), white (CC), chard white (BL), chard and (BL), red and chard red (LC) chard (n (LC)= 9). (n = 9).

PlantPlant Mode Mode of of Inhibition Inhibition EC EC5050 [mg[mg FWFW/mL]/mL] Ki Ki [mg [mg FW FW/mL]/mL] Vmax Vmax [∆AU [ΔAU/min]/min] WhiteWhite beetroot beetroot uncompetitive uncompetitive 72.29 ± 0.850.85bb 72.01 72.01 ±2.11 2.11bb 416.66 416.668.32 ± 8.32bb ± ± ± RedRed beetroot beetroot uncompetitive uncompetitive 56.97 ± 1.051.05cc 55.99 55.99 ±1.21 1.21cc 412.58 412.587.25 ± 7.25cc ± ± ± White chard uncompetitive 53.23 2.23 d 49.13 1.02 d 400.02 5.55 d White chard uncompetitive 53.23 ±± 2.23d 49.13± ± 1.02d 400.02± ± 5.55d Red chard uncompetitive 78.29 1.13 a 78.61 3.22 a 434.32 6.25 a Red chard uncompetitive 78.29± ± 1.13a 78.61± ± 3.22a 434.32± ± 6.25a * The values are expressed as the mean SD; means with different letter superscripts (a–d) in the columns are * The values are expressed as the mean ± SD; means with different letter superscripts (a–d) in the significantly different (α = 0.05). ± columns are significantly different (α = 0.05).

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0.01 0.01 no… SK 1/V no… LC A B1/V 0.005 0.005

0 0 -10121/S -2 -11/S 0 1 2 0.006 0.01 no… CC no… BL 1/V 0.004 D C 1/V 0.005 0.002

0 0 -2 -11/S 0 1 2 -2 -11/S 0 1 2

FigureFigure 5. 5. ModeMode of oflipoxygenase lipoxygenase inhibition inhibition by byextracts extracts from from red redchard chard (A), (whiteA), white beetroot beetroot (B), red (B), beetrootred beetroot leaves leaves (C), (andC), and white white chard chard (D ().D LC—red). LC—red chard; chard; SK—white SK—white beetroot; beetroot; CC—red CC—red beetroot beetroot leaves;leaves; BL—white BL—white chard. chard.

2.5.2.5. Xanthine Xanthine Oxidase Oxidase Inhibition Inhibition As presented in Table3, the highest XO inhibitory potential was found for the SK extract As presented in Table 3, the highest XO inhibitory potential was found for the SK extract (EC50 =(EC 81.0450 = mg81.04 FW/mL), mg FW whereas/mL), whereas the lowest the lowestvalue was value determined was determined in the case in the of the case CC of extract the CC (205.09 extract mg(205.09 FW/mL). mg FW As/mL). shown As in shown Table in 3 Table and 3Figure and Figure 6A,C,6 A,C,a noncompetitive a noncompetitive type typeof XO of inhibition XO inhibition was obtainedwas obtained in both in bothextracts extracts from from the thered redleaves leaves (CC (CC and and LC), LC), whereas whereas an an uncompetitive uncompetitive mode mode of of inhibitioninhibition was was noted noted in in the the case case of of the the white white leaf leaf extracts extracts (SK (SK and and LC) LC) (Figure (Figure 6B,D).6B,D). Based on these observations,observations, it it can can be be assumed assumed that that the the presence presence of of betanin betanin influences inﬂuences the the XO XO inhibition inhibition mechanism; mechanism; however,however, confirmation conﬁrmation of of this this thesis thesis requires requires further research.

Table 3. EC value, Ki value, Vmax value, and mode of xanthine oxidase inhibition by extracts from Table 3. EC5050 value, Ki value, Vmax value, and mode of xanthine oxidase inhibition by extracts from n leavesleaves of of white white beetroot beetroot (SK), (SK), beetroot beetroot ( (CC),CC), white white chard chard (BL), (BL), and and red red chard (LC) (n = 9).9). Plant Mode of Inhibition EC [mg FW/mL] Ki [mg FW/mL] Vmax [∆AU/min] Plant Mode of Inhibition EC5050 [mg FW/mL] Ki [mg FW/mL] Vmax [ΔAU/min] WhiteWhite beetroot beetroot uncompetitive uncompetitive 81.04 81.04 ± 2.22d2.22 d 24.62 24.62 ±0.14 0.14dd 0.42 0.42 0.01± 0.01cc ± ± ± Red beetroot noncompetitive 205.09 4.58 a 1020.48 13.58 a 1.5 0.02 a Red beetroot noncompetitive 205.09 ±± 4.58a 1020.48± ± 13.58a 1.5± ± 0.02a White chard noncompetitive 147.28 5.32 b 229.71 8.23 b 1.11 0.4 b White chard noncompetitive 147.28 ±± 5.32b 229.71± ± 8.23b 1.11± ± 0.4b Red chard uncompetitive 105.32 7.41 c 39.156 1.54 c 0.49 0.01 c Red chard uncompetitive 105.32 ±± 7.41c 39.156± ± 1.54c 0.49± ± 0.01c * The values are expressed as the mean SD; means with different letter superscripts (a–d) in the columns are *significantly The values di arefferent expressed (α = 0.05). as the mean± ± SD; means with different letter superscripts (a–d) in the columns are significantly different (α = 0.05).

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25 20 18 A B 20 no inhibitor SK 16 no inhibitor LC 1/V 14 1/V 12 15

8 10

4 5

0 0 -0.500.511.52 -0.500.511.521/S 1/S 25 25 C D no inhibitor CC 20 1/V 20 no inhibitor BL 1/V

15 15

10 10

5 5

0 0 -0.5 0 0.5 1 1.5 2 -0.5 -0.1 0.3 0.7 1.1 1.5 1.9 1/S 1/S Figure 6. ModeMode of of xanthine xanthine oxid oxidasease inhibition inhibition by by extr extractsacts from from red red chard chard (A), ( Awhite), white beetroot beetroot (B), (redB), redbeetroot beetroot leaves leaves (C), ( Cand), and white white chard chard (D (D). ).LC—red LC—red chard; chard; SK—white SK—white beetroot; beetroot; CC—red beetrootbeetroot leaves;leaves; BL—whiteBL—white chard.chard.

3. Discussion Almost all thethe plant-derivedplant-derived foods, which constituteconstitute a large part of the human diet, are richrich inin phenolicphenolic acids.acids. The The average average intake intake of phenolic of phenolic acids hasacids been has reported been reported to reach 200to reach mg/day 200 depending mg/day ondepending the diet habitson the anddiet preferenceshabits and preferences [12]. [12]. As presented in in Table Table 11,, the the red red chard chard leaves leaves were were the the richest richest sources sources of phenolic of phenolic acids acids (1.81 (1.81mg/g mgFW)./g Unexpectedly, FW). Unexpectedly, the lowest the content lowest of content total PA of was total determined PA was determined in the leaves in of the red leaves beetroot of red(116.89 beetroot mg/100 (116.89 g FW). mg /The100 gtotal FW). PA The contents total PA in contents the white in thechard white and chard SK leaves and SK were leaves quite were similar. quite similar.Interestingly, Interestingly, the PA theprofile PA profileof the SK of theand SK LC and leav LCes leaveswas the was same, the same,but the but amounts the amounts of phenolic of phenolic acids acidsdiffered diff eredsignificantly. significantly. Protocatechuic, Protocatechuic, p-hydroxybezoic,p-hydroxybezoic, caffeic, caffeic, syryngic, syryngic, ferulic, ferulic, p-coumaric, synaptic,synaptic, and salicylicsalicylic acidsacids werewere identified.identified. The results are in accordanceaccordance withwith thosethose obtainedobtained inin earlierearlier investigationsinvestigations [[12,13].12,13]. Gallic, ferulic, chlorogenic, caffeic, caffeic, vanillic, syringic, and ellagicellagic acidacid werewere identifiedidentified inin rootsroots andand stemsstems ofof redred beetbeet [[13].13]. The water extract of chard leaves had higher levels of vanillic,vanillic, cacaffeic,ffeic, andand ellagicellagic acidsacids (85.91,(85.91, 28.45,28.45, andand 12.5812.58 ppm,ppm, respectively),respectively), whilewhile thethe methanolicmethanolic extractextract of chardchard leavesleaves waswas richerricher inin vanillic,vanillic, salicillic,salicillic, and protocatechuicprotocatechuic acids (103.32, 62.32, 22.6, and 12.72 ppm, respectively). In contrast, water and methanolic extracts of beetroot leaves was higher inin vanillic,vanillic, ellagic,ellagic, andand protocatechuicprotocatechuic acidsacids [[14].14]. The totaltotal concentrationconcentration of phenolic compounds in beetroots and Swiss chard has beenbeen widelywidely studied;studied; however,however, investigation investigation results results di ffdifferer significantly. significantly. As shownAs shown by Pyo by et Pyo al. [2et], theal. [2], total the content total ofcontent phenolics of phenolics (red; 128.1 (red; mg 128.1/100 mg g FW,/100 white;g FW, white; 101.5 mg101.5/100 mg/100 g FW) g wasFW) substantiallywas substantially higher higher in leaf in extractsleaf extracts than than in stem in stem extracts extracts (red; (red; 29.7 29.7 mg/ 100mg/100 g FW, g FW, white; white; 23.2 /23.2/100100 g FW). g FW). Sacan Sacan and and Yanardag Yanardag [15] demonstrated 11.88 1.46 µg epicatechin equivalents of phenolics in 1 mg of water extracts of chard. [15] demonstrated 11.88± ± 1.46 µg epicatechin equivalents of phenolics in 1 mg of water extracts of Ninfalichard. Ninfali and Angelino and Angelino [16] showed [16] showed that the that TPC the content TPC content in Beta in vulgaris Beta vulgaris cicla leaves cicla leaves was 11.12 was mg11.12/g DW,mg/g while DW, thewhile average the average flavonoid flavonoid level was level 7.92 was mg 7.92/g DW.mg/g For DW. comparison, For comparison,Beta vulgaris Beta vulgaris rubra leaves rubra containedleaves contained 12.76 mg 12.76 TPC mg per TPC g DW per and g DW 11.64 and mg 11.64 flavonoids mg flavonoids per g DW. per However, g DW. However, it should be it notedshould that be noted that the authors did not provide accurate systematic data or the extraction method; hence,

Plants 2020, 9, 944 8 of 14 the authors did not provide accurate systematic data or the extraction method; hence, comparison of the results is difficult. Noteworthy, the content of phenolics is significantly influenced by the cultivation conditions and the methodology used. Flavonoids are well-known natural antioxidants. Therefore, dietary intake of foods rich in flavonoids—has been suggested to contribute to protection from free radical damage. Our study indicated that all analyzed materials contained comparable amounts of flavonoids. In the paper by Sacan and Yanardag [15], the methanol-water (8:2) extracts of green and yellow Swiss chard cultivars were compared in terms of their flavonoid composition and content. The total flavonoid contents were 2.76 and 1.26 mg/g of fresh weight, respectively. A phenolic fraction obtained from the ethanol-water (4:6) Swiss chard extract was analyzed and 2”-xylosylvitexin, isorhamnetin 3-gentiobioside, and vitexin 2O-rhamnoside were identified as major components, whereas rutin, quercetin 7-glucuronide, isorhamnetin, and apigenin 7-rutinoside were minor compounds [16]. Similarly, Chandra et al. [17] reported flavonoid content of 11.08 mg QE/g DW in chard, which is generally consistent with our results. In turn, significantly lower values were obtained by Zein, Hashish, and Ismaiel [14], who analyzed the phenolic contents in white chard and beetroot leaves. As demonstrated by the authors, the total flavonoids content was on average 0.19 and 0.48 mg/g FW, respectively. Due to their antioxidant activity, betalains protect from free radical-related disorders. Therefore, regular consumption of betalain-rich products or foods colored with betalains, which are considered to be safe natural food dyes, can exert a positive effect on the consumer’s organism. Betanin which is listed as food additive E162, is commonly used in a multiple of food products. Red beetroots were found to be a rich source of betanin (about 300–600 mg/kg) [18]. The results obtained in our laboratory are generally in agreement with those reported by Kugler et al. [19]. As shown in their study, red-purple petioles contained 50.6 mg of betalains per kg of FW and yellow stems—49.7 mg/kg of FW. In turn, the lowest values were obtained in the case of yellow-orange Swiss chard petioles—33.6 mg/kg FW. It worth mentioning that the leaves were analyzed in our study. As reported by Ali et al. [20] the 1 highest content of betacyanin was found in the vein in red beet (271.61 µg betanin equivalents g− FW), 1 while green amaranth exhibited the lowest content (17.35 µg betanin equivalents g− FW) under 12 and 24 h photoperiods, respectively. Plants usually contain mixtures of phenolic compounds. Therefore, their interactions must be borne in mind when discussing the antioxidant activity of phenolics. Many researches have tried to determine the antioxidant potential of a given food based on its total phenolics content and have failed in most cases due to the differences between the theoretical and the calculated antioxidant activity of the analyzed product. Red beetroot leaves are a good source of natural antioxidants. In these leaves ROS induce the synthesis of betacyanin, which can neutralize free radicals and reduce damage caused by bacterial infection and wounding [1]. Antioxidant properties, especially antiradical activities, are very important due to the harmful effect of free radicals in biological systems, including food. A commonly used method to evaluate antiradical activities is the model of scavenging stable ABTS radicals [21]. In our study, the highest antiradical activity was observed for LC (EC50 = 41.65 mg FW/mL). Extracts from SK and BL showed similarly low activity (EC50 = 65.20 and 69.97 mg FW/mL, respectively) (Figure4A), probably it is a consequence of a lack of betanin. Sacan and Yanardag [15] found that the EC50 values of the ABTS radical scavenging activity in chard extracts and rutin were 1093.14 52.02 and 113.70 3.24 µg/mL, respectively. As shown by ± ± Burri et al. [22], the antioxidant potential of beetroot leaves (measured as ABTS radical scavenging activity) was on average from 8.4 to 10.0 mmol TEAC/100 g DW) [22]. The OH radical is a highly reactive oxygen species which is commonly formed in vivo and • can attack many cell macromolecules, including nucleic acids, lipids, and proteins. It is involved in inflammation-related diseases, e.g., chronic inflammation, neurodegeneration, and cancer [5]. Our results demonstrated highly that the investigated plants exhibited a high ability to neutralize OH • radicals. The highest activity was observed for the LC extracts (EC50 = 50.00 mg FW/mL), whereas SK Plants 2020, 9, 944 9 of 14

showed the lowest activity (EC50 = 29.78 mg FW/mL) (Figure4B). Studies reported by other researchers confirmed the antiradical potential of beetroots and chard leaves. As demonstrated by Sacan and Yanardag [15], chard extract neutralized OH radicals at a level of 12.03 1.37% at 0.02 mg/mL and • ± 20.24 6.28% at 0.06 mg/mL. Trolox and BHA exhibited good antiradical activity of 54.31 4.30% and ± ± 52.00 3.43% at a concentration of 0.06 mg/mL, respectively. The OH radical scavenging activity of ± • water extract (20.24 6.28%) was nearly equal to that of ascorbic acid (26.47 2.86%) with EC values ± ± 50 of the water extract (0.158 0.041 mg/mL) lower than that of Trolox (46.76 4.17 µg/mL), ascorbic acid ± ± (116.04 4.75 µg/mL), and BHA (51.54 2.24 µg/mL). ± ± Due to the susceptibility of membrane phospholipids to oxidative attack, the process of lipid peroxidation after iron overload has been widely investigated [23]. However, to the best of our knowledge, there are no data on the chelating properties of Beta vulgaris leaves. Our investigation showed that the CC extract exhibited the highest chelating power (EC50 = 79.98 mg FW/mL), whereas the lowest activity was noticed for BL (EC50 = 143.46 mg FW/mL) (Figure4C). Hydroxyl radical, i.e., the most reactive free radical in vivo, is generated in the reaction of O2•− with H2O2 in the presence of Fe2+ or Cu+. This process is known as the Fenton reaction [9]. Therefore, the ability of phenolics to chelate strongly prooxidant metal ions such as Cu or Fe contributes to their antioxidant properties. Many plant flavonoids are able to form stable metal complexes through their multiple OH groups and the carbonyl moiety [24,25]. Lipoxygenase (LOX) are enzymes involved in conversion of arachidonic acid to leukotrienes and prostaglandins, which are inflammatory mediators. Several studies indicate a correlation between 5-LOX expression and viability of cancer cells, proliferation, cell migration, invasion, metastasis, and apoptosis pathway induction [26]. The LOX-inhibitory potential of plant extracts and pure chemicals is well known and extensively studied [27,28]. However, to the best of our knowledge, no data on the anti-LOX activity and inhibition mechanisms of Beta vulgaris subspecies cycla and white beetroot leaves (SK) have been reported so far. As presented in Table2, the highest LOX inhibitory potential was found for the BL and CC leaf extracts (EC50 = 53.23 mg FW/mL and 56.97 mg FW/mL, respectively), whereas the lowest value was determined in the case of the LC extract (78.29 mg FW/mL). The uncompetitive type of LOX inhibition was detected in all cases. As demonstrated by Miguel [29], betanidin and betanin were able to inhibit soybean LOX, and this activity was higher than that of catechin. The anti-inflammatory effects of betalains were evidenced in a study conducted by Vidal et al. [30]. In addition to suppression of COX-2 synthesis, betanidin extracted from beetroot inhibited LOX in a dose-dependent manner [4]. However, our results clearly show that betanin does not play a major role in the LOX-inhibitory potential of the tested extracts. This was clearly visible in the SK and BL samples, which exhibited higher inhibitory potential than LC (Table2). Although the ability of plant extracts to inhibit LOX is widely documented in the literature [26–28] to the best of our knowledge, there are no such studies on beetroot and chard leaves. Dietary polyphenols are able to reduce uric acid synthesis via xanthine oxidase blocking, suppress renal urate reabsorption, and ameliorate uric acid secretion [31]. As reported by Vulićet al. [32], beetroot pomace extract induced XO inhibition in rat liver homogenates due to the presence of a high amount of phenolic compounds and betalain. However, to the best of our knowledge, there is no similar research of beetroot leaf extracts in the recent literature. The highest XO inhibitory potential was found for the SK leaves extract (EC50 = 81.04 mg FW/mL), whereas the lowest value was determined in the case of the CC extract (205.09 mg FW/mL) (Table3). A noncompetitive type of XO inhibition was detected in both extracts from the red leaves, whereas an uncompetitive mode of inhibition was observed in the case of the white leaf extracts. Thus, it can be assumed that the presence of betanin influences the XO inhibition mechanism. Medicinal plants are rich sources of phytochemicals with XO-inhibitory activity. Kong et al. [33] investigated 122 traditional Chinese medicinal plants used for treatment of gout and other hyperuricemia-related diseases. Among 122 methanol extracts from these plants, 69 exhibited inhibitory activity at 100 mg/mL, with 29 exerting more than 50% inhibition. In turn, Chen et al. [34] reported Plants 2020, 9, 944 10 of 14 higher XO inhibitory activities in extracts from Koelreuteria henryi, Prunus campanulata, and Rhodiola rosea. However, data on XO inhibitors derived from common food are limited. Dew et al. [35] found potent XO inhibitors in black tea, and juices from cranberry and purple grape.

4. Materials and Methods

4.1. Chemicals Xantine oxidase (E.C. 1.17.3.2), xanthine, soybean lipoxygenase (E.C. 1.13.11), linoleic acid, ABTS (2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), sodium salicylate, quercetin, gallic acid, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, salicylic acid, and ferulic acid were purchased from Sigma-Aldrich (Poznan, Poland). All other chemicals were of analytical grade.

4.2. Plant Material and Extract Preparations The Growth of Plant Samples. Seeds for beetroot (Beta vulgaris L. “Czerwona Kula” (CC) and “Snie˙znaKula”(SK))´ and Beta vulgaris subsp. vulgaris, Cicla-Group and Flavescens-Group; red—Rhubarb Chard (LC) tested in this study were provided by the Torseed SA corporation (Toru´n,Poland). Beta vulgaris subsp. vulgaris, Cicla-Group, and Flavescens-Group white—Lukullus (BL) was purchased in PNOS O˙zarów Mazowiecki (Poland). The plants were cultivated from April to June of 2019 on an experimental farm belonging to the University of Life Sciences in Lublin, Poland. The soil was characterized by a very low level of phosphorus, potassium, and magnesium content, mean content of humus, and was acidic in reaction. The leaves of all species were collected after 60 days of vegetations, moisture content was 76.2%. Each sample was fresh, previously cleaned, and washed with distilled water, and ground chard and beetroot leaves (2 g) were extracted by mixing (using a rotator Bio RS-24, Józefów near Otwock, Poland) with 15 mL of 1:1 methanol/water (v/v) and centrifuged at 10,000 rpm for 15 min [36]. Extraction was repeated twice, the supernatants were combined.

4.3. Total Phenolics (TPC) Estimation Total phenolics content was estimated according to the Folin–Ciocalteau method [37]. A 10 µL of the extract was mixed with 10 µL of H2O, 40 µL of Folin reagent (1:5 H2O), and after 3 min with 200 µL of 10% Na2CO3. After 30 min, the absorbance of mixture was measured at a wavelength of 725 nm. TPC was expressed as gallic acid equivalents (GAE) per gram of fresh weight (FW) on the basis of a standard curve of gallic acid.

4.4. Total Flavonoids (TFC) Estimation Total ﬂavonoids content (TFC) was estimated according to Bahorun et al. [38]. One hundred microlitters of sample was mixed with 100 µL 2 g/100 AlCl 6H O. After 10 min, absorbance at 3 × 2 430 nm was measured. TFC was expressed as the quercetin (QE) equivalent (mg/g FW) on the basis of a standard curve of quercetin.

4.5. Phenolic Acids (PA) Proﬁle Samples were analyzed with a Shimadzu Prominence Auto Sampler HPLC (SIL-20A; Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector, and LC solution 1.22 SP1 software (Shimadzu). The column used was a 250 4.6 mm id, 5 µm Varian ChromSep C18 SS, with a guard column × ChromSpher 5 C18 SS 10 2 mm at 40 C. The mobile phase consisted of 4.5% acetic acid (solvent A) × ◦ and 50% acetonitrile (solvent B) at a ﬂow rate of 0.8 mL/min. At the end of the gradient, the column was washed with 50% acetonitrile and equilibrated to the initial condition for 10 min. Quantitative determinations were performed with the external standard calculation, using calibration curves. Plants 2020, 9, 944 11 of 14

The gradient elution was used as follows: 0 min 92% A, 30 min 70% A, 45 min 60%, 80 min 61% A, 82 min 0% A, 85 min 0% A, 86 min 92% A, 90 min 92% A [39]. Identiﬁcation of PA was conducted by comparing retention time and UV absorption spectra with those of pure chemical patterns. The chromatography peaks were conﬁrmed by comparing the retention times with those of reference standards and by the DAD spectra (200 to 500 nm).

4.6. Quantitation of Betalain Content The total betalains content was determined according to Nillson [40] and was expressed as betanin equivalent (BE) per kg of FW.

4.7. Antioxidant Assay

4.7.1. Ability to Scavenge ABTS Radicals The experiments were carried out using the ABTS decolorization assay [41]. The aﬃnity of test material to quench the ABTS free radical was evaluated according to the following equation:

scavenging% = [(A A )/A )] 100, (1) C− A C × where AC is the absorbance of the control and AA is the absorbance of the sample. The scavenging activity was expressed as EC50—extract concentration providing 50% of activity based on a dose-dependent mode of action. To determine EC50 extracts concentrations ranging from 20 to 50 mg FW/mL were used. As a positive control trolox (TA) was used.

4.7.2. Ability to Scavenge the Hydroxyl (OH ) Radicals • The OH scavenging ability was performed according to Su et al. [42]. The scavenging activity • was calculated using the following equation:

scavenging rate% = [1 (A A )/Ac] 100, (2) − 1− 2 × where Ac is the absorbance of the control (without tested sample), A1 is the absorbance of the tested sample addition, and A2 is the absorbance without sodium salicylate. The scavenging activity was expressed as EC50—extract concentration providing 50% of activity based on a dose-dependent mode of action. To determine EC50 extracts concentrations ranging from 20 to 50 mg FW/mL were used. As a positive control trolox (TA) was used.

4.7.3. Metal Chelating Activity (CHP) Chelating power was determined by the method of Guo et al. [43]. The activity was calculated according to the formula: % inhibition = [1 (As/Ac)] 100, (3) − × where Ac is the absorbance of the control and As is the absorbance of the sample. The chelating activity was expressed as EC50—extract concentration providing 50% of activity based on a dose-dependent mode of action. To determine EC50 extracts concentrations ranging from 20 to 50 mg FW/mL were used. As a positive control ethylenediaminetetraacetic acid (EDTA) was used.

4.8. Inhibition of Lipoxygenase Activity (LOXI) Inhibition of soybean 15-LOX is generally regarded as a predictive for inhibition of the mammalian enzyme [44]. The LOX activity was determined spectrophotometrically at 20 ◦C by measuring the increase of absorbance at 234 nm over a 2 min period [45]. One unit of the LOX activity [U] was deﬁned as an increase of absorbance by 0.001 after 1 min at λ = 234 nm. Plants 2020, 9, 944 12 of 14

The LOXI activity was expressed as EC50—extract concentration providing 50% of activity based on a dose-dependent mode of action. The mode of inhibition on the enzyme was performed using the Lineweaver–Burk plot (at EC50 extract concentration).

4.9. Inhibition of Xanthine Oxidase Activity (XOI) XOI activities with xanthine as a substrate were estimated according to Sweeney et al. [46]. One unit of the XO activity [U] was deﬁned as an increase of absorbance by 0.001 after 1 min at λ = 295 nm. The XOI activity was expressed as EC50—extract concentration providing 50% of activity based on a dose-dependent mode of action. The mode of inhibition on the enzyme was performed using the Lineweaver–Burk plot (at EC50 extract concentration).

4.10. Statistical Analysis Experiments were performed in four repetitions. The one-way analysis of variance and Tukey’s test were used for data analysis with a signiﬁcance level of α = 0.05.

5. Conclusions The white beetroot is a new plant gaining popularity especially in Eastern Europe. Beetroot leaves are an underestimated part of the plant. As shown in this paper, SK leaves have a multidirectional antioxidant activity. They contain significant amounts of phenolic acids, and the content of flavonoids and total phenolic compounds is slightly lower than that of chards. Particularly noteworthy is the fact that SK leaves are rich sources of xanthine oxidase inhibitors and compounds capable of chelating transition metal ions. Therefore, they may be a valuable component of the diet of patients suffering from hyperuricemic disorder; however, confirmation of this thesis requires further research involving determination of the bioavailability of biologically active compounds.

Author Contributions: Conceptualization, U.G.-D.; methodology, U.G.-D.; validation, U.G.-D. and D.D.; formal analysis, U.G.-D. and D.D.; investigation, L.D., J.A., M.S., and E.H.-K.; data curation, E.H.-K. and D.D.; writing—original draft preparation, U.G.-D., L.D., and J.A.; writing—review and editing, U.G.-D. and D.D.; visualization, U.G.-D.; supervision, U.G.-D. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: Authors are thankful to the TORSEED S.A. for providing seeds of white beetroot. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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