Rev. bras. farmacogn. vol.25 número5

w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Evaluation

of

limonoid

production

in

suspension

cell

culture

of

Citrus

sinensis

Elisângela

Fumagali

Gerolino

_,

_Talita

_Perez

_Cantuaria

_Chierrito

_,

_Arquimedes

_Santana

_Filho

_,

Eliezer

Rodrigues

Souto

_,

_Regina

_Aparecida

_Correia

_Gonc¸

_alves

_,

_Arildo

_José

_Braz

_de

_Oliveira

a_{ProgramadePós-graduac¸ãoemCiênciasFarmaceuticas,DepartamentodeFarmacia,UniversidadeEstadualdeMaringá,Maringá,PR,Brazil} b_{LaboratoriodeQuímicadeCarboidratos,DepartamentodeBioquímica,UniversidadeFederaldoParaná,Curitiba,PR,Brazil}

c_{DepartamentodeAgronomia,UniversidadeEstadualdeMaringá,Maringá,PR,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5November2014 Accepted22May2015 Availableonline23June2015

Keywords: Citrussinensis Biotechnology Limonoidaglycones Limonin

Plantcellculture

a

b

s

t

r

a

c

t

Theuseofcellandplanttissueculturetechniquestoproduceeconomicallyimportantactivemetabolites

hasbeengrowing.Amongthesesubstancesaretotallimonoidaglycones,whichareproducedby“pera”

orange(Citrussinensis(L.)Osbeck,Rutaceae)andhavereceivedconsiderableattentionbecauseoftheir

anticanceractions.Themainobjectiveofthepresentstudywastoanalyzeandcomparethelevelsof

limonoidaglyconesinseeds,calluscultures(originatingfromseeds),calluscultures(originatingfrom

hypocotyls),cellsuspensionsfromhypocotylscells,andcellsuspensionsfromcotyledons.Thecell

cul-turesorC.sinensiswereobtainedbyinoculatingtwostrainsofcallusinMSmediumsupplementedwith

2.0␮M2,4-dichlorophenoxyaceticacid,7.0␮Mbenzylaminopurine,and3%(w/v)sucroseinthedark.

Thehighestconcentrationsoflimonoidaglyconethatwereobtainedwereobservedincotyledoncell

lines(240mg/100gdryweight)thatwereproducedonday21ofcultureandhypocotylcelllinesonday

7(210mg/100gdryweight).Explantsofdifferentoriginsunderthesamecultureconditionshaddifferent

limonoidaglyconecontent.Thepresentresultsmaysuggeststrategiesforenhancingtheproductivityof

biologicallyimportantlimonoidaglyconesandinvestigatingthecomplexpathwaysofthesesecondary

metabolitesinplanttissuecultures.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Biotechnologyusestechniquesandprocessesthatinvolve liv-ingorganismstoobtainspecificproductsand/ormodificationsthat increasetheproductionofchemicalsubstancesofinterestinless timeandlesscapitalinvestment(DaviesandDeroles,2014). Sec-ondarymetabolitesthatarefoundinplantsaregenerallyproduced inlowconcentrationscomparedwithprimarymetabolites. There-fore,differentstrategies,includinginvitroculturesystems,have beenextensivelystudiedtoincreasetheproductionofsecondary metabolitesinplants(Smetanska,2008;MuranakaandSaito,2010; Gilletal.,2013).Invitrocellculturesrepresentaninteresting alter-nativebecausesecondarymetabolitesofinterestareobtainedin acontrolledenvironmentthatisnotinfluencedbychangesin cli-mateorsoilconditions(Gonc¸alvesandRomano,2013;Collin,2001;

Fumagalietal.,2008).Plantsthataregrownintheirnaturalhabitat

generallyhavevaryingconcentrationsofcompoundsofinterest,

∗ Correspondingauthor.

E-mail:ajboliveira@uem.br(A.J.B.deOliveira).

depending onthe particular cropseason(Salmore and Hunter,

2001;Puricellietal.,2002;RalphsandGardner,2001).Moreover,

theirexploitationintheirnaturalenvironmentcancausegradual geneticerosion(SidhuandBel,1996).

Citrusplantsaregrownworldwide.Therearebasicallyfour com-mercialspeciesofCitrus,amongwhichCitrussinensis(L.)Osbeck, Rutaceae(sweetorange)isnotable.Variousmetaboliteshavebeen isolatedfromspeciesoftheCitrusgenus.Amongthemajor second-arymetabolitesareflavonoidsandlimonoids,andpectinsarethe primarymetabolites(Khaliletal.,2002;Berhowetal.,2000;Okwu,

2008).

Limonoidsarehighlyoxygenatedmodifiedtriterpenesthatare derivedfromaprecursorwitha4,4,8-trimethyl-17-furanylsteroid skeletonthatisbiosynthesizedbytheacetate–mevalonate path-way in Citrus (Hasegawa and Hoagland, 1977). All naturally occurringCitruslimonoidscontainafuranringthatisattachedto ad_{-ringatC-17andoxygen-containingfunctionalgroupsatC-3,} C-4,C-7,C-16,andC-17(Fig.1).Oxidativedegradationonthe C-17sidechainofeitheroftheseskeletonsresultsinthelossoffour carbonatomsand theformationof ␤-substitutedfuran.Further oxidationandskeletalrearrangementsinoneormoreofthefour

http://dx.doi.org/10.1016/j.bjp.2015.05.008

O

O

O

O O

O O

O O

O O

O O

H O

A B

C D

O

AcO

O O

O O O

H O O

2

3

5

4

6

1

O O

O O O

H HO HO₂C O

O

O O O

H O O

HO HO

OH

O O O

CO₂H

H

H H

H A

A' C

B D O

O

O

Fig.1.LimoninbiosynthesispathwayproposedbyBreskaetal.(2007).

rings(designatedA,B,C,andD;Fig.1)giverisetodifferent skele-tonsofCitruslimonoids.Fig.1showsthebiosyntheticpathwayof limonin(1),themostabundantaglyconeinCitrusspecies.

Limonoidshavepropertiesthatinhibittheformationof chem-ically induced tumors in the mouth, stomach,small intestines, colon,lungs,andskininexperimentalanimals(Berhowetal.,2000). Anotherinterestingpropertythathasbeendescribedforlimonoid aglyconeisitsantimalarialactivity(Khaliletal.,2002).

Callusculturesandcellsinsuspensionhavebeenusedtostudy thebiosynthesisofeconomicallyimportantsecondarymetabolites

(Gonc¸alvesetal.,2010),enablingthepropagationofcelllineages

thatcontainalterationsinbiosyntheticcapabilities.

Theproductionofdifferentcompoundsinplantsisgenerally mediatedbyenvironmentalfactorsthatvaryaccordingto physio-logicalconditionsandseasonalvariations(Gilletal.,2013).Thus, cellculturesensurecontrolledconditionsthatcircumvent environ-mentalchangesandallowustoguide,inacontrolledmanner,the synthesisofthesecompounds.

Onlyafewstudieshaveutilizedcallusand/orcellculturesofC. sinensistoobtainsecondarymetabolites.Forexample,Niedzetal.

(1987)soughttoproducevolatilecompoundsfromcalluscultures,

andEndoetal.(2002)usedacellculturethatwasobtainedfrom

embryogeniccallustoproduceCitruslimonoids.

Thepurposeofthepresentstudywastoevaluatetheinvitro productionoflimonoidaglycones usingnon-embryogeniccallus culturesandcellsinsuspensionthatoriginatedfromcotyledons andhypocotylsofBrazilian“laranja-pêra”oranges(Citrussinensis).

Materialandmethods

Instrumentationandgeneralprocedures

Absorption spectra in the infrared regionwere obtained on aKBrtabletinaFouriertransforminfraredspectrometer (FTIR-Bomen, model MB-100C26). The analysis was performed on a diskcomposedof1mgofsteepedsampleswith250mgKBr.The one-dimensional(1D)/two-dimensional(2D)1_{H and}13_Cnuclear

magneticresonance(NMR)spectrawereobtainedusingaVarian MercuryPlusspectrometerat300MHzwithCDCl3asthesolvent

andTMSastheinternalstandard.Thespectraofhigh-resolution masseswereobtainedusingBrukerDaltonicsequipment(model microTOF-QII-ESI-TOF).Theplatesthatwereusedforanalytical thin-layerchromatography(TLC)were60F254aluminumsilicagel

sheets(Merck).Thechromatoplatesweredevelopedusing ultravi-oletlight(254and 365nm)andasulfuricvanillin solution.The solvents (hexane, acetone,and acetonitrile) that were used for thepreparationoftheextractsandchromatographicfractionation wereofanalyticalgrade.

Plantmaterial,calluscultures,andcells

inoculatedinbottledMSculture(MurashigeandSkoog,1962) sup-plementedwith30gsucrose,2.0␮M2,4-dichlorophenoxyacetic acid(2,4-D), 7.0␮Mkinetin,and 0.8%(w/v)agarandincubated at28◦_{Cinthedarktoinducecallusformation.Aftergermination}

andseedlingdevelopment,thehypocotylsandsterilecotyledons wereusedasexplants.TheyweretransferredtoMSmedium sup-plementedwith 2.0␮M2,4-dichlorophenoxyacetic acid(2,4-D), 7.0␮Mkinetin,and0.8%(w/v)agarandthenincubatedat28◦_C

inthedarktoinducecallusformation.Afterward,totallimonoid aglyconesweremeasuredinthethird-generationsubculture,and thegrowthprofilewasstudiedinaliquidcellculturesuspension.

Establishmentofcellculturesinsuspension

Freshcallus(0.6g)weretransferredtoaflaskthatcontained 50mlofMSliquidmediumsupplementedwith2.0␮M2,4-Dand 7.0␮Mkinetinandthencultivatedat28◦_{Cinthedarkonanorbital}

shakerat120rotationsperminuteforfourweeks.Thesuspended cellswereremovedeverysevendays(intriplicate),weighed(fresh weight),andfrozenforsubsequentlyophilizationandverification ofthedryweightatintervalsof7,14,21,28,and35daysofculture. Thisprocedurewasperformedforthetwotypesofcallusthat wereinducedunderlaboratoryconditions:callusthatarosefrom hypocotylsandcallusthatarosefromseeds.

Obtaininglimonoidaglycones

Theisolationoflimonoidaglyconesoccurredafterextracting seedsfromthe“laranja-pêra”orange(C.sinensis,287.72g),which weredried,crushedbyturbolysis,andextracted,firstunderreflux inhexane(toeliminateoils)andtheninacetonefor4htoobtain limonoidaglycones.Theacetoneextractoftheseeds(AES;1.31g) was concentrated and resuspended in acetonitrileand filtered. Afterevaporationofthesolvent,weobservedcrystalformation. Thesamplewaspurifiedusingpreparativethin-layer chromatog-raphy(PTLC)usingCH2Cl2:acetone(9:1,v/v)asthemobilephase

insilicagel60,whichallowedisolationofthelimonoidaglycone limonin(1).Thestructureofthislimonoidwasdeterminedby1D and2D1_{H and}13_{CNMR,mass spectrometry,infrared}

spectros-copy, and comparisonswith theliterature(Khalil et al.,2002): limonin(1):IVmax(KBr)3488,2986,1758(lactone),1708(C O),

1391cm−1_.The1_Hand13_{CNMRdatawereinagreementwiththe}

literature(Khaliletal.,2002;EM-IES493,2071[M+Na+_],C 26H30O8

(calculatedmass,470.19).

O O

O O

O

O O

O

H H

1

A A'

B C

D 21

20 22 23

1 2 3

10

4

25

26 6

5 7 8 9 24 11

12 18

1317 16

15 14

Extractionoftotallimonoidaglyconesfromseedsandcallusand suspensionculturesofC.sinensis

CallusandsuspensionculturesofC.sinensiswerelyophilized andthensubjectedtoextractionbyrefluxinacetonefor4h(Ohta etal.,1993).Theacetonicextractswerefiltered,concentrated,and storedat−20◦_{Cuntilanalysis.}

Theseedshadpreviouslyundergoneextractionbyrefluxin hex-anetoremovetheoilandsubsequentlysubjectedtoextractionby refluxinacetonetoobtainlimonoidaglyconesextract(Ohtaetal., 1993).Afterevaporationofthesolvent,themasswasdetermined, andthesampleswerestoredat−20◦_{Cuntilanalysis.}

Quantificationoftotallimonoidaglycones

Total limonoid aglycones were quantified in a spectropho-tometer(VarianCary100UV-VIS).Afterdilutionoftheextracts inacetonitrileandreactionwithfreshlypreparedErlichreagent (0.1g4-dimethyl-amino-benzaldehyde,2.4mlperchloricacid,and 3.0mlaceticacid),thesampleswerehomogenizedandleft undis-turbedwithoutstirringfor30min.Readingswerethenperformed

at503nm(BreskaandIbarra,2007).

Limonin(1)thatwasisolatedfromtheseedswasusedasthe standard to constructtheanalytical (calibration)curve.From a 500␮g/mlstocksolutioninacetonitrile,10–100␮g/mlofthe solu-tionswasprepared,andtheanalyticalcurvewasconstructedby linearregression.

Theanalysesoftotallimonoidaglyconeswereperformedwith the following samples: seeds, callus cultures (originating from seeds),calluscultures(originatingfromhypocotyls),cell suspen-sionfromhypocotylscells,andcellsuspensionfromcotyledons, whichwereobtainedondays7,14,21,28,and35ofcultureafter lyophilizationtoobtainthedryweight.

Statisticalanalysis

Thedatawereanalyzedbyanalysisofvariance,andmeanswere comparedusingDuncan’stestandOriginMicrocal9.0software. Valuesofp<0.05wereconsideredstatisticallysignificant.Forthe determinationof totallimonoids,theresultswerebasedonthe averageofthreeindependenttestswiththreereplicationseach. Theresultsareexpressedasthemean±standarddeviation.

Results

Fromtheacetoneextractoftheseeds,weusedPTLCtoisolate andsubsequentlyidentifylimonoidaglycone(1),aprocedurethat wasmonitoredbyananalyticalcharge-coupleddevice.The extrac-tionmethodthatwasproposedbyOhtaetal.(1993)allowsoneto obtainanextractthatisrichinlimonoidaglyconesandonlytraces oflimonoidglucosides.

The mass spectra of compound 1 showed a molecular ion [M+Na]+ _{atm/z=493.2171,equivalenttothemolecularformula}

C26H30O8,and12degrees ofunsaturation.Thespectraldatafor

compound1indicatedthepresenceoftwolactones,oneketone, fourmethyls,andonefuranring.Thephysicalandspectraldata (infraredspectroscopy,1_Hand13_{CNMR,DEPT,and2Dcorrelation}

techniques)showninattachmentthatwereconsistentwiththose reportedforlimonincompound1inKhaliletal.(2002).

Thedeterminationoftotallimonoidaglyconeswasperformed usingthemethodthatwasdevelopedbyBreskaandIbarra(2007), whichestimatestotallimonoidaglyconeconcentrationsinsamples ofCitrusinlimonin(1)equivalents.

Underthepresentconditions,theformationofacolorimetric complexwaslinearwithincreasingconcentrationsoflimonin,even withconcentrationsthatexceeded100␮g/ml.Thecorrelation coef-ficient(R2_{)fortheanalyticalcurvewas0.9984(Fig.2).}

0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 20 40 60 80 100

[18/4/2008 14:41 "/graph1" (2454574)] Linear regression for data1_B: Y = A+B * X

Error Value Parameter

---1.01045 –12.53021 A

3.25581 236.42961 B

---

-P N SD R

---<0.0001 10 1.24984 0.99924

---Lim

o

ni

n

co

nce

ntr

a

tion

(

μ

g/m

l)

Absorbance

Fig.2.Analyticalcurveforthequantificationoftotallimonoidaglyconesusing limonin(1)asthestandard.Thestandardoflimonin(10–100␮g/ml)wasdissolved inacetonitrileandreactedwithacolorimetricreagent.

calluscultureswhenconsideringtheirreadyavailabilityandrate ofmultiplication(Fig.3).

Thedeterminedquantityoflimonoidaglyconesintheextractof C.sinensis(Pera-Rio)seeds(Table1)washigherthantheonefound intheseedsofothervarietiesofCitrussinensis,including Valen-ciaorange(213.6mg/100g),Rubybloodorange(219.3mg/100g), Queenorange(173.1mg/100g),andJaffaorange(224.2mg/100g;

RouseffandNagy,1982).

Consideringthegrowthrateandproductionoflimonoid agly-conesfromthetwocelllinesinsuspension,theresultingcellsfrom cotyledonshadaslowergrowthratethanthecellsfromhypocotyls butwithhigherconcentrationsoflimonoidaglycones(Fig.4).

Agreateramountoflimonoidaglycone(240mg/100gofcelldry weight)wasaccumulatedinthecallusculturesfromcotyledons, butthedifferencewassmallcomparedwithcallusculturesfrom

0 50 100 150 200 250 300 350

35 28

21 14

7 0

0 50 100 150 200 250 300

35 28

21 14

7 0

Cell growth (mg/fflask)

Cell growth (mg/fflask)

TLA (mg/100 g of dried mass)

TLA (mg/100 g of dried mass)

Culture time (days)

Culture time (days)

A

B

Fig.4.Cellgrowth(mg, )andproductionoftotallimonoidaglycones(mg/100g dry weight,---)in calluscultureinsuspension ofCitrussinensisfrom:(A) hypocotylsand(B)cotyledons.CallusweregrowninMSmedium(Murashigeand Skoog,1962)supplementedwith2.0␮M2,4-dichlorophenoxyaceticacid,7.0␮M kinetin,and3.0%(w/v)sucrose.

Table1

Contentoftotallimonoidaglyconesinseeds,callusandcellssuspensioncultureof Citrussinensis.

Samplea _{LTA(mg/100gdryweight)}

Seeds 400±7.0

Callus(cotyledons) 10.0±0.5

Callus(hypocotyls) 200±1.0

Cellsuspension(hypocotyls)b ₂₁₀ ±3.0 Cellsuspension(cotyledons)b ₂₄₀

±5.0

a_{Analyzeswereperformedintriplicate.}

b_{ThevaluesrefertothepeakproductionofLTAonthegrowthcurve.}

hypocotyls(210mg/100gcelldryweight),eventhoughthiscell linehasagreatergrowthrate.

Discussion

Limonin(1)wasusedasthestandardforthespectrophotometric analysistodeterminethelevels oflimonoid aglyconesbecause itis themajorcomponentof Citrusspecies, themostabundant limonoidaglycone,andcommerciallyavailable(Bilaletal.,2013).

Furthermore,formostCitruslimonoids,thestructureofBCDrings (Fig.1)remainsthesame,whereasbiosyntheticreactionsare con-centratedontheconversionoftheAringofnomalin(2)totheA–A′

two-ringstructureoflimonin(1)(BreskaandIbarra,2007).Mostof theotheraglyconesthathavebeenisolatedfromCitruswere inter-mediates,withvariationsinthestructureoftheAring,including obacunone(3)andichangin(5)(RoyandSaraf,2006).

Althoughthis colorimetricmethodisnot currentlythemost widelyusedforCitruslimonoidanalyses(Tianetal.,2003;Vikram

etal.,2007;Solemainetal.,2005),wechosethismethodologyto

determinelimonoidaglyconecontentbecauseitdoesnotrequire expensiveequipment,thusincreasingthepotentialforits applica-tionintheanalysisoflimonoidaglyconesincallusculturesandcell suspensionsofCitrusspecies.

The combinationsof phytoregulators that wereused in this studyweresimilartothoseusedbyMansellandMcIntosh(1991), whousedacombinationof2,4-Dandkinetin,whichdifferfrom

Endoetal.(2002),whousedonlycytokinin.

Thelimonoidaglyconesconcentrationwasthehighestinthe C.sinensisseedextractcomparedwithextractsofcallusthatwere obtainedfromhypocotylandcotyledonexplants(Table1).

Squalene

Nomalin (2)

Ichangin

Limonoid A-ring lactone (LARL)

Limonin _Limonin

17-β-D-glucopyranoside LGT

LLH

O O

O

O O

O O

O

H H

O O

O O O

H

2

1

O O

AcO

CO2H O

O O

O

O

O

O

O H

H

OH OH OH

HO

LGBG

Mechanical harvest demage and acid pH (there are only in seeds)

Intermediate stored in the leaves, fruits and seedlings

There are only in seeds Obacunone

Fig.5.MajorenzymesinvolvedinthekeystepsoflimonoidbiosynthesisinCitrus.LGT,limonoidglucosyltransferase;LLH,limonoidd-ringlactonehydrolase;LGBG,limonoid

Importantly, callus from both hypocotyls and cotyledons wereabletoproducelimonoidaglycones. However,callusfrom hypocotylshada greaterability tobiosynthesizelimonoid agly-cones. This increased biosynthesis of callus from hypocotyl explantscanbeexplainedbythefactthatnomalin(2)isthemost likelyinitialprecursorofallknownlimonoidsinCitrus.Nomalin(2) hasbeenshowntobebiosynthesizedbytheacetate–mevalonate pathwayinthephloemregionofthestem,whichispresentinthe hypocotylexplantsthatareutilizedforcallusinduction(Moriguchi etal.,2003).Thisprecursorthenmigratestoothertissues,suchas leaves,fruittissues,andseeds,whereotherlimonoidsare biosyn-thesizedindependently(Fig.5).Thistoocanexplainthesmaller quantitiesof limonoidaglycones in the callus from seeds that requirenomalin(2)to biosynthesizelimonoidaglycones (Endo

etal.,2002).

Plantcellsuspensionsculturesnormallyareinitiatedwithcallus culturelinesthatproducehighamountsofinterestingsecondary metabolite,suchaslimonoids(Ravaletal.,2003).Callusorcell cul-turescanbeundercatabolicrepression,whichpreventsthemfrom producinglimonoidsorcausesthemtoproducelimonoidsinvery smallamounts.Underfavorableconditions,however,theycan pro-ducelimonoidsagain(Ravaletal.,2003).Thecallusculturesfrom hypocotylsofC.sinensisexhibitedthischaracteristic(Table1).Thus, wealsoevaluatedlimonoidaglyconeproductionincalluscultures fromcotyledonsandcellsuspensions.

Asshown in Fig.4, both cultures had a lag phase of 14–21 daysafterinoculation,followedbyarapidgrowthphasethat sta-bilizedafter28daysofcultivation.Inthecallusculturethatwas obtainedfromhypocotyls,thelevelsoftotallimonoidaglycones increased,togetherwithanincreaseinbiomass.Thus,the concen-trationsofthesecompoundsremainedatconstitutivelylowlevels forthebiosynthesisofthesecompoundsundercultureconditions

(MansellandMcIntosh,1991).

The suspension culturefrom cotyledons however showed a dramatic increase in limonoid aglycone concentrations on day 7,which maybe attributableto theconversion of

limonin-17-␤-d-glucopyranoside (7) to limonin aglycone (1) by limonoid glucoside ␤-glucosidase (Fig. 5) that probably was induced by adaptation to suspension culture stress through a shear effect or aeration, mimicking the same conditions and damage that occur with mechanical harvest in orange juice processing or seed germination. This conversion of limonoid glucosides to limonoidaglyconesoccurredonlyintheseedscells(Berhowetal.,

2000).

Inthis first momentoccurreda conversion oflimonoid gly-cosidesstockedtolimonoidaglycones,showedbyincrementof TLAconcentrationfromcotyledonscellline(onday7),afterthe TLAconcentrationwasdrasticallyreducedbut withmeasurable concentrationsbycolorimetricassay(day14)asshowninFig.5. Howeverinthehypocotylscellssuspensionatday14,theTLA con-centrationwasreducedtoalevelbelowthedetectionlimitofthe assay(day14).

Theresultssuggest thatde-differentiatedcallustissuesfrom cotyledonshavealimitedcapacitytoproducelimonoidaglycones. However,callusfromhypocotylsexplantsappeared tobemore promisingforthispurpose.Ontheotherhandwhenworkingwith suspensioncellslinesthatareobtainedfromcotyledonscallusare themostappropriatebecausetheyhavethecapacitytoproduceTLA bydirectbiosynthesisandfromhydrolyzeoflimonoidglucosidesto TLA.Clearly,tissueculturesandcellsuspensionsarevaluabletools forstudyingthebiosynthesisoflimonoidaglyconesfromCitrus.The analysisandevaluationoftechniquesthatareusedtosuppressor enhancelimonoidaglyconesbiosynthesisinplantsarepossible.The resultsthatwereobtainedusingthesetechniques,combinedwith moleculartechniques,willaidinthedevelopmentofpracticaluses ofthesecultures.

Authors’contributions

EFG(Ph.D.student)andERScontributedtotherunningofthe plantcellculturelaboratoryworkandanalysis.EFGandAPSF con-tributedtoMS,NMRandcolorimetricdateanalysisanddiscussion. TCPC(undergraduatestudent)contributedtomaintenanceand cul-tivationofplantcellculturesandphytochemicalwork.AJBOwrote manuscript.RACGcontributedtocriticalreadingofthemanuscript. AJBOandRACGdesignedthestudy,supervisedthelaboratorywork andcontributedtothecriticalreadingofthemanuscript.Allthe authorshavereadthefinalmanuscriptandapprovedsubmission. Allauthorsreadandapprovedthefinalmanuscriptsubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WethankCNPqandCAPESforfinancialsupport.Wealsothank Dr.EliezerRodriguesSouto,DepartmentofAgronomy,State Uni-versityofMaringá,Maringá,PR,Brazil,forallowingustousehis laboratoryfortheinductionandmaintenanceofcallusculturesof C.sinensis.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2015.05.008.

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