Rev. bras. farmacogn. vol.27 número4

w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Impact

of

light

quality

on

flavonoid

production

and

growth

of

Hyptis

marrubioides

seedlings

cultivated

in

vitro

Rita

Cassia

N.

Pedroso

_,

_Núbia

_Angelica

_A.

_Branquinho

_,

_Alessandra

_C.B.A.M.

_Hara

_,

Alan

Carlos

Costa

_,

_Fabiano

_Guimarães

_Silva

_,

_Leticia

_P.

_Pimenta

_,

_Marcio

_Luiz

_A.

_Silva

_,

Wilson

Roberto

Cunha

_,

_Patricia

_M.

_Pauletti

_,

_Ana

_Helena

_Januario

a_{InstitutoFederaldeEducac¸ão,CiênciaeTecnologiaGoiano,CâmpusRioVerde,RioVerde,GO,Brazil} b_{NúcleodePesquisaemCiênciasExataseTecnológicas,UniversidadedeFranca,Franca,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2November2016 Accepted22December2016 Availableonline28March2017

Keywords:

Flavonoid

Hyptismarrubioides Invitroculture Lightquality

a

b

s

t

r

a

c

t

HyptismarrubioidesEpling,Lamiaceae,aspeciesfromBrazilianCerrado,hasbeenusedagainst gastroin-testinalinfections,skininfections,pain,andcramps.Herein,H.marrubioidesseedlingswerecultured invitrounderdifferentwavelengths(white,blue,green,red,andyellow)with50␮molm−2_s−1

irra-dianceanda16-hphotoperiod.After20and30daysofcultivation,shootlength,leafnumber,fresh mass,anddrymasswereevaluated.TheflavonoidrutincontentwasdeterminedbytheHPLC-DAD method.Theshootswerelongerinplantscultivatedunderyellow(16.603±0.790cm,1.8-fold),red (15.465±0.461cm,1.7-fold),andgreen(14.677±0.737cm,1.6-fold)lightsthanincontrolplantsexposed towhitelight(9.203±0.388cm).Thenumberofleavesincreasedinplantsexposedtored(23.425±1.138, 1.1-fold)andgreen(22.725±1.814,1.1-fold)lights,comparedtocontrolplants(20.133±0.827).Fresh (0.665±0.048g,1.2-fold)and dry (0.066±0.005g,1.3-fold) mass ofseedlingswere thehighestin seedlingsgrownunderredlight,comparedtoseedlingsgrownunderwhitelight(0.553±0.048and 0.028±0.004,respectively).However,rutinproductionwashigherunderwhite(0.308mgg−1_ofdry

weight)andbluelights(0.298mgg−1_{ofdryweight).Thus,redlightinducesplantgrowthandincreases}

leafnumberanddryweightininvitro-cultivatedH.marrubioides,whereasblueandwhitelightspromote thegreatestrutinaccumulation.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

The genus Hyptis, Lamiaceae, comprises about 300 species, widely distributed, occurring mainly in tropical regions of the AmericasandAfrica.HyptismarrubioidesEpling,commonlyknown asmint-of-field,isaspeciesoftheBrazilianCerradotraditionally usedtotreatgastrointestinalandskininfections,pain,andcramps (McNeiletal.,2011).ThepharmacologicalpotentialofH. marru-bioideshasbeenpreviously investigated,andmoststudieshave focusedon examination of thechemical composition of essen-tialoils, themaincomponentsof which arethesesquiterpenes caryophylla-4(14),8(15)-dien-5␤-ol,eudesma-4(15),7-dien-1␤-ol, caryophyllene oxide, and ␤-caryophyllene (Sales et al., 2007; McNeiletal.,2011).EssentialoilsofH.marrubioideshavealsobeen studiedtoinvestigatetheirpotentialforpestcontrolinagriculture, suchasthepreventionandcontrolof Asiansoybeanrust(Silva

∗ _{Correspondingauthor.}

E-mail:ana.januario@unifran.edu.br(A.H.Januario).

etal.,2012b)andtreatmentofColletotrichumtruncatum-infected soybeanseeds(Silvaetal.,2012a).

Theuseofmicropropagationtechniqueshasgreatadvantages intheproductionofhigh-qualityseedlings,astheyimprovetheir pharmacologicalpotential(RaoandRavishankar,2002)by ensur-ingthereproductionofidentical,high-qualityplants(Serafinietal., 2001),whichinturnenhancesthebiosynthesisofspecial metabo-lites(BhuiyanandAdachi,2003;Zhaoetal.,2005).However,the accumulationofsecondarymetabolitesinplantsisinfluencedby variousenvironmentalfactorssuchaslightquality,UVirradiation, temperature,irrigation,nutrientdeficiency,pathogenattack,and heavymetalstress(DixonandPaiva,1995;Winkel-Shirley,2002; KopsellandKopsell,2008).

Lightisakeyabioticelicitorinplantsthataffects,directlyor indirectly,growthanddevelopmentofplants,mainlyofthose dis-tributedinhigh-latitudeareas(MoriniandMuleo,2003;OuYang etal.,2015).Plantresponsesdonotdependonlyontheabsenceor presenceoflightbutalsoonthevariationinlightquality(Felippe, 1986).Theactionoflightonplantsoccursmainlyintwoaspects: first,thelightsourceprovidestheenergyrequiredbytheplantfor

http://dx.doi.org/10.1016/j.bjp.2016.12.004

photosynthesis,especiallyredandbluelight,andsecond,asa sig-nalreceivedbyphotoreceptorsitregulatesgrowth,differentiation, andplantmetabolism(Wangetal.,2001).

Lightisoneofthemostsignificantenvironmentalfactors affect-ingtheaccumulationofflavonoidsinplants(Huché-Thélieretal., 2016). However, the biosynthesis of flavonoids in response to lightqualityneedtobebetterunderstood,especiallyin invitro culture.

Studiesinvolvingtheinfluenceoflight ontheproduction of secondarymetabolitesandgrowthparametersinH.marrubioides seedlingscultivatedinvitroarenon-existent.Inapreviousstudy, wehaveevaluatedthephytochemicalprofileof H.marrubioides microplantsinoculatedwithisolatesofbacteriaand endophytic fungi(Vitorinoetal.,2013).PhotosyntheticstudiesinH. marru-bioidesinvitrocultureinvolvingmeasuringgasexchangewerealso conductedbyourresearchgroupandtheirradianceeffects,flow rate,andairhumidityparameterswereinvestigated.Inaddition, thechlorophyllafluorescenceand chloroplastidicpigment con-tentwerealsoassessed(Costaetal.,2014).Hence,theaimofthe presentstudywastoassesstheeffectoflightqualityongrowthof H.marrubioidesseedlingsinvitroandtheirflavonoidproduction. Thisstudyfurthersourunderstandingofthefactorsthatpromote thegrowthandincreasetheresistanceofthismedicinalspecies, throughtheproductionofflavonoids.

Materialsandmethods

Plantmaterialandinvitrocultivation

Seedlings previously established via in vitro germination of Hyptis marrubioidesEpling,Lamiaceae,seedssuitable for inocu-lation obtained onthe experimental field of the Laboratory of PlantTissue Cultureof theInstitutoFederal deEducac¸ão, Ciên-ciaeTecnologiaGoiano,CampusRioVerde.Avoucherspecimen (HRV71) hasbeen deposited at the Herbarium of the Institute (HerbariumHRV).

Theseedsweredisinfectedwith0.2%Bendazol(carbendazim) and0.2%Alterno(tebuconazole)for1h,followedbytreatmentwith 1%sodiumhypochlorite for30min,andthenrinsedthrice with steriledistilledwater.Seedsweregerminatedandmaintainedon Murashige&Skoogmedium(MurashigeandSkoog,1962) supple-mentedwith30gl−1_{ofsucroseandsolidifiedwithagarat3.5gl}−1_; thepHwasadjustedto5.8beforeautoclaving.Thecultureswere incubatedinagrowthchamberfor30daysat50␮molm−2_s−1 pho-tosyntheticallyactiveradiation(PAR),atanaveragetemperature of23±1◦_{C,andaphotoperiodof16h.Thereafter,theseedlings}

weresub-culturedonthesamemediumandincubatedinagrowth cabinetfor10daysat50␮molm−2_s−1_PAR,23±1◦_C,and16-h

pho-toperiod.Allexperimentswerecarriedoutinglassflaskscontaining 50mlofsemisolidmediumwithfiveseedlingsperflaskwithatotal offortyseedlings.

Lightconditions

After 10 days, the cultures were transferred to a cabinet andexposed tocontinuingirradiationofdifferentlightspectra: white(300–750nm),blue(400–490nm),green(490–560nm),red (600–700nm),andyellow(560–590nm).Thespecificlight condi-tionsweremaintainedusingTP40Wlamps(Taschibra® Indaial, SantaCatarina,Brazil),atalightintensityof50␮molm2_s−1 _and aphotoperiodof16h.Thecultureswereevaluatedafter20and 30daysoflightexposure.Culturesgrownunderwhitelightwere usedasacontrol.Forweightmeasurements,thewholeseedlings withoutrootswasconsidered.

Chemicalanalysis

The seedlingswere driedat 35◦_{C in a forcedair circulation}

ovenand thedrybiomass(200mg)fromtheinvitrocultureof H.marrubioides wasextractedwith4ml methanol ofhigh per-formanceliquid chromatography(HPLC)gradeusingultrasound bath(30min).Thesampleshadbeenpreviouslyfilteredthrough a0.2-␮msyringefilterwithhydrophilicPTFEmembrane (Advan-tec,Dublin,CA,USA)andtransferredinto1-mlHPLCvials.These procedureswereconductedintriplicate.

Forthequantitativeanalysis,solutionsweredoneinHPLCgrade methanoltoobtainsolutionscontaining0.500,0.250,0.125,and 0.063mgml−1 _{rutin.Eachstandardsolutionwasinjectedin} trip-licate. The calibration curve wasconstructed to determine the linearityofthemethodbyplottingthepeakareaversusthe con-centrationofthesubstanceinmgml−1_{.Thequantitativeanalysis} byHPLCwithdiode-arraydetection(DAD)wascarriedoutona ShimadzuProminenceLC-20AD binarysystemequippedwitha DGU-20A5 degasser, an SPD-20A series diode arraydetector, a CBM-20A communicationbus module,anSIL-20A HT autosam-pler,andaCTO-20Acolumnoven(Shimadzu,Kyoto,Japan).The analyseswereconductedonaGeminiODScolumn(250×4.6mm, 5␮m; Phenomenex,Aschaffenburg, Germany) equippedwith a pre-columnwiththesamematerialunderthefollowingconditions: injectionvolume setat20␮l,flow rateof 1.0mlmin−1_{, mobile} phasewasCH3OH/H2O/HOAc(5:94.9:0.1,v/v/v)deliveredina lin-eargradientuntil100%CH3OHin30min,10minat100%CH3OH, and20mintoreturntotheinitialconditions.TheUVdetectionwas setat254nmand40◦_C.

MethanolusedintheexperimentswasHPLCgradeanditwas obtainedfromJ.T. Baker (AvantorPerformance Materials, Cen-ter Valley, PA, USA). Ultrapure water was obtainedby passing redistilledwaterthrougha Direct-QUV3 systemfromMillipore (Billerica,MA,USA).Theflavonoidrutinusedasexternalstandard wasacquiredfromthestandardbankoftheNaturalProductsGroup oftheUniversidadedeFranca.

Statisticalanalyses

Thestatisticalanalysiswasperformedin Sisvar5.3software (Ferreira,2011).Theexperimentwasconductedinacompletely randomizeddesignwithfourreplications.Themulti-factorial anal-ysisofvariancefollowedbyTukeymultiplecomparisontestswere usedforstatisticalcomparisons(p≤0.05).

Results

Seedlingsgrowth

ThemeasuredgrowthparametersofH.marrubioidesseedlings cultivated in vitro under different wavelengths showed signif-icant difference (Table 1). During the 20-day cultivation, the seedlingsexposedtoredlightshowedthehighestshootlengths (10.175±0.669cm), which was 1.69-fold higher than the cor-responding lengths of control seedlings (6.035±0.511cm). In addition,after30daysofirradiationwithgreen,red,andyellow lights, thelengthof the shoots increased1.8-fold compared to seedlingsexposedtowhitelight.Thenumberofexpandedleaves wasnotaffectedbylightconditionsafter20daysoftreatment; however,after30daysofculture,greenandredlightpromoted themosttheincreaseinthenumberofleaves(22.725±1.814and 23.425±1.138,respectively).

Table1

ShootlengthandaveragenumberofexpandedleavesofHyptismarrubioidesseedlingssubmittedto20and30daysoflighttreatment.

Lighttreatments Evaluationtime(days) Evaluationtime(days)

20 30 20 30

Averageshootlength(cm) Averageleafnumber

White 6.035a_Bb±0.511b _{9.203Ca±0.388 12.475Ab±0.927 20.133Ba±0.827}

Blue 8.400Ab±0.453 13.030Ba±0.865 13.143Ab±0.667 17.750Ba±1.293 Green 9.900Ab±0.823 14.677Aa±0.737 13.514Ab±0.676 22.725Aa±1.814 Red 10.175Ab±0.669 15.465Aa±0.461 12.600Ab±1.223 23.425Aa±1.138 Yellow 9.871Ab± 0.462 16.603Aa± 0.790 11.143Ab± 1.166 20.067Ba± 0.935

CV(%) 15.46 18.87

Freshweight(g) Dryweight(g)

White 0.311Ab± 0.059 0.553Aa± 0.048 0.028Ab± 0.003 0.052Ba± 0.004 Blue 0.306Ab±0.024 0.549Aa±0.042 0.033Ab±0.002 0.057Ba±0.004 Green 0.334Ab±0.046 0.534Aa±0.053 0.034Ab±0.003 0.053Ba±0.004 Red 0.404Ab±0.067 0.665Aa±0.048 0.035Ab±0.004 0.066Aa±0.005 Yellow 0.246Ab±0.018 0.426Aa±0.025 0.025Ab±0.001 0.045Ba±0.002

CV(%) 29.77 22.14

a_{MeansfollowedbythesameuppercaseletterinthecolumnandlowercaseletterintherowdonotdiffersignificantlyaccordingtotheScott-Knotttestat5%probability.}

b _{Mean±standarderror.}

CV,coefficientofvariation.

Table2

Accumulationofrutin(mgg−1_{dryweight)inHyptismarrubioidesseedlings}

irradi-atedwithwhite,blue,green,red,andyellowlightsfor20and30days.

Lighttreatment Evaluationtime

Rutincontent(20days) (mgg−1_dryweight)

Rutincontent(30days) (mgg−1_dryweight)

White 0.243a_A±0.012b _{0.308A±0.012}

Blue 0.084D±0.004 0.298A±0.007 Green 0.101C± 0.002 0.198C± 0.002 Red 0.105C± 0.006 0.192C± 0.002 Yellow 0.126B±0.002 0.262B±0.006

a_{Meansfollowedbythesameletterdonotdiffersignificantlyaccordingtothe}

Tukeytestat5%probability.

b _{Mean±standarderror.}

seedlings increased1.27-fold (0.066±0.005g) compared to dry weightofthecontrolseedlings(0.052±0.004g).

Accumulationofrutinflavonoid

Methanolextracts ofrutin obtainedfrom seedlings cultured under different lights for 20 and 30 days was quantitatively assessedusingHPLC-DADanalysis(Table2).Theresponseofthe UVdetectorat254nmwaslinearfrom0.063a0.500mgml−1_for rutin.Theobtainedregressionequationwasy=4.0×107_x−69,442 withacorrelationcoefficient(R2_{)of0.9998.After20daysof} cul-ture,theaccumulationofrutininallsampleswaslowerthanthat inthecontrol(0.243mgg−1_{dryweight(DW));thelowestamount} ofrutinwasdetectedinplantsexposedtobluelight(0.084mgg−1 DW).However,after30daysoftreatment,theamountofrutinin seedlingsexposedtobluelight(0.298mgg−1_DW)wasnot signif-icantlydifferenttothatin controlseedlings(0.308mgg−1 _DW), whereasitscontentinseedlingsexposedtogreen(0.198mgg−1 DW),red(0.192mgg−1_{DW),andyellow(0.262mgg}−1_DW)lights was reduced significantly compared to that in control plants. Overalltheresultsdemonstratedthattherutincontentin micro-propagatedplantswaslowerthanthatfoundinwildplantsofH. marrubioides(1.259mgg−1_{DW)obtainedinpreviousstudies} con-ductedbyourresearchgroup.

Discussion

Lightqualityhasimportanteffectsonplantgrowthand develop-ment,especiallyforphotosynthesisandphotomorphogenesis,the twoprocessesthat,throughsignalsreceivedbythephotoreceptors, regulateplantgrowth,differentiation,andmetabolism(Guoetal., 2007).Plantgrowthcanbeaffectedbyendogenoushormonelevels interactionswithlightquality.Theinteractionoflightqualitywith planthormonepathwaysinregulationgrowthofvegetalspecies includesgibberellins(GAs),auxinsbeingthemostcommonisthe indoleaceticacid(IAA),cytokinins(CKs),andabscisicacid(ABA) (OuYangetal.,2015).

AnotherstudyfoundthatthelevelsofendogenousCKs,IAA,and GAsaremarkedlyincreasedinresponsetolow-intensityredto far-redlightatlowornormalPAR,promotingthegrowthofhypocotyls inHelianthusannuus(Kurepinetal.,2007).

Transcriptome analysis of Norway spruce (Picea abies) by OuYang and collaborators revealed that red and blue light are associated with plant growth and developmentand phytohor-monemetabolism(OuYangetal.,2015).Inaddition,theyreported thatbluelightpromotesIAAaccumulationandphenylpropanoid biosynthesis.Incontrast,redlightaffectsstemgrowthby regulat-ingbiosynthesisofGAs.Inlinewiththeseresults,Thweetal.(2014) reportedthatirradiationwithbluelightincreasespolyphenol con-tentinlettuceandinducestheproductionofrutinandcyanidin 3-O-rutinosideinFagopyrumtataricum.

The evaluation of growth parameters in H. marrubioides seedlingsculturedinvitroinourstudyshowthatredlightalmost doubledthelengthofshootsandincreasedthenumberofleaves anddrybiomasscomparedtothecontrol.Concerningthe produc-tionofsecondarymetabolites,wefoundthatblueandwhitelight favoredthebiosynthesisoftheflavonoidrutin.

Jeong et al. (2012) studied the influences of four differ-ent light-emitting diode lights on flowering and polyphenol variationsintheleavesofchrysanthemum(Chrysanthemum mori-folium).Theluteolin-7-O-glucoside,luteolin-7-O-glucuronideand quercetagetin-trimethylethershowedhighestyieldundergreen light.Ontheotherhand,dicaeoylquinicacidisomer,dicaeoylquinic acidisomer,naringenin,andapigenin-7-O-glucuronidewere great-est under red light. Taulavuori et al. (2016) evaluated the differences in synthesis offlavonoids and phenolic acids under increasing periodsof enhanced blue light in leaves red lettuce andbasil.Flavonoidsandphenolicacidswerefoundtobespecies dependent. Dicaffeoyltartaric acidwas the majorcompound in lettuce while rosmaric acid was the prodominant compound inbasil.

Theseexamplessuggestthatthepolyphenolproductionis influ-encedbylightqualitycomposition.

Ourresultsonthegrowthandmetabolismofinvitro-cultured seedlings of H. marrubioides are in accordance with the data describedbyOuYangetal.(2015)andThweetal.(2014)aswell thedatareportedforotherplantspecies(Vreugdenhiletal.,1998; Kvaalen andAppelgren, 1999;Leal-Costaet al.,2010;Ouzounis etal.,2015).

In summary, we concludethat for the invitro cultureof H. marrubioides light quality is an important parameter that can be utilized to optimize seedlings growth and accumulation of rutin,inparticulartheredandbluelights,whicharebeneficialto growthandrutinmetabolism,respectively.Therefore,ourresults may be useful as theoretical basis for studies related to qual-itycontrolofgrowthandproductionofsecondarymetabolismof H.marrubioides.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

RCNPandNAABperformedtheinvitroexperiments,ACBAMH contributed to the supervision of laboratory work,and critical reading of the manuscript, RCNP and LPP contributed to the chromatographicanalysis,ACC,FGS,MLAS,WRC,PMPandAHJ con-tributedtotheanalysisofexperimentaldataandcriticalrevision. Allauthorshelpedinpreparingthepaperandapprovedthe sub-missionofthefinalversion.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsaregratefultoCNPq(Grant#305783/2012-2)and FAPESP(Grant#2011/00631-5)forfinancial support.Fundac¸ão de Amparo a Pesquisa do Estado de Goiás (FAPEG, Grant # 201310267000257)arealsoacknowledgedforfellowship.

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