antioxidant and antiradical activity of beetroot

Research ArticleAntioxidant and Antiradical Activity of Beetroot (Beta vulgarisL var conditiva Alef) Grown Using Different Fertilizers

Aynur Babagil1 Esen Tasgin2 Hayrunnisa Nadaroglu 13 and Haluk Caglar Kaymak4

1Faculty of Engineering Department of Nano-Science and Nano-Engineering Ataturk University 25240 Erzurum Turkey2Faculty of Health Sciences Department of Nutrition and Dietetics Ataturk University 25240 Erzurum Turkey3Erzurum Vocational Training School Department of Food Technology Ataturk University 25240 Erzurum Turkey4Faculty of Agriculture Department of Horticulture Ataturk University 25240 Erzurum Turkey

Correspondence should be addressed to Hayrunnisa Nadaroglu hnisa25yahoocom

Received 13 November 2017 Revised 6 January 2018 Accepted 11 January 2018 Published 5 March 2018

Academic Editor Patricia Valentao

Copyright copy 2018 Aynur Babagil et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Fertilizers in different nitrogen forms (calcium ammonium nitrate (CAN) urea ammonium sulfate (AS) and ammonium nitrate(AN)) and their doses (50 100 and 150) for beetroot (BT) (Beta vulgaris L var conditiva Alef) and the antioxidant and antiradicalactivities in the lyophilized water and alcohol extracts of BT were evaluated In order to evaluate antioxidant and radical removingactivities of BT roots total phenolic compound amount assignment total flavonoids amount assignment method of Fe3+-Fe2+reduction activity using ferric cyanate reduction cupric ions (Cu2+) reducing capacity with CUPRAC method Fe3+ reducingcapacity according to FRAP method ferrous ions (Fe2+) chelating activity superoxide anion radical (O

2

∙minus) removing activity and221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS∙+) radical removing activity were determined In the study BHAand 120572-tocopherol were used as standard antioxidants It was determined that water and alcohol extracts obtained from BT rootsindicated reduction activities effectively In addition it was also determined that these reduction activities were indicated in mostBT roots grown in fertilizermedia at lower percentage and that they hadhigher antioxidative level than that of standard antioxidants

1 Introduction

Beta vulgaris L var conditiva Alef is used for the protectionof the balance in the tissues and cells in all living creaturesand performing of functions Breakdown of the balanceleads to the occurrence of oxidative stress and free radicalsin living metabolism Free radicals are generally reactiveoxygen species (ROS) superoxide anion radical (O

2

∙minus) andhydroxyl (OH∙minus) radicals Oxygen types which are not freeradicals include the derivatives which are not radical suchas hydrogen peroxide (H

2O2) singlet oxygen (1O

2) ozone

(O3) hypochlorous acid (HOCl) nitric oxide (NOminus) and

peroxynitrite (ONOOminus) [1]Occurring ROS components lead to a number of types of

damage such as mutation and damage of DNA breakdown ofnucleotide structured coenzymes changes in enzyme activi-ties and lipid metabolism mucopolysaccharides breakdownoccurring of structural damage in proteins lipid peroxidationand depending on this breakdown of membrane structure

damage inmembrane proteins and breakdown ofmembranetransport systems and steroid and accumulation of somesubstance called age pigment These types of damage haveplayed great role in process of biologic aging cancer or a lot ofdiseases such as hypertension and immune failure of senilityFor this reason the fact that living creatures should feed withthe foods having high levelled antioxidative activity in orderto fight against these negativeness has a great importance asregards their nutrition [2ndash5]

Beetroot (BT) having common usage among public (Betavulgaris L var conditiva Alef) is a vegetable roots of whichare traditional and popular Having rich fiber structure itfacilitates digestion It is very rich as regards B vitamins(B1 B2 B3 and B6) and folic acid [6] BT roots includeboth dissolved and phenolics compounds taking place inthe structure of cell wall and betalain compounds [7] Thepigments giving red colour to the BT roots are bioactivecompounds and provide antioxidative activity for humanhealth [8 9] It is revealed that BT roots may be useful

HindawiJournal of ChemistryVolume 2018 Article ID 7101605 10 pageshttpsdoiorg10115520187101605

2 Journal of Chemistry

for improving blood sugar level and hypertension [10] Inaddition betalains have great importance for cardiovasculardiseases by lowering the level of homocysteine [11ndash13]

According to the foodstuffs not available sufficiently inthe soil in which they are produced in order to removethe lack and increase yield the fertilization has been used[14 15] With the correct fertilization applied in agriculturalapplication yield increasing up to 60 has been obtained[16 17] In addition to the usefulness fertilization hassome negativeness as regards environment In addition toexcessive and wrong fertilization application there are theaccumulation of some substances in the plant and occurrenceof some harmful gases as a result of evaporation with soilapplication of nitrogenous manure and especially occurrenceof greenhouse effect as a result of participation of nitrogenoxides Moreover in some fertilizersrsquo production this leadsto the formation of soil pollution of heavy metal ions such asCd2+ and the increasing of the amount of nitrogen in watersas a result of excessive irrigation As a result of the pollution innature it may increase risk of cancer in humans as well as therisk for plant and animal life It is proved that the fertilizersled to ovarian and prostate cancers and there was mortalityrate at 41 It is detected that some fertilizers had effects onnervous system and even mutation may occur [18 19] Dueto negativeness like these excessive fertilizations should beavoided

In this study we tried to investigate the effect of fertil-ization with different nitrogen forms (CAN urea AS andAN) and their doses (50 100 and 150 kg haminus1) on chemicalstructure and antioxidant or antiradical activities of beetroot(BT) (Beta vulgaris L var conditiva Alef)

2 Materials and Methods

21 Chemicals 221015840-Azino-bis(3-ethylbenzothiazoline-6-sul-fonic acid) (ABTS) neocuproine (29-dimethyl-110-phe-nanthroline) riboflavin methionine nitroblue tetrazolium(NBT) 11-diphenyl-2-picrylhydrazyl (DPPH) 3-(2-pyridyl)-56-bis(4-phenyl-sulfonic acid)-124-triazine (ferrozine) 120572-tocopherol linoleic acid gallic acid quercetin Folin-Ciocalteu reagent and trichloroacetic acid (TCA) were pur-chased from Sigma-Aldrich GmbH (Steinheim Germany)The other chemicals were obtained fromMerck

22 Beetroot (BT) (Beta vulgaris L var conditiva Alef)The Growth of Plant Samples The field experiment wasconducted during MayndashOctober of 2015 in Erzurum TurkeySeeds for beetroot (Beta vulgaris L var conditiva Alef cvldquoBikoresrdquo) tested in this study were provided by the MetgenSeed Corporation (Istanbul Turkey) The soil of the experi-mental area was clay loam texture (clay 3546 silt 3499and sand 2955) ustorthent great soil group with neutralpH (756) and EC 315 120583mhos cmminus1 It had 187 organic mat-ter 2430 cmol kgminus1 Ca 235 cmol kgminus1Mg 124 cmol kgminus1 K027 cmol kgminus1Na 4067mg kgminus1 P and 0083 total N Seedswere sown on plots of 5m2 in field on 20 May in 2015 inrows 230 cm long at a separation between rows of 40 cmand between plants of 20 cm The plants were thinned afterthe emergence when they formed 4-5 true leaves Irrigation

was on a need basis about twice a week after emergenceExperimental plots were kept weed-free using hand weeding

The plots were fertilized with different doses of nitrogenforms Four forms of nitrogen fertilizer [urea (46 N)calcium ammonium nitrate (26 N) ammonium nitrate(33 N) and ammonium sulfate (21 N)] and three doses ofthem (50 100 and 150 kg haminus1) were appliedThe same P dose(100 kg haminus1 P

2O5) was the accepted usual dose according

to Swiader et al [20] and Kaymak et al [21] reports whichapplied all fertilized plots All of the P

2O5and half of the

nitrogen fertilizer were applied uniformly prior to plantingonto soil surface by hand and incorporated The remaininghalf of the nitrogen was given 20 days after emergence [21]Plots not exposed to nitrogen fertilizer served as controlBeetroot plots were harvested on 9 October 2015 accordingto the suggestions of Seed Corporation for tested cultivar andthey were stored at +4∘C until use

23The Preparation of LyophilizedWater and Alcohol Extractsof Beetroot (BT) (Beta vulgaris L var conditiva Alef) 100 gbeetroot (BT) (Beta vulgaris L var conditiva Alef) wasweighted and split with blender device for each treatmentThen samples were divided into two parts In the first part100mL purified water was added to the split sample It wasextracted at the room temperature for a night Then it wasfiltered and the operation was repeated After the extractswere combined they were filtered into filter paper then thefiltrates were taken to balloons and frozen in deepfreezeThe frozen extracts were lyophilized under the pressure of50mm-Hg until it dried in lyophilizer After alcohol wasadded to the second part of split beetroot cells extracted itwas filtered and removed by evaporator dissolver

24 Assigning of Total Amount of Phenolic Compound Theamount of phenolic compounds available in lyophilizedwater and alcohol extracts of BT roots for all treatments(0 50 100 and 150 kg haminus1) of all nitrogen sources wasdetermined totally by using Folin-Ciocalteu reactivity [22]Gallic acid was used as a standard and standard graphicwas prepared 1000 120583g extract was taken from stock solutionand it was taken into a measure container and volume wascompleted to 23mL by means of distilled water 05mLFolin-Ciocalteu reagent was added to the mixture and itwas incubated for three minutes and then 15mL 2 (wv)Na2CO3was added Then after the samples were stirred at

the room temperature for two hours absorbances at 760 nmwere determined against the blend consisting of distilledwater Gallic acid equivalent amount accounting for obtainedabsorbance values was calculated by help of the equationobtained from standard graphic The results obtained weregiven as gallic acid equivalent (GAE)

25 The Determination of the Amount of Total FlavonoidCompounds Total flavonoid amounts in lyophilized waterand alcohol extracts of BT roots for all sources of nitrogendoses were determined according to the method by Parket al [23] for all doses (0 50 100 and 150 kg haminus1) ofall nitrogen sources So 43mL ethanol solution including1000 120583g extract medium 01mL 1M CH

3COOK and 01mL

Journal of Chemistry 3

10 Al(NO3)3was added to an experiment tube and stirred

with vortexThen after it was incubated for 40minutes at theroom temperature its absorbances were read at 415 nm Byusing the equation obtained from standard graphics preparedby using quercetin total flavonoid concentrations of waterand alcohol samples of all BT roots were determined asmicrogram quercetin equivalent (QE)

26 Fe3+-Fe2+ Reducing Capacity Total reducing assignmentwas done according to Oyaizu method [24] So firstly stocksolution at 1mgmL concentration was prepared 10 20 and30 120583gmL samples from this stock solution were transportedto the experiment tubes and the volume was completed up to1mL by distilled waterThen 25mL phosphate buffer (02MpH66) and 25mLK

3Fe(CN)

6(1 (wv)) were added to each

tube and the mixture was incubated at 50∘C for 20 minutesAfter these processes 25mL TCA (10 (wv)) was added toreaction mixture 25mL was taken from the upper phase ofsediment So 25mL distilled water and 05mL FeCl

3(01

(wv)) were added and the absorbance of all samples wasdetermined against the blank at 700 nm and water was usedinstead of sample in the control

27 Cu2+-Cu+ Reducing Capacity (CUPRAC Method) Cu2+reducing activities of lyophilized water and alcohol extractsof BT roots grown in different fertilization media weredetermined according to the reducing method of copperions [25] From lyophilized water and alcohol extracts of BTroots preparedwith 10 and 30 120583gmL concentrations 025mLCuCl2solution (001M) 025mL ethanolic neocuproine

solution (75 times 10minus3M) and 025mL CH3COONH

4buffer

solution (1M)were added to the tubes respectively and at theroom temperature for 30min they were incubated and theirabsorbances were determined against the blank occurringwith pure water at 450 nm

28 Ferrous Ions and (Fe2+) Chelating Activity Metal chelat-ing activity of lyophilized water and alcohol extracts of BTroots were determined according to a method applied byDinis et al [26] This process and solution medium included005mL FeCl

2sdot4H2O (2mM) and 035mL distilled water

then the solution including BT root samples at 30 120583gmL con-centration and 02mL lyophilized water and alcohol extractswas added and the last volume was completed to the 4mLby ethanol Then the solution of ferrozine (02mL 5mM)was added to the reactionmedium and it was stirred stronglywith vortex After reaction mixtures were incubated at roomtemperature for 10 minutes the absorbance was determinedat 562 nm against the blank occurring with ethanol solutionAs control solution contents formedwithout BT root extractswere used

29 Removing Activity of Super Oxide Anion Radicals (O2minus)Superoxide anion radicals removing activity of all BT rootswater and alcohol extracts was determined by means ofspectrophotometric measuring occurring in the medium asa result of reaction of nitroblue tetrazolium (NBT) Forthis aim the method utilized by Zhishen et al [27] wasused Stock solution which had been prepared before was

used for this purpose For this different concentrations ofBT root extracts and standards (BHA and 120572-tocopherol)were prepared with phosphate buffer (005M and pH 78)To the reaction mixture including samples the amounts ofriboflavin methionine and NBT (133 times 10minus5 446 times 10minus5and 815 times 10minus8M concentrations) were stimulated at roomtemperature for 40 minutes with 20W fluorescent light Theabsorbances of reaction mixtures were recorded against theblend occurring at water at 560 nm

210 11-Diphenyl-2-picrylhydrazyl (DPPH) Free Radicals Re-moving Activity In lyophilized water and alcohol extracts ofall BT root samples DPPH free radical removing activity wasdetermined according to Blois method [28] As free radicalthe solution of DPPH∙ (1mM) was used To the experimentstubes at concentrations of 10 20 and 30120583g120583L water andalcohol extracts obtained from BT roots were transportedand their total volumes were completed to 3mLwith ethanolThen to each sample tube 1mL from stock DPPH solutionwas added and incubated at room temperature for 30 min-utes and the absorbances were determined against the blankoccurring with ethanol at 517 nm As controls 3mL ethanoland 1mL DPPH∙ solution were used Reduced absorbancesgave remainingDPPH∙ solution quantity namely free radicalremoving activity

211 22-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) Radical Removing Activity In water and alcoholextracts of BT samples ABTS radical removing activity wasdetermined according to themethod used by Re et al [29] Byadding persulphate solution (245 nM) to the ABTS (7mM)solution the formation of ABTS radicals in reaction mediumwas provided At 734 nm the absorbance of control solution(01M pH 74) was adjusted to 0700 plusmn 0025 nm usingphosphate buffer 1mL ABTS radical solution was added tothe lyophilized water and alcohol extracts at 10ndash30 120583gmL ofBT roots and incubated at room temperature for 30 minutesThe absorbances of all samples were determined against theblank occurring from ethanol at 734 nm

3 Results and Discussion

In the fertilization process carried out for increasing theyield in agriculture organic and inorganic fertilizers leadto soil and environmental pollution As well as leadingto death of living creatures in both soil and water theycause some diseases in humans such as bronchitis nervoussystem disorders and some cancers (stomach cancers gallbladder cancers intestinal cancers etc) Due to havingnegative effects of fertilizers on a number of health subjectsantioxidative and antiradical activities of BT roots grown indifferent fertilization media have been examined

31 Assignment of Total Flavonoid Amount Flavonoids arepolyphenolic compounds available in plants It is knownthat these compounds have strong antioxidant features andthey also have metal connection and keeping of free radicalfeatures [30] Standard graphic was created using gallic acid(GAE) and total phenolic amounts available in lyophilized


Table 1 Total phenolic content and total flavonoids amounts of water extracts alcohol extracts 120572-tocopherol and BHA

Total phenolics Total flavonoids(120583g of GAEamg dw) (120583g of QEbmg dw)

Water Alcohol Water AlcoholBHA 326 plusmn 57 665 plusmn 34 289 plusmn 85 626 plusmn 24120572-Tocopherol 254 plusmn 36 855 plusmn 48 215 plusmn 64 813 plusmn 48CAN 50 kg haminus1 273 plusmn 42 1474 plusmn 38 236 plusmn 61 1436 plusmn 89CAN 100 kg haminus1 376 plusmn 52 1491 plusmn 06 339 plusmn 32 1459 plusmn 11CAN 150 kg haminus1 179 plusmn 88 661 plusmn 18 143 plusmn 26 618 plusmn 26Urea 50 kg haminus1 245 plusmn 51 965 plusmn 44 208 plusmn 16 925 plusmn 46Urea 100 kg haminus1 314 plusmn 34 805 plusmn 14 277 plusmn 93 767 plusmn 73Urea 150 kg haminus1 251 plusmn 15 737 plusmn 69 213 plusmn 44 720 plusmn 97AS 50 kg haminus1 384 plusmn 39 1093 plusmn 33 349 plusmn 27 1057 plusmn 74AS 100 kg haminus1 319 plusmn 18 1121 plusmn 08 292 plusmn 18 1084 plusmn 36AS 150 kg haminus1 296 plusmn 24 1064 plusmn 38 256 plusmn 57 1031 plusmn 16AN 50 kg haminus1 214 plusmn 39 1174 plusmn 32 179 plusmn 97 1138 plusmn 77AN 100 kg haminus1 436 plusmn 18 1048 plusmn 78 398 plusmn 84 1012 plusmn 15AN 150 kg haminus1 326 plusmn 58 664 plusmn 35 289 plusmn 85 626 plusmn 66aDetermined as gallic acid equivalent (GAE) bDetermined as quercetin equivalent (QE)

water and alcohol extracts of beetroot (Beta vulgaris L varconditivaAlef) were calculated bymeans of standard graphicTotal phenolic matter amounts obtained by using all differentfertilizes for BT samples were given in Table 1 From theresults obtained it was determined that all fertilizers had thehighest phenolicmatter rate at 1 concentration of fertilizersIt was also determined that the highest matter content wasin beetrootrsquos alcohol extracts at fertilization with 50 kg haminus1CAN and urea 150 kg haminus1 AS and 100 kg haminus1 AN It wasfound that fertilization with 100 kg haminus1 AN had maximumphenolic content of 436 plusmn 18 120583gmg GAE and 1491 plusmn06 120583gmg GAE for water and alcohol samples respectively

32 The Assignment of Total Phenolic Compound AmountFor assignment standard graphic was created using quercetin(QE) and total phenolic amounts in lyophilized water andalcohol extracts of BT roots (Beta vulgaris L var conditivaAlef) were calculated using standard graphic and all resultswere given in Table 1

It was determined that water extracts of BT roots hadthe highest flavonoid matter amount as 398 plusmn 84 120583gmgQE in fertilization with 100 kg CAN 50 kg urea 100 kg ASand 50 kgANhaminus1 When alcohol extracts were comparedas regards flavonoid matter amount it was seen that CAN(100 kg haminus1) urea (50 kg haminus1) AS (100 kg haminus1) and AN(100 kg haminus1) had the highest flavonoid matter amount In allsamples it was detected that fertilization with 100 kg CANhaminus1 had maximum amount of flavonoid matter at the valueof 1439 plusmn 11 120583gmg QE From the results obtained the factthat BT roots have high phenolic and flavonoid content inlower fertilizer concentrations was evaluated as a positiveresult [31]

33 Superoxide Anion Radical Removing Activities Super-oxide radicals from free radicals in both enzymatic and

nonenzymatic reactions aremost easily producedThese radi-cals lead to lipid peroxidation and depend on the breakdownof the structure of membrane [32] In addition superoxideanion radicals can reduce Fe3+ ions to Fe2+ In addition itis known that Fe2+ ions by using hydrogen peroxide withFenton reaction led to formation of OH radicals which arevery highly reactive in a number of disorders For thesereasons removing superoxide anion radicals in medium isneeded

It is found that in the study in which BT roots were usedwith lyophilized water extract at 30 120583gmL concentrationssuperoxide anion radical removing activity became the high-est at fertilization with 150 kg haminus1 urea gt BHA gt AN gt CANgt 120572-tocopherol gtAS respectivelyThese values were shown ina way as 771 plusmn 34 gt 664 plusmn 12 gt 641 plusmn 98 gt 593 plusmn 33 gt554 plusmn 73 gt 480 plusmn 32 respectively In alcohol extractsof BT roots the highest superoxide anion radical removingactivities at fertilizationwith 150 kg haminus1 areureagtANgtBHAasymp CAN gtAS gt 120572-tocopherol respectivelyThese values were846plusmn35 gt 703plusmn91 gt 664plusmn12 asymp 657plusmn69 asymp 643plusmn68 gt

554 plusmn 73 respectively As is seen in Table 1 when the resultswere compared to standard it was observed that BT rootsremoved effectively superoxide anion radicals in both waterand alcohol extracts at fertilization with 150 kg of all nitrogensources haminus1

34 Ferrous Ions Chelating Capacity The activities of ferrousions chelating of lyophilized water and alcohol extracts of BTplants grown in different media and comparison with BHAand 120572-tocopherol being a standard antioxidant were given inTable 2When chelating activities of ferrous ions (Fe2+) for 120572-tocopherol BHA and water and alcohol extracts of BT plantat 30 120583gmL concentration were measured it was observedthat high ferrous ions chelating activities were observed forE-Urea 150 kg haminus1 W-AS-50 kg haminus1 and E-CAN 150 kg haminus1according to standards These values were determined as


Table 2The results of superoxide anion radical scavenging ferrous ion chelating andhydrogenperoxide scavenging activity ofwater extractsalcohol extracts 120572-tocopherol and BHA

Superoxide scavenging activity () Ferrous ion chelating activity () H2O2scavenging activity ()

Water Alcohol Water Alcohol Water AlcoholBHA 664 plusmn 12 614 plusmn 36 386 plusmn 15120572-Tocopherol 554 plusmn 73 485 plusmn 49 416 plusmn 56CAN 50 kg haminus1 213 plusmn 29 337 plusmn 63 309 plusmn 96 559 plusmn 23 386 plusmn 38 523 plusmn 22CAN 100 kg haminus1 469 plusmn 88 586 plusmn 55 219 plusmn 19 512 plusmn 19 426 plusmn 95 586 plusmn 19CAN 150 kg haminus1 593 plusmn 33 657 plusmn 69 212 plusmn 16 831 plusmn 26 401 plusmn 33 536 plusmn 28Urea 50 kg haminus1 270 plusmn 30 358 plusmn 47 614 plusmn 13 339 plusmn 71 554 plusmn 36 632 plusmn 57Urea 100 kg haminus1 492 plusmn 21 582 plusmn 12 578 plusmn 77 116 plusmn 40 583 plusmn 44 662 plusmn 11Urea 150 kg haminus1 771 plusmn 34 846 plusmn 35 286 plusmn 27 928 plusmn 84 563 plusmn 05 651 plusmn 53AS 50 kg haminus1 213 plusmn 29 336 plusmn 33 883 plusmn 32 607 plusmn 36 653 plusmn 76 723 plusmn 42AS 100 kg haminus1 324 plusmn 39 566 plusmn 25 630 plusmn 02 206 plusmn 59 692 plusmn 25 715 plusmn 14AS 150 kg haminus1 480 plusmn 32 643 plusmn 68 583 plusmn 23 89 plusmn 86 631 plusmn 81 703 plusmn 33AN 50 kg haminus1 488 plusmn 76 532 plusmn 27 524 plusmn 36 133 plusmn 27 681 plusmn 23 756 plusmn 51AN 100 kg haminus1 584 plusmn 35 623 plusmn 36 461 plusmn 94 294 plusmn 39 766 plusmn 46 782 plusmn 69AN 150 kg haminus1 641 plusmn 98 703 plusmn 91 272 plusmn 41 236 plusmn 24 772 plusmn 89 743 plusmn 78

Water

0

05

1

15

2

Abso

rban

ce (7

00 n

m)

300 10 20 Concentration (mgmL)

BHAa-TocopherolW-ControlW-CAN 50 kg Ｂminus1

W-CAN 100 kgW-CAN 150 kgW-Urea 50 kgW-Urea 100 kgW-Urea 150 kgW-AS 50 kgW-AS 100 kgW-AS 150 kgW-AN 50 kgW-AN 100 kgW-AN 150 kg

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

Abso

rban

ce (7

00 n

m)


BHAa-TocopherolE-ControlE-CAN 50 kg Ｂminus1

E-CAN 100 kg Ｂminus1


E-Urea 50 kg Ｂminus1



E-AS 50 kg Ｂminus1



E-AN 50 kg Ｂminus1



(b)

Figure 1 The Fe3+-Fe2+ reducing activity of different concentrations (10ndash30120583gmL) of water extracts alcohol extracts 120572-tocopherol andBHA


Water

0

04

08

12Ab

sorb

ance

(450

nm

)

10 20 300Concentration (gmL)



Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol


0

04

08

12

16

Abso

rban

ce (4

50 n

m)













(b)

Figure 2 The Cu2+-Cu+ reducing activity of water extracts alcohol extracts BHA and 120572-tocopherol at different concentrations (10ndash30120583gmL)

614 for BHA and 485 for 120572-tocopherol and also for E-Urea 150 kg haminus1 W-AS 50 kg haminus1 and E-CAN 150 kg haminus1values were 928 883 and 831 respectively

35 Hydrogen Peroxide Scavenging Activity During the reac-tion when oxygen is reduced in the cell by taking electronin the case complete reducing is not obtained the formationof H2O2and OH∙minus which is very reactive is done true The

reaction of reduction from oxygen to water is shown asfollows

minus minus minus minus

2∙minus2 （∙minus（22 （2

(1)

For this reason H2O2should be removed by means of

antioxidative substances In both water and alcohol extractsof BT roots grown at different fertilizer media hydrogen per-oxide scavenging activity was carried out according to Ruchet al [33] and the results were compared with standard BHAand120572-tocopherol and theywere given inTable 2 At 15 120583gmL

concentration while hydrogen peroxide scavenging activitywas 386 for BHA and 416 for 120572-tocopherol in E-AN-150 kg haminus1 for example the highest activity was observedwith 782 (Table 2) It was determined that both waterand alcohol extracts plant samples grown in all fertilizersmedia indicated much higher hydrogen peroxide scavengingactivity than BHA and 120572-tocopherol standards These resultsshowed that BT samples which grew with all used fertilizershad an effective hydrogen peroxide scavenging activity

36 The Fe3+-Fe2+ Reducing (FRAP) Activity The reducingpower of a compound is known as the capacity of giving elec-tron of that compound and can be measured with differentmethodsThe Fe3+-Fe2+ reducing method is the one in whichantioxidants give electrons and indicate antioxidant activityIt was found that ferrous ions (Fe3+) reducing capacity wasincreased with increasing fertilizer concentrations (50 100and 150 kg haminus1) according to FRAP method It was alsoobserved that alcohol extracts had higher FRAP activity in allsamples At 30 120583gmL concentrations the samples of W-AN


Water

0

05

1

15

2

25

3Ab

sorb

ance

(517

nm

)

10 20 300Concentration (mgmL)



Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

3

Abso

rban

ce (5

17 n

m)














(b)

Figure 3 The DPPH∙ scavenging effect of water extracts alcohol extracts BHA and 120572-tocopherol at different concentrations (10ndash30120583gmLminus1)

150 kg haminus1 and E-AN-150 kg haminus1 had the highest FRAPactivity In addition according to BHA and 120572-tocopherolused standardly it was detected that all samples indicatedhigher FRAP activity (Figure 1)

37 The Cu2+-Cu+ Reducing Activity Another method usedfor determining the reducing capacity is CUPRAC methodReducing capacity of cupric ions of lyophilized water andalcohol extracts of BT roots (Cu2+) was determined bymeansof spectrophotometric method at different concentrations inthe samples which contain extract (10ndash30 120583gmL) Reduc-ing capacity of cupric ions of water and alcohol extractsobtained from red beetroots growth at different fertilizer andconcentrations media (Cu2+) was compared with BHA and120572-tocopherol a standard antioxidant Related results wereshown in Figure 2 As seen in Figure 2 both water andalcohol extracts samples exhibited higher reducing capacitythan both BHA and 120572-tocopherol antioxidant standards At30 120583gmL concentration when compared with the standardsof reducing capacity of cupric ions (Cu2+) the highest ones of

them were E-AN-100 kg haminus1 gt W-CAN-50 kg haminus1 gt BHAgt 120572-tocopherol respectively

38 The DPPH∙ Scavenging Activity DPPH∙ (11-diphenyl-2-picrylhydrazyl) is an organic structured radical givingabsorbance at 517 nm In our study as to removing of DPPH∙radical activity absorbance reducing at 517 nm and residinginDPPH∙ solution amount bymeasuring namely free radicalremoving activity were determined In order to assign ofDPPH∙ radical removing activity firstly standard graphic wasformed and used for calculations It is clearly seen fromFigure 3 that lyophilized water and alcohol extracts of BTroots grown in different fertilizer media exhibited higherDPPH∙ radical removing activity than standard antioxidantcompounds such as BHA and 120572-tocopherol At 30 120583gmLconcentrations the highest activities in water and alcoholextracts of BT roots were compared with standard antioxi-dants they are exhibited in the way of W-AN-100 kg haminus1 gtE-AN-100 kg haminus1 gt BHA gt a-tocopherol DPPH∙ radicalremoving activity These values were calculated as 844


Water

0

03

06

09Ab

sorb

ance

(734

nm

)


BHAa-TocopherolControlW-CAN 50 kg Ｂminus1


Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

03

06

09

Abso

rban

ce (7

34 n

m)


BHAa-TocopherolControlE-CAN 50 kg Ｂminus1












(b)

Figure 4 The stable ABTS∙+ scavenging effect of water extracts alcohol extracts BHA and 120572-tocopherol at different concentrations(10ndash30 120583gmLminus1)

528 and 167 respectively That water and alcoholextracts of BT roots grown in all different fertilizer mediaindicated higher DPPH radical removing activity than thatof control samples which was shown in Figure 3

When the previous studies were examined it was foundthat in the extracts whose contents of C vitamin andpolyhydroxy aromatic compounds are high excessive DPPH∙scavenging activity was high We could say that these studiessupported our results Also BT roots obtained from fer-tilization with 150 kg haminus1 of nitrogen sources giving highyield indicate that there is no need for excessive fertilizationapplication

39 The ABTS∙+ Scavenging Activity ABTS∙+ radical is acoloured compound giving absorbance at 734 nm ABTS∙+radical participates in chemical reaction with antioxidantsubstances transfers one electron and turns into a unradicalABTS substance Related reaction was given as follows

４３+∙ + ４３+∙ + +∙ (2)

In the study carried out first spectrophotometric measuringwas conducted and then was followed by reducing theabsorbance value at 734 nm and ABTS∙+ radical removingactivity was calculated

ABTS∙+ removing activity has been commonly usedin the radical removing activities from watered mixturesbeverages and extracts and pure substances [34] Firstlystandard graphic was formed to assign the ABTS removingactivities of lyophilized water and alcohol extracts of BT rootsgrown at different fertilizer media and standard antioxidantcompounds such as BHA 120572-tocopherol this standard graphicwas used for ABTS∙+ removing activity calculation in allsamples According to the results obtained at 30 120583gmLconcentrations it was detected that BHA indicated ABTSradical removing at the rate of 810 and 120572-tocopherol atthe rate of 876 (Figure 4) In this study it was found thatlyophilized water and alcohol extracts of BT roots removedABTS radical stronger than standard antioxidants

Nitrogen is an indispensable component of proteins usedto form cell materials and plant tissues but high nitrogen


levels are toxic to plant growth NH4

+ toxicity probablyindicates that excessive production of ROS can cause anamount of oxidative damage to proteins lipids and DNAresulting in lipid peroxidation cell damage and cell death[35] In this study urea CAN AN and AS fertilizers wereutilized as the most used organic nitrogen source in thecultivation of BT

Although urea CAN AN and AS are generally known tohave low toxicity to organisms they have indirect and long-term harm to ecosystems such as eutrophication ground-water pollution and soil acidification [36 37] Ammoniumformed as a result of the hydrolysis of the urine CAN ANand AS is more toxic to plants [38] Higher amounts of ureacause decreased biological efficiency of the plants and causephysiological disorders [39 40] However the effects of plant-induced oxidative stress on plants are not clear [41]

In low concentration urea application (100 mg L-1) inplant oxidative stress has been reduced due to decreasedROS (superoxide and hydrogen peroxide) formation andlipid peroxidation At high concentration urea leads to thedepletion of a low molecular weight antioxidant pool Itis thought to be associated with increased oxidative stressand increased antioxidative protection of the plant [41 42]Similar results were obtained in the application of fertilizersof nitrogen origin such as CAN AN and AS to the plant Inlowdoses of nitrogen fertilizers good growthwas observed inthe plant while high-dose plant growth caused unnecessaryand lethal outcomes

Also nitrogen application at higher rates negativelyaffected the antioxidant activities such as ferric cyanatereduction cupric ions (Cu2+) reducing capacity withCUPRACmethod Fe3+ reducing capacity according to FRAPmethod ferrous ions (Fe2+) chelating activity superoxideanion radical and 21015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radical removing activity Thereforebased on these results it had been concluded that lownitrogen was effective in plant growth and high antioxidativeactivity and it also caused decrease of oxidative stress in BT

4 Conclusion

On the basis of the results of this study it is clearly indicatedthat BT roots growth by using low doses of CAN ureaAS and AN fertilizers has a powerful antioxidant activityagainst various oxidative systems in vitro For this reason asthe concentration of applied fertilizer lowers environmentalpollution and threat factors of human health also lower andso the rate of cost of the products will lower

Disclosure

This work was previously submitted at 4th InternationalISEKI-Food Conference (6ndash8 July 2016 Vienna Austria) asa poster presentation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this study

References

[1] A Pavlov P Kovatcheva V Georgiev I Koleva and MIlieva ldquoBiosynthesis and radical scavenging activity of betalainsduring the cultivation of red beet (Beta vulgaris) hairy rootculturesrdquo Zeitschrift fur Naturforschung - Section C Journal ofBiosciences vol 57 no 7-8 pp 640ndash644 2002

[2] H Celik K Kucukoglu H Nadaroglu and M Senol ldquoEvalu-ation of antioxidant antiradicalic and antimicrobial activitiesof kernel date (fructus dactylus)rdquo Journal of Pure and AppliedMicrobiology vol 8 no 2 pp 993ndash1002 2014

[3] H Celik H Nadaroglu and M Senol ldquoEvaluation of antioxi-dant antiradicalic and antimicrobial activities of olive pits (Oleaeuropaea L)rdquo Bulgarian Journal of Agricultural Science vol 20no 6 pp 1392ndash1400 2014

[4] HNadaroglu YDemir andNDemir ldquoAntioxidant and radicalscavenging properties of Iris germanicardquoPharmaceutical Chem-istry Journal vol 41 no 8 pp 409ndash415 2007

[5] HNadaroglu NDemir andYDemir ldquoAntioxidant and radicalscavenging activities of capsules of caper (Capparis spinosa)rdquoAsian Journal of Chemistry vol 21 no 7 pp 5123ndash5134 2009

[6] M E Latorre P Narvaiz A M Rojas and L N GerschensonldquoEffects of gamma irradiation on bio-chemical and physico-chemical parameters of fresh-cut red beet (Beta vulgaris L varconditiva) rootrdquo Journal of Food Engineering vol 98 no 2 pp178ndash191 2010

[7] M N Gasztonyi H Daood M T Hajos and P Biacs ldquoCom-parison of red beet (Beta vulgaris var conditiva) varieties onthe basis of their pigment componentsrdquo Journal of the Scienceof Food and Agriculture vol 81 no 9 pp 932-933 2001

[8] S J Schwartzieber and J H Von Elbe ldquoQuantitative determi-nation of individual betacyanin pigments by high-performanceliquid chromatographyrdquo Journal of Agricultural and FoodChem-istry vol 28 no 5 pp 540ndash547 1980

[9] G J Kapadia H Tokuda T Konoshima and H NishinoldquoChemoprevention of lung and skin cancer by Beta vulgaris(beet) root extractrdquo Cancer Letters vol 100 no 1-2 pp 211ndash2141996

[10] A Gliszczynska-Swigło ldquoAntioxidant activity of water solublevitamins in the TEAC (trolox equivalent antioxidant capacity)and the FRAP (ferric reducing antioxidant power) assaysrdquo FoodChemistry vol 96 no 1 pp 131ndash136 2006

[11] A Pavlov P Kovatcheva D Tuneva M Ilieva and T BleyldquoRadical scavenging activity and stability of betalains from Betavulgaris hairy root culture in simulated conditions of humangastrointestinal tractrdquo Plant Foods for HumanNutrition vol 60no 2 pp 43ndash47 2005

[12] M R Olthof T Van Vliet E Boelsma and P Verhoef ldquoLowDose Betaine Supplementation Leads to Immediate and LongTerm Lowering of Plasma Homocysteine in Healthy Men andWomenrdquo Journal of Nutrition vol 133 no 12 pp 4135ndash41382003

[13] T Nagai S Ishizuka H Hara and Y Aoyama ldquoDietary sugarbeet fiber prevents the increase in aberrant crypt foci inducedby 120574-irradiation in the colorectum of rats treated with animmunosuppressantrdquo Journal of Nutrition vol 130 no 7 pp1682ndash1687 2000

[14] W Aktar D Sengupta and A Chowdhury ldquoImpact of pesti-cides use in agriculture their benefits and hazardsrdquo Interdisci-plinary Toxicology vol 2 no 1 pp 1ndash12 2009

[15] R Sima D Maniutiu A S Apahidean M Apahidean V Lazarand C Muresan ldquoThe influence of fertilization on greenhouse


tomatoes cultivated in peat bags systemrdquo Bulletin UASVMHorticulture vol 66 no 1 pp 455ndash460 2009

[16] D Tilman K G Cassman P A Matson R Naylor and SPolasky ldquoAgricultural sustainability and intensive productionpracticesrdquo Nature vol 418 no 6898 pp 671ndash677 2002

[17] J R Purman and F R Gouin ldquoInfluence of compost aging andfertilizer regimes on the growth of bedding plants transplantsand poinsettiardquo Journal of Environmental Horticulture vol 10pp 52ndash54 1992

[18] J Dich S H Zahm A Hanberg and H-O Adami ldquoPesticidesand cancerrdquoCancer Causes amp Control vol 8 no 3 pp 420ndash4431997

[19] G Van Maele-Fabry and J L Willems ldquoOccupation relatedpesticide exposure and cancer of the prostate A meta-analysisrdquoOccupational and Environmental Medicine vol 60 no 9 pp634ndash642 2003

[20] J M Swiader GWWare and J P Collum Producing VegetableCrops Interstate Publishes Inc Danville Ill USA 1992

[21] H C Kaymak S Ozturk S Ercisli and I Guvenc ldquoIn vitroantibacterial activities of black and white radishes (RaphanusSativus L)rdquoComptes Rendus de LrsquoAcademie Bulgare des SciencesSciencesMathematiques et Naturelles vol 68 no 2 pp 201ndash2082015

[22] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1999

[23] Y K Park M H Koo M Ikegaki and J L Contado ldquoCom-parison of the flavonoid aglycone contents of Apis melliferapropolis from various regions of Brazilrdquo Arquivos de BiologiaeTechnologia vol 40 pp 97ndash106 1997

[24] M Oyaizu ldquoStudies on products of browning reactionsldquoAntioxidative activities of products of browning reaction pre-pared from glucosaminerdquordquo Japanese Journal of Nutrition vol103 pp 413ndash419 1986

[25] RApakKGucluM Ozyurek S EsinKarademir andE ErcagldquoThe cupric ion reducing antioxidant capacity and polyphenoliccontent of some herbal teasrdquo International Journal of FoodSciences and Nutrition vol 57 no 5-6 pp 292ndash304 2006

[26] T C P Dinis V M C Madeira and L M Almeida ldquoActionof phenolic derivatives (acetaminophen salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidationand as peroxyl radical scavengersrdquo Archives of Biochemistry andBiophysics vol 315 no 1 pp 161ndash169 1994

[27] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[28] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199-1200 1958

[29] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[30] A Saija M Scalese M Lanza D Marzullo F Bonina andF Castelli ldquoFlavonoids as antioxidant agents importance oftheir interaction with biomembranesrdquo Free Radical Biology ampMedicine vol 19 no 4 pp 481ndash486 1995

[31] T S Kujala M S Vienola K D Klika J M Loponenand K Pihlaja ldquoBetalain and phenolic compositions of fourbeetroot (Beta vulgaris) cultivarsrdquo European Food Research andTechnology vol 214 no 6 pp 505ndash510 2002

[32] B Halliwell and J M Gutteridge Free Radicals in Biology andMedicine Clarendon Press Oxford UK 1989

[33] R J Ruch S-J Cheng and J E Klaunig ldquoPrevention ofcytotoxicity and inhibition of intercellular communication byantioxidant catechins isolated fromChinese green teardquoCarcino-genesis vol 10 no 6 pp 1003ndash1008 1989

[34] D D Miller ldquoMineralrdquo in Food Chemistry O R Fennema Edpp 618ndash649 Dekker New York NY USA 1996

[35] P Flores J M Navarro C Garrido J S Rubio and VMartınezldquoInfluence of Ca2+ K+ and NO3- fertilisation on nutritionalquality of pepperrdquo Journal of the Science of Food and Agriculturevol 84 no 6 pp 569ndash574 2004

[36] K Finlay A Patoine D B Donald M J Bogard and P RLeavitt ldquoExperimental evidence that pollution with urea candegrade water quality in phosphorus-rich lakes of the NorthernGreat Plainsrdquo Limnology and Oceanography vol 55 no 3 pp1213ndash1230 2010

[37] W J Ng T S Sim S L Ong et al ldquoThe effect of Elodea densaon aquaculture water qualityrdquo Aquaculture vol 84 no 3-4 pp267ndash276 1990

[38] O M Usenko A E Sakevich and P D Klochenko ldquoThe par-ticipations of photosynthetic hydrobionts in urea degradationrdquoHidrobiologicheskii Jurnal vol 36 pp 20ndash29 2000

[39] A T Mokronosov Z G Ilinykh and N I ShukolyukovaldquoAssimilation of urea potato plantsrdquo Fiziologicheskii Rastenii(Soviet Plant Physiology) vol 13 pp 798ndash806 1966

[40] M J Krogmeier GWMcCarty and JM Bremner ldquoPhytotoxi-city of foliar-applied ureardquo Proceedings of the National Acadamyof Sciences of the United States of America vol 86 no 21 pp8189ndash8191 1989

[41] M DrsquoApolito X Du H Zong et al ldquoUrea-induced ROSgeneration causes insulin resistance in mice with chronic renalfailurerdquo The Journal of Clinical Investigation vol 120 no 1 pp203ndash213 2010

[42] M Maleva G Borisova N Chukina and M N V PrasadldquoUrea-induced oxidative damage in Elodea densa leavesrdquo Envi-ronmental Science and Pollution Research vol 22 no 17 pp13556ndash13563 2015

TribologyAdvances in

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Advances inPhysical Chemistry

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Analytical Methods in Chemistry

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Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of


NanotechnologyHindawiwwwhindawicom Volume 2018

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SpectroscopyAnalytical ChemistryInternational Journal of


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nom

ate

ria

ls


Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom


for improving blood sugar level and hypertension [10] Inaddition betalains have great importance for cardiovasculardiseases by lowering the level of homocysteine [11ndash13]

According to the foodstuffs not available sufficiently inthe soil in which they are produced in order to removethe lack and increase yield the fertilization has been used[14 15] With the correct fertilization applied in agriculturalapplication yield increasing up to 60 has been obtained[16 17] In addition to the usefulness fertilization hassome negativeness as regards environment In addition toexcessive and wrong fertilization application there are theaccumulation of some substances in the plant and occurrenceof some harmful gases as a result of evaporation with soilapplication of nitrogenous manure and especially occurrenceof greenhouse effect as a result of participation of nitrogenoxides Moreover in some fertilizersrsquo production this leadsto the formation of soil pollution of heavy metal ions such asCd2+ and the increasing of the amount of nitrogen in watersas a result of excessive irrigation As a result of the pollution innature it may increase risk of cancer in humans as well as therisk for plant and animal life It is proved that the fertilizersled to ovarian and prostate cancers and there was mortalityrate at 41 It is detected that some fertilizers had effects onnervous system and even mutation may occur [18 19] Dueto negativeness like these excessive fertilizations should beavoided

In this study we tried to investigate the effect of fertil-ization with different nitrogen forms (CAN urea AS andAN) and their doses (50 100 and 150 kg haminus1) on chemicalstructure and antioxidant or antiradical activities of beetroot(BT) (Beta vulgaris L var conditiva Alef)

2 Materials and Methods

21 Chemicals 221015840-Azino-bis(3-ethylbenzothiazoline-6-sul-fonic acid) (ABTS) neocuproine (29-dimethyl-110-phe-nanthroline) riboflavin methionine nitroblue tetrazolium(NBT) 11-diphenyl-2-picrylhydrazyl (DPPH) 3-(2-pyridyl)-56-bis(4-phenyl-sulfonic acid)-124-triazine (ferrozine) 120572-tocopherol linoleic acid gallic acid quercetin Folin-Ciocalteu reagent and trichloroacetic acid (TCA) were pur-chased from Sigma-Aldrich GmbH (Steinheim Germany)The other chemicals were obtained fromMerck

22 Beetroot (BT) (Beta vulgaris L var conditiva Alef)The Growth of Plant Samples The field experiment wasconducted during MayndashOctober of 2015 in Erzurum TurkeySeeds for beetroot (Beta vulgaris L var conditiva Alef cvldquoBikoresrdquo) tested in this study were provided by the MetgenSeed Corporation (Istanbul Turkey) The soil of the experi-mental area was clay loam texture (clay 3546 silt 3499and sand 2955) ustorthent great soil group with neutralpH (756) and EC 315 120583mhos cmminus1 It had 187 organic mat-ter 2430 cmol kgminus1 Ca 235 cmol kgminus1Mg 124 cmol kgminus1 K027 cmol kgminus1Na 4067mg kgminus1 P and 0083 total N Seedswere sown on plots of 5m2 in field on 20 May in 2015 inrows 230 cm long at a separation between rows of 40 cmand between plants of 20 cm The plants were thinned afterthe emergence when they formed 4-5 true leaves Irrigation

was on a need basis about twice a week after emergenceExperimental plots were kept weed-free using hand weeding

The plots were fertilized with different doses of nitrogenforms Four forms of nitrogen fertilizer [urea (46 N)calcium ammonium nitrate (26 N) ammonium nitrate(33 N) and ammonium sulfate (21 N)] and three doses ofthem (50 100 and 150 kg haminus1) were appliedThe same P dose(100 kg haminus1 P

2O5) was the accepted usual dose according

to Swiader et al [20] and Kaymak et al [21] reports whichapplied all fertilized plots All of the P

2O5and half of the

nitrogen fertilizer were applied uniformly prior to plantingonto soil surface by hand and incorporated The remaininghalf of the nitrogen was given 20 days after emergence [21]Plots not exposed to nitrogen fertilizer served as controlBeetroot plots were harvested on 9 October 2015 accordingto the suggestions of Seed Corporation for tested cultivar andthey were stored at +4∘C until use

23The Preparation of LyophilizedWater and Alcohol Extractsof Beetroot (BT) (Beta vulgaris L var conditiva Alef) 100 gbeetroot (BT) (Beta vulgaris L var conditiva Alef) wasweighted and split with blender device for each treatmentThen samples were divided into two parts In the first part100mL purified water was added to the split sample It wasextracted at the room temperature for a night Then it wasfiltered and the operation was repeated After the extractswere combined they were filtered into filter paper then thefiltrates were taken to balloons and frozen in deepfreezeThe frozen extracts were lyophilized under the pressure of50mm-Hg until it dried in lyophilizer After alcohol wasadded to the second part of split beetroot cells extracted itwas filtered and removed by evaporator dissolver

24 Assigning of Total Amount of Phenolic Compound Theamount of phenolic compounds available in lyophilizedwater and alcohol extracts of BT roots for all treatments(0 50 100 and 150 kg haminus1) of all nitrogen sources wasdetermined totally by using Folin-Ciocalteu reactivity [22]Gallic acid was used as a standard and standard graphicwas prepared 1000 120583g extract was taken from stock solutionand it was taken into a measure container and volume wascompleted to 23mL by means of distilled water 05mLFolin-Ciocalteu reagent was added to the mixture and itwas incubated for three minutes and then 15mL 2 (wv)Na2CO3was added Then after the samples were stirred at

the room temperature for two hours absorbances at 760 nmwere determined against the blend consisting of distilledwater Gallic acid equivalent amount accounting for obtainedabsorbance values was calculated by help of the equationobtained from standard graphic The results obtained weregiven as gallic acid equivalent (GAE)

25 The Determination of the Amount of Total FlavonoidCompounds Total flavonoid amounts in lyophilized waterand alcohol extracts of BT roots for all sources of nitrogendoses were determined according to the method by Parket al [23] for all doses (0 50 100 and 150 kg haminus1) ofall nitrogen sources So 43mL ethanol solution including1000 120583g extract medium 01mL 1M CH

3COOK and 01mL





3Fe(CN)



3(01




4buffer




























Water

0

05

1

15

2

Abso

rban

ce (7

00 n

m)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

Abso

rban

ce (7

00 n

m)














(b)



Water

0

04

08

12Ab

sorb

ance

(450

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol


0

04

08

12

16

Abso

rban

ce (4

50 n

m)













(b)







(1)






Water

0

05

1

15

2

25

3Ab

sorb

ance

(517

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

3

Abso

rban

ce (5

17 n

m)














(b)







Water

0

03

06

09Ab

sorb

ance

(734

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

03

06

09

Abso

rban

ce (7

34 n

m)














(b)





４３+∙ + ４３+∙ + +∙ (2)










4 Conclusion


Disclosure




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








3Fe(CN)



3(01




4buffer




























Water

0

05

1

15

2

Abso

rban

ce (7

00 n

m)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

Abso

rban

ce (7

00 n

m)














(b)



Water

0

04

08

12Ab

sorb

ance

(450

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol


0

04

08

12

16

Abso

rban

ce (4

50 n

m)













(b)







(1)






Water

0

05

1

15

2

25

3Ab

sorb

ance

(517

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

3

Abso

rban

ce (5

17 n

m)














(b)







Water

0

03

06

09Ab

sorb

ance

(734

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

03

06

09

Abso

rban

ce (7

34 n

m)














(b)





４３+∙ + ４３+∙ + +∙ (2)










4 Conclusion


Disclosure




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




















Water

0

05

1

15

2

Abso

rban

ce (7

00 n

m)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

Abso

rban

ce (7

00 n

m)














(b)



Water

0

04

08

12Ab

sorb

ance

(450

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol


0

04

08

12

16

Abso

rban

ce (4

50 n

m)













(b)







(1)






Water

0

05

1

15

2

25

3Ab

sorb

ance

(517

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

3

Abso

rban

ce (5

17 n

m)














(b)







Water

0

03

06

09Ab

sorb

ance

(734

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

03

06

09

Abso

rban

ce (7

34 n

m)














(b)





４３+∙ + ４３+∙ + +∙ (2)










4 Conclusion


Disclosure




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





Water

0

04

08

12Ab

sorb

ance

(450

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol


0

04

08

12

16

Abso

rban

ce (4

50 n

m)













(b)







(1)






Water

0

05

1

15

2

25

3Ab

sorb

ance

(517

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

3

Abso

rban

ce (5

17 n

m)














(b)







Water

0

03

06

09Ab

sorb

ance

(734

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

03

06

09

Abso

rban

ce (7

34 n

m)














(b)





４３+∙ + ４３+∙ + +∙ (2)










4 Conclusion


Disclosure




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





Water

0

05

1

15

2

25

3Ab

sorb

ance

(517

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

05

1

15

2

25

3

Abso

rban

ce (5

17 n

m)














(b)







Water

0

03

06

09Ab

sorb

ance

(734

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

03

06

09

Abso

rban

ce (7

34 n

m)














(b)





４３+∙ + ４３+∙ + +∙ (2)










4 Conclusion


Disclosure




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





Water

0

03

06

09Ab

sorb

ance

(734

nm

)




Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

Ｂminus1

(a)

Alcohol

0

03

06

09

Abso

rban

ce (7

34 n

m)














(b)





４３+∙ + ４３+∙ + +∙ (2)










4 Conclusion


Disclosure




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls










4 Conclusion


Disclosure




References


















































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




antioxidant and antiradical activity of beetroot

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