TheEffectofNitrogenandBlueRedLightRationon

AnthocyaninContentofRedCrispLettuce(Lactucasativa
var.crispa)

AhmadFaizal,KatarinaIkaKurniawati,RinaMSianipar,FeryFebriansyah
SchoolofLifeSciencesandTechnology,InstitutTeknologiBandung
JalanGanesa10,Bandung40132,Indonesia

afaizal@sith.itb.ac.id

Abstract. Red crisp lettuce (Lactuca sativa var. crispa) contains anthocyanin and other
phenoliccompoundderivativesthatresponsibleforitsantioxidantproperties.Therefore,recent
efforthasbeenallocatedtoevaluatetheeffectofnitrogenandblueredlightratioonredcrisp
lettuceanthocyanincontentanditsantioxidantactivity.WecombinedblueandredLEDlight
withdifferentratioredtoblue1:2,1:1,and2:1.LettucewasgrowninHoaglandmediumusing
deep water hydroponics system supplemented by different concentration of nitrate.
Subsequently,plantsweresubjectedtodifferentcompositionsandcombinationsofbothblue
andredLEDlight.AnthocyanincontentwasmeasuredbyusingdifferentialpHmethod,while
theactivityofantioxidantwasdeterminedasH 2O2scavengingactivity.Theresultshowedthat
the increasing of nitrate concentration positively correlated to higher lettuce anthocyanin
content and their respective antioxidant activities. In addition, similar results were also
obtainedupontreatmentwithhigherratioofbluetoredlightasmuchas5foldcomparedto
control. Therefore, we concluded that the production of anthocyanin in red crisp lettuce
requiresfinetuningoflightandnutrientconcentration.

Keywords:Lactucasativavar.crispa;nitrate;blueredlight;anthocyanin;antioxidant

1.Introduction

Redlettuce(Lactucasativavar.crispa)originatedfromItaly,ithasfringedandcrinkledleavesthat
aretingeddeepredattheirends,withafairlytender,yetcrisptexture.Lettuceismostoftenusedfor
saladsalthoughitisalsoseeninotherkindsoffood,suchassoups,sandwichesandwraps;itcanalso
begrilled.InIndonesia,therearefewerredlettucecultivationcomparedtogreenlettuce,thus,itis
wellknowntopeople1.

Redlettucecontainsanthocyanin,apigmenwhichgiveredpigmenttotheleaves.Anthocyaninisa
polyphenol derivatives. In red lettuce, the dominant anthocyanin is in the form of cyanidin3O
glucosidewhichisverybeneficialforhealth,especiallytheantidiabeticproperties 2. Redlettuceis
knowntocontaintotalantioxidantactivityasmuchas3.5foldhigherthangreenlettuce 3.Antioxidant
is a compound that prevent and inhibit other molecular oxidation by preventing the initiation or
propagationofoxidativereaction4,5.Mostnaturalantioxidantsuchaspolyphenol,shownawiderange
ofbiologicaleffects,includingantibacteria,antivirus,antiinflammation,antialergic,antitrombotic,and
vasodilatory.Somestudiesalsoshownthatredlettucehasapotentialtoreducetheriskcardiovascular
diseases6.

Recently,redlettucecultivationisdoneinoutdoor(outdooragriculture),butthisissusceptibleto
diseaseandpests. Variousindooragriculturaltechnologieshavebeendevelopedrecently.Oneof
technology that could substitute outdoor agriculture is indoor agriculture with close system
hydroponicsequipedwithLEDsasourceoflighting.LEDshavebeenusedassourcesofartificial
lighting in closedtype plant production systems, where environmental conditions are controlled,
allowingcropstobeproducedthroughouttheyearregardlessofexternalweatherconditions.

Environmentfactorsuchasnutritionandlightingreallyaffectstheplantgrowthandchemicalquality
insideit.Nitrogenisanutritionthatisrequiredbyplantinhighamountandanimportantcomponen
forprotein,nucleicacid,chlorophyll,andotherorganicmoleculesformation.Inaddition,nitrogenalso
holdanimportantroleinplantgrowth 7andproductionofsecondarymetabolitespolyphenol 8.While
lightingisanimportantfactorforplantgrowth,asitisusedforsourceofenergyforphotosynthesis.

2.MaterialandMethods

Plant material and growth condition

Seeds of red lettuce crisp (Lactuca sativa var. crispa) were germinated in rockwool cubes (2.5 cm2.5 cm2.5
cm) and acclimatized using Hoagland medium half strength for 28 days since seedling. Then, plants were grown
in Hoagland medium using deep water hydroponics system supplemented by different concentration of nitrate.
Other plants were subjected to different compositions and combinations of both blue and red LED ligh t for 28
days after cultivating. The Hoagland solution was adjusted to pH 66.5 every 3 days. The air temperature and
relative humidity for all treatments were respectively maintained throughout the experiment at 27C, 59%, , and
4000-6000 lux.

2.2Nitratetreatment
TherearefourvarietiesofnitrateconcentrationinHoaglandmedium.Theconcentrationis532.79
mg.L1(1/2N,control);853.67mg.L 1(1N);1918.14mg.L1(2N);and3981.78mg.L1(3N).Lettuces
wassupplementedbythesedifferentconcentrationnitratefor28daysaftercultivating.Then,lettuce
waslocatedunderwhiteLED(4000600lux)withphotopheriod12/12hday/night.

2.3Lighttreatment
Thecompositionofred(R)andblue(B)LEDis0:0(whiteLED);1:2;1:1;and2:1. Lettucewas
locatedundertheseLEDs(40006000lux)for28daysaftercultivationgwithphotoperiod12/12h
day/night.

2.4Analysisofanthocyaninandactivityantioxidant

2.4.1Extractionofanthocyanin.LettuceswereextractedbythemethodLestario 9.Lettucesweredried
inadryingovenfor4daysat40C.Then,lettucesweregroundwithamortarandpestleandextracted
withacifiedmethanol(1%HCl)(1:4,w/v)withshakingfor24hindarkcondition.Theextractswere
filtratedbyWhatmanfilterpapernumber1andusedtoanalyzeofanthocyanininredcrisplettuce.

2.4.2Analysisofanthocyanin.AnthocyanincontentwasmeasuredbyusingdifferentialpHmethod 10.
Extractofanthocyaninwasdilutedto1/5(v/v)withpH1.0buffer(KCl)andpH4.5buffer(NaOAc)
and incubated for 15 minutes at room temperature. Then, the absorbance was measured by
spectrophotometer at both 520 and 700 nm. Total anthocyanin concentration anthocyanin was
expressed as a g of cyanidin3gluycocide equivalents per g of dry weight (DW) lettuce and
calculatedbyEq.(1)
Totalanthocyaninconcentration(mg/gDW)=
A MW DF 10 6 (1)
L DW

WhereA= [(A520A700)pH1 (A520A700)pH 4.5];MW(molecularweight)=449,2g/molfor

cyanidin3glucoside;DF=dilutionfactor;L:pathlength; =26,900molarextinctioncoefficient;
DW=dryweightoflettuce(g);and106=factorforconversionfromgtog.

2.4.3 Hydrogen peroxide scavenging capacity. The ability of the extract to scavenge hydrogen
peroxide was determined by the method Ruch 11. A solution of hydrogen peroxide (40 mM) was
preparedinphosphatebuffer(pH7.4).Extractofanthocyanin(100g/ml)indistilledwaterwasadded
toahydrogenperoxidesolution(0.6ml;40mM).Theabsorbanceofhydrogenperoxideat230nm
was determined after 10 minutes against a blank solution containing phosphate buffer without
hydrogenperoxide.Ascorbicacidwasusedasarelativecomparison.Thepercentageofhydrogen
peroxidescavengingwascalculatedbyEq.(2).
%Scavenged[H2O2]=[(AcAs)/Ac]100(2)
whereAc=theabsorbanceofsolutionofphosphatebufferaddedhydrogenperoxide;As=absorbance
ofextract.

2.5Staticticalanalysis.Experimentalresultsareexpressedasmeans SD.Allmeasurementswere
replicatedfivetimes.Thedatawereanalyzedbyananalysisofvariance(p<0.005).Thisanalysisused
Duncansmultiplerangetest.

3.ResultandDiscussion

3.1AnthocyaninContent

Effect of nitrate and light treatment on anthocyanin content of lettuce is showed by Fig.1. This
study shows that every treatment resulting in an increase of anthocyanin content. In nitrogen
treatment,anthocyanincontentincreasedasthenitrogenconcentrationhigher.Thiscouldhappened
becausethereisphenylalanineaccumulation,whichisaprecursorinphenolicmetabolicpathway 12.In
red lettuce, a dominant phenolic compound, cyanidin3glucoside. Previous study shows that the
highernitrateconsumptionresultingindecreasinganthocyanincontent 13.Thedifferentresultmightbe
caused by different plant used and the amount of nitrogen given. In this study, a certain range
concentrationofnitrogencouldbetoxictotheplant,iniatiatingaplantdefencemechanism.
Inthelight treatment,thehigher blueratio,thehigher anthocyanincontentinredlettuce. Inthe
treatmentwithhighestredratioshowsanonsignificantresultcomparedtocontrol.Thiscausedby
bluelightregulatethegenexpressionwhichcodethechalconesynthaseenzyme(CHS)14.
CHSisakeyenzimincatalyzingthefirstreactioninanthocyaninbiosynthesis 15.Bluelightirradiation
leadtohigherCHSproduction,thushigheranthocyaninconcentration 16.

3.2 Antioxidant activity

Effect of nitrate and light treatment on activity antioxidant lettuce is showed by Fig.2. In this
study, antioxidant activity in red lettuce show an increase in every treatment, eventhough the
activity still lower than the ascorbic acid antioxidant activity used as standard in this study. In
the treament of nitrate, the antioxidant activity to H2O2 is significantly different between three
other treatment and control. The different in antioxidant activity could be caused by
increasing anthocyanin content, as well as higher nitrate concentration in medium. In light
treatment, antioxidant activity in 2B : 1R, 1B : 1R, 1B : 2R, and control are not significantly
different. The total anthocyanin content could increase with the irrradiation of red and blue
light, but in term of antioxidant activity is not different, because, it is basically the same kind
of antioxidant tested.

180
a
160

140
b
120
b
100
Anthocyanin content (g/g DW) c
c 80

60
d
d 40

20

Control 1N 2N 3N 2B : 1R 1B : 1R 1B : 2R

Figure1.AnthocyanincontentonLactucasativavar.crispawithnitrateandlighttreatment.Values
designatedbythedifferentlettersaresignificantlydifferent(p<0.05).

40

35

30

25

% H2O2 scavenged 20
15

10

Control 1N 2N
3N 2B : 1R 1B : 1R
1B : 2R Ascorbic acid
Figure2.ActivityantioxidantonLactucasativavar.crispawithnitrateandlighttreatment.Values
designatedbythedifferentlettersaresignificantlydifferent(p<0.05).

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