Production of commercially of important secondary metabolites and antioxidant activity in cell suspension cultures of Arthemisia absinthium L.

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ContentslistsavailableatSciVerseScienceDirect

Industrial

Crops

and

Products

j o u r n al ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / i n d c r o p

Production

of

commercially

important

secondary

metabolites

and

antioxidant

activity

in

cell

suspension

cultures

of

Artemisia

absinthium

L.

Mohammad

Ali

a

_,

_Bilal

_Haider

_Abbasi

a,∗

,

Ihsan-ul-haq

b

a_Department_of_{Biotechnology,}_Quaid-i-Azam_University,_Islamabad_45320,_Pakistan b_Department_of_Pharmacy,_Quaid-i-Azam_University,_Islamabad_45320,_Pakistan

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15February2013

Receivedinrevisedform22May2013 Accepted27May2013

Keywords:

Callus Cellsuspension Phenolics Flavonoids Antioxidant

Artemisia

a

b

s

t

r

a

c

t

AninvitrocultureofArtemisiaabsinthiumL.wasestablishedforproductionofcommercially impor-tantsecondarymetabolites.CalluscultureswereobtainedbyinoculatingleafexplantsonMurashige andSkoog(MS)mediumsupplementedwithThidiazuron(TDZ;0.5–5.0mgl−1₎_alone_or_in_combination witheither␣-naphthaleneaceticacid(NAA;1.0mgl−1)orIndoleaceticacid(IAA;1.0mgl−1).The cal-lusobtainedinresponseto1.0mgl−1 _TDZ_and_1.0_mg_l−1_NAA_was_subcultured_on_the_same_medium toinvestigateitsbiomassaccumulationandsecondarymetabolitesproductiononweeklybasisfor7 weeks.Forsubmergedcultivation,35dayoldcalliwereculturedonMSbasalmediasupplemented with1.0mgl−1_TDZ_and_1.0_mg_l−1_NAA._Growth_kinetics_and_secondary_metabolites_production_were investigatedin3dayoldsuspensionculturesfor42days.Additionally,highperformanceliquid chro-matography(HPLC)basedquantificationofgallicacid,caffeicacidandcatechinwascarriedoutincell suspensioncultures.Seedgerminatedplantletswereusedascontrol.Maximumlevelsoftotalphenolic content3.57mgGAE/gDW(control:2.75mgGAE/gDW),totalflavonoidcontent1.89mgQE/gDW (con-trol:1.20mgQE/gDW),andantioxidantactivity82.7%(control:72.3%)weredisplayedbysuspension cultures.Amongthephenoliccompounds,maximumlevelofgallicacid104␮gg−1(control:21.3␮gg−1), caffeicacid27.40␮gg−1 (control:28.5␮gg−1)andcatechin 92.0␮gg−1 (control:68.10␮gg−1)were detectedinsuspensioncultures.TheresultsindicatethatcellsuspensionculturesofA.absinthiumL. havethepotentialforenhancedproductionofphenolicsand,hence,highestantioxidantactivitythan calluscultureandseedderivedplantlets.

1. Introduction

Secondarymetabolitesproduced inplantsarelowmolecular weight(LMW)naturalproducts(Jooetal.,2010).Plantcell cul-tureisanattractivealternativetechnologyforenhancingsecondary metabolitesthatare eitherdifficulttosynthesizechemically or areproduced in limited quantitiesinwildplants. In particular, plant cell suspension cultures containing undifferentiatedcells areattractiveindustrially,especially compared todifferentiated cultures,duetotheirrelativesimilaritytomicrobialcellculture systems(Koleweetal.,2008).

Among different classes of secondary metabolites, plant polyphenolsconstitutethelargestgroupofnaturalantioxidants

(Cie´slaetal.,2012).Phenolicsareconsideredmorepotent

antioxi-dantsthanVitaminCandEandcarotenoids(Rice-Evansetal.,1995,

∗Correspondingauthor.Tel.:+925190644121;fax:+925190644121.

E-mailaddresses:bhabbasi@qau.edu.pk,chinabilal@yahoo.com(B.H.Abbasi).

1996).Thesecompoundsareconsideredtoplayaprotectiverole againstawiderangeofdiseasessuchascoronaryheartdiseaseand certaintypesofcancers(Ibrahimetal.,2012).Additionally,astrong relationshipexistsbetweenthephenoliccompoundsproducedby theinvitroculturesofdifferentplantsandtheirantioxidant activ-ities(Amidetal.,2011;Girietal.,2012;AlKhateebetal.,2012;

Diwanetal.,2012).

Artemisia absinthium L., commonly known as “Wormwood” has been used as herbal medicine throughout Europe, Middle East, North Africa, and Asia (Sharopov et al., 2012). A very populardrinkabsinthe, prepared fromA. absinthium L., is con-sidered to stimulate creativity and excitement (Gambelunghe

andMelai,2002).Theplanthastraditionallybeenusedas

anti-helmintic,choleretic,antiseptic, balsamic, depurative, digestive, diuretic, emmenagogueand intreating leukaemia andsclerosis

(Canadanovic-Brunetetal.,2005).Recently, theaerialpartofA.

absinthium L. has shown to possess anti-snake venom activity

(Nalbantsoy et al., 2013). Antimalarial and anticanceractivities

areamongtheprominentbiologicaleffectsreportedfordifferent

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speciesofthegenusArtemisia(Hernandezetal.,1990;Zafaretal.,

1990;Canadanovic-Brunetetal.,2005;Irshadetal.,2011;Shafi

etal.,2012).

In vitro studies of the genus Artemisia have mainly been focusedonenhancedproductionofartemisinin;strategiesshould be adopted to enhance medicinally important phenolics and flavonoidsin thespeciesofthis commerciallyimportantgenus. Thisstudyaimedtoestablishcellsuspensioncultureandto inves-tigatephenolicsandflavonoidsandantioxidant potentialinthe suspensionculturesofA.absinthiumL.

2. Materialsandmethods

2.1. Seedgerminationandexplantinoculation

SeedsofA. absinthium L.were obtainedfromNational Agri-culture Research Centre (NARC) Islamabad, Pakistan. Following a singlewashwithrunningtapwater seedswereimmersedin ethanol (3min), followed by treatment with mercuric chloride (2min)andthenwashedwithautoclavedwateranddriedon ster-ilized filter papers. MS0 (Murashige and Skoog basal medium; PhytotechnologyLabs,USA;MurashigeandSkoog,1962) contain-ing3%sucroseand0.8%(w/v)agar(PhytotechnologyLabs,USA) wasusedforseedgermination.

2.2. Callusinductionandbiomassyield

Tostudytheeffectsofplantgrowthregulatorsoncallus induc-tion,approximately1.5cmoftheleafsectionsfrom28-daysold seed germinated plantlets were incubated on Murashige and Skoog(MS) (1962)mediasupplementedwithTDZ(0.5,1.0,2.0, 3.0, 4.0, 5.0mgl−1₎ _alone _and _in _combination _with _1.0_mg_l−1 of NAA or 1.0mgl−1 _IAA. _Before _autoclaving ₍₁₂₁◦_C, ₂₀_min,

Systec VX 100, Germany), pH of all media was adjusted to 5.8 (Eutech Instruments pH 510, Singapore). All cultures were placedin16hphotoperiodwithlightintensityof40␮molm−2s−1 and temperature of the growth room was maintained at 25±1◦_C._Murashige_and_Skoog_basal_medium_(MS0)_was_used_as

control.

Differentialcallusformationfrequencyand changesincallus morphologywererecordedonweeklybasis.Furthermore,growth curvewasestablishedforthebiomassaccumulationoftherapidly growingfriablecallus,obtainedinresponseto1.0mgl−1_TDZ_and 1.0mgl−1_NAA._For_this_purpose,_1.0_g_of_the_callus_sections_were subculturedonMSmediacontaining1.0mgl−1_TDZ_and_1.0_mg_l−1 NAA.Investigationsofthebiomassaccumulationwereperformed withanintervalof7daysfor49daysperiod.Triplicateflaskswere usedinallexperiments.

2.3. Cellsuspensioncultureandgrowthkinetics

To establishcell suspension culture,35-day old proliferated calliweretransferredto500mlErlenmeyerflaskscontainingMS basalmediasupplementedwithcombinationof1.0mgl−1_TDZ_and 1.0mgl−1_NAA._The_cultures_were_placed_in_gyratory_shaker₍₂₅◦_C,

120rpm) in dark for thedevelopment of stock cellsuspension culturesasinoculumcultures.Finecellsuspensioncultureswere collectedafteraperiodof14days.Subsequentexperimentswere carriedoutin250mlErlenmeyerflaskscontaining50mlMSmedia with30gl−1_sucrose,_1.0_mg_l−1_TDZ_and_1.0_mg_l−1_NAA_in combi-nationand1.5gfreshcellsuspensionwasinoculatedineachflask. Observationsanddatarecordingofthegrowthkineticswere per-formedwithanintervalofthreedaysfor42daysperiod.Triplicate flaskswereusedinallexperiments.

2.4. Analyticalmethods

For fresh weight (FW) determination, calli were harvested fromthemediaandweighedwhilecellsuspensionswerefiltered through0.45␮mstainlesssteelsieve(Sigma),washedwith dis-tilledwater,pressedgentlyonfilterpapertoremoveexcesswater andweighed.Subsequently,calliandcellsuspensioncultureswere ovendried(60◦_C,₂₄_h)_for_dry_weight_(DW)_{determination.}

Extractionofcalli,cellsuspensionculturesamplesandinvitro seedderivedplantlets(control)wasperformedaccordingtothe protocoldescribedbyGirietal.(2012)withminormodification. Briefly,eachfinelygrounddriedsample(100mg)wasmixedwith 80%(v/v)methanol(10ml).Themixturesweresonicated(10min; Toshiba,Japan)3timeswitharestingperiodof30mininbetween andcentrifuged(8000rpm,10min).Thesupernatantswere col-lectedandeitherimmediatelyusedforanalysisorstoredat4◦_C.

For total phenolic content determination, Folin-Ciocalteu reagentwasusedaccordingtotheprotocolofVeliogluetal.(1998). Absorbancewasmeasuredat725nmbyusingUV/VIS–DAD spec-trophotometer(HaloDR-20,UV–VISspectrophotometer, Dynam-ica Ltd., Victoria, Australia). The calibrationcurve (0–50␮g/ml, R2₌_0.968)_was_plotted_by_using_gallic_acid_as_standard_and_the_TPC wasexpressedasgallicacidequivalents(GAE)/gofdryweight.

Fortotalflavonoidcontentdetermination,thealuminium chlo-ride colorimetric method as described by Chang et al. (2002) wasused.Absorbanceofthereactionmixtureswasmeasuredat 415nmbyusingUV/VIS–DADspectrophotometer.Thecalibration curve(0–40␮g/ml,R2=0.998)wasplottedbyusingquercetinas standard.TheTFCwasexpressedasquercetinequivalents(QE)/g ofdryweight.

Quantitativeanalysisofphenoliccompoundswascarriedoutby usingHPLC–DADattachedwithDiscoveryC-18analyticalcolumn, bythemethoddescribedbyZuetal.(2006)withminor modifi-cation.Methanol–acetonitrile–water–aecticacid(10:5:85:1)were used as mobile phase A and methanol-acetonitrile-acetic acid (60:40:1)wereusedasmobilephaseB.Agradientoftime0–20min for0–50%B,20–25minfor50–100%Bandthenisocratic100%Btill 30minwasused.Injectionvolumewas20␮landflowratewas 1.0ml/min.Gallicacidwasanalyzedat257nm,catechinat279nm andcaffeicacidat325nm.Everytimecolumnwasreconditioned for10minbeforethenextanalysis.

Forantioxidantactivitydetermination,theDPPHfree radical scavengingassay(FRSA)asdescribedbyAbbasietal.(2010)was used.Absorbanceofthemixtureswasrecordedat517nmby spec-trophotometer.Forbackgroundcorrection,amethanolicsolution ofDPPHthathaddecayedwithnoresultantpurplecolour(2mgof butylatedhydroxyanisole(BHA)dissolvedin4mlofmethanolwith 0.5mlofDPPHsolutionadded)wasusedinsteadofpuremethanol. Theradicalscavengingactivitywascalculatedbythefollowing for-mulaandexpressedas%ageofDPPHdiscoloration:

%scavengingDPPHfreeradical=100×

1−AE

AD

whereAEisabsorbanceofthesolutionwhenanextractwasadded ataparticularconcentrationandADistheabsorbanceoftheDPPH solutionwithnothingadded.

2.5. Experimentaldesignanddataanalysis

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Table1

EffectsofdifferentconcentrationsofTDZaloneandincombinationwithIAAandNAAoncallusformationfrequency,callusmorphologyandcallusgrowth(freshweightand dryweight).Valuesaremean±standarderrorofthreereplicates.

S.no. Growthregulator(mgl−1₎ _Callus_(%) _Callus_morphology _FW_(g_l−1₎ _DW_(g_l−1₎

(Windowsversion7.5.1,SPSSInc.,Chicago)wasusedtodetermine thesignificanceatP<0.05.

3. Resultsanddiscussion

3.1. Callusformationandgrowthkinetics

TheleafexplantsofA.absinthiumL.treatedwith1.0mgl−1_TDZ incombinationwith1.0mgl−1 _NAA_resulted_into_highest_callus formationfrequency(83.3%)andmaximumcallusbiomass(FW: 132gl−1₎_with_yellowish_friable_features_after₅_weeks_of_culture

(Table1).WhenTDZwasusedalone,maximumcallusformation

frequencyof80.3%wasobservedon1.0mgl−1_TDZ._The_callus_was greenandfriable.However,greencompactorbrownishcompact calliwereproducedinresponsetocombinationofTDZandIAA.NAA incombinationwithTDZwasmoreresponsiveforcallogensisthan IAAincombinationwithTDZ.Furthermore,combinationofTDZ andNAAwasmoreefficientforcallusformationcomparedtoTDZ alone.Previously,bestcallogenicresponseshavebeenobservedon BAincombinationwithNAA(Ninetal.,1997;Ziaetal.,2007a,b) which,inaccordancewithourresults,showthatthecombination ofcytokininsandauxinsissuperiorininducingfriablecalliinleaf explantsofA.absinthiumL.

Biomassformation of thecallus cultureshowed a 7-daylag phase with relatively slow growth (Fig. 1). Almost threefold increaseindrybiomass(DW:5.67gl−1₎_was_achieved_on_day₁₄ and,asawhole,fourandhalf-foldincrease(DW:8.73gl−1₎_in_dry biomasswasobservedonday42of culture.Declinephasewas observedafter42daysofculture,characterizedbydecreaseindry biomass(DW:7.77gl−1₎_on_day_49.

3.2. Cellsuspensionculturedevelopmentandgrowthkinetics

BiomassformationofthecellsuspensioncultureofA.absinthium L.displayedarelativelyquickgrowthcurveandwascharacterized byalagphaseof6daysforfreshanddrybiomass,followedbya longlogphaseof21days,andasubsequentstationaryphase dur-ing42dayperiodofstudy(Fig.2).Almostdoublinginfreshweight (63gl−1₎_and_dry_weight_(4.07_g_l−1₎_were_recorded_on_day₉_of cul-ture.However,maximumfreshweightanddryweightdisplayed bycultureatday27were171gl−1_and_9.20_g_l−1_,_{respectively.} Fur-thermore,cellsuspensioncultureswerefoundtobemilkywhite, greenandbrownishincolourduringlog,stationaryand decline phases,respectively(Fig.3).

0 7 14 21 28 35 42 49

Culture time (days)

F

Fig.1.GrowthkineticsofcalluscultureofArtemisiaabsinthiumL.onMSmedium supplementedwith 1.0mgl−1_TDZ₊_1.0_mg_l−1_NAA._Values _are _mean_±_standard

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Fig.3. Cellsuspensionculturesduring(A)logphase,(B)stationaryphaseand(C)declinephase.

3.3. Totalphenoliccontent(TPC)andtotalflavonoidcontent (TFC)incallusandcellsuspensioncultures

Phenolics and flavonoids induction in callus culture of A. absinthiumL.wasnotfoundtobestrictlygrowthdependent(Fig.4). Initialincrease in TPC and TFC wasobserved in the log phase (day21),withrespectivepeakvaluesof1.48mgGAE/gDW (4.9-foldincrease)and0.48mgQE/gDW(4.1-foldincrease)onday35. Earlier studies have been undertaken on the investigations of totalphenoliccontentincalluscultureofvariousmedicinalplants

(Schmeda-Hirschmann et al.,2005; Nazet al.,2008; Giri etal.,

2012).Ourresultswereconsistentwiththepreviousreportsfor highestlevelsofflavonoidsproductionincallusculturesof differ-entmedicinalspeciesduringthelogphaseoftheculture(Fuetal.,

2005;Antognonietal.,2007;Andreazza etal.,2009;Tanetal.,

2010).

A considerable increase with more than doubling in TPC andTFCwasobservedin6-dayoldsuspensioncultures(Fig.5). On contrary to callus profile, peak values of total phenolics with 3.57mgGAE/gDW (day 30) and total flavonoids with 1.89mgQE/gDW(day33)wereobservedintheearlystationary phaseofthegrowthcurve.Previously,thecellculturesofArtemisia fragidaandSilybummarianumwerereportedtobemore produc-tiveforphenolicacidsamongninedifferentplantspecies(Riedel et al., 2010). Furthermore, thepresence of different secondary

0 7 14 21 28 35 42 49

Fig.4. Totalphenoliccontent(mggallicacid/gdryweight)andtotalflavonoid con-tent(mgquercetin/gdryweight)incallusculturewithrespecttogrowthcurve (DW).Valuesaremean±standarderrorofthreereplicates.

metabolites including phenolics and flavonoids in the in vitro culturesofA.annuahasbeenreviewed(Bhakunietal.,2001).

3.4. Antioxidantactivity

TheDPPHfreeradicalscavengingactivityincalluscultureswas foundtobeindependentofcallusbiomassaccumulation.However, itwasfoundtobedependentonsecondarymetabolites produc-tionduringtheculturegrowth.Highestantioxidantactivity(63.3%) andmaximumaccumulationoftotalphenolics(1.48mgGAE/gDW) andtotalflavonoids(0.48mgQE/gDW) wererecordedin35day oldcalli(Fig.6).Declineinantioxidantactivitywasrecordedafter 35 daysof culture.Theseresultsshowa positive correlationof phenoliccompoundsandantioxidantactivityincallusculturesof A.absinthiumL.

Ontheotherhand,theDPPHradicalscavengingactivityincell suspensioncultureswasnotfoundtobestrictlyculturegrowth associated.Maximumbiomassaccumulation(DW:9.03gl−1₎_was observedonday27whilehighestantioxidantactivity(82.7%)was foundin30-dayoldsuspensioncultures,instationaryphase. How-ever,antioxidantactivitywasfoundtobeassociatedwithtotal phenolics production (Fig.7). Maximum total phenolic content (3.57mgGAE/gDW)inconjunctionwithhighestantioxidant activ-itywasrecordedin30-dayoldsuspensionculturesinstationary phase.Declineinactivitywasrecordedafter39daysand59%of

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42

Fig.5. Totalphenoliccontent(mggallicacid/gdryweight)andtotalflavonoid con-tent(mgquercetin/gdryweight)incellsuspensioncultureofArtemisiaabsinthium

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0 7 14 21 28 35 42 49

Fig.6. DPPHradicalscavengingactivity(%)withrespecttototalphenoliccontent andtotalflavonoidcontentincallusculturesofArtemisiaabsinthiumL.Valuesare mean±standarderrorofthreereplicates.Columnswithsimilaralphabetsarenot significantlydifferentatP<0.05.

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42

Fig.7. DPPHradicalscavengingactivity(%)withrespecttototalphenoliccontent andtotalflavonoidcontentincellsuspensionculturesofArtemisiaabsinthiumL. Valuesaremean±standarderrorofthreereplicates.Columnswithsimilaralphabets arenotsignificantlydifferentatP<0.05.

radicalscavengingactivitywasobservedin42dayoldsuspension cultures.

3.5. HPLCbasedquantificationofphenoliccompoundsincell suspensionculture

Gallic acid was found to be the major phenolic compound whichaccumulatedinconjunctionwithbiomassaccumulationin cellculture(Table2).Overallaccumulationofgallicacidranged from 43.3␮gg−1 to 104␮gg−1DW (control: 21.3␮gg−1DW) and maximumaccumulation wasobserved during theonset of stationaryphase(day-30)ofculturewhichissignificantlyhigher thanthecontentfoundincontrol.Theseresultsareincorrelation withourfindingsofmaximumTPCaccumulationin30dayoldcell suspensioncultures.Caffeicacidwasthesecondmajorphenolic compoundobservedintermsofitsaccumulationinthelogphase of cultureand ranged from1.03␮gg−1DW to27.40␮gg−1DW (control:28.4␮gg−1DW). However,itsmaximumaccumulation wasrecordedinthemidlogphase(day24).Catechinwas accu-mulatedonlyinthestationaryphase(day36and42)ofculture

Table2

Quantificationofgallicacid,caffeicacidandcatechinincellsuspensionculturesof

ArtemisiaabsinthiumL.Valuesaremean±standarderrorofthreereplicates. Phenoliccompounds(␮gg−1)

Day Gallicacid Caffeicacid Catechin

Control(SDPa₎ _21.3

a_SDP,_seed_derived_plantlets. b_ND,_not_detected.

with the values 75.3␮gg−1DW and 92.0␮gg−1DW (control: 68.1␮gg−1DW)onday-36andday-42,respectively.

The phenolic compounds detectedin the present study are medicinallyimportantphytochemicals.Gallicacidisalow molec-ularweightantioxidant(LMWA)havingantiapoptotic(Sohietal., 2003),neuroprotective(Luetal.,2006)andanticarcinogenic prop-erties(Tomas-BarberanandClifford,2000).Similarly,Caffeicacid hasexhibitedpharmacologicalantioxidant,anticancerand antimu-tagenic activities(Okutan et al., 2005) and catechin prevented oxidativeinjuryinhumangastricepithelialcells(Grazianietal.,

2005).

3.6. Relationshipbetweentotalphenoliccontent,totalflavonoid content,phenoliccompoundsandantioxidantactivityincell suspensionculture

Manyreportsareavailableontheprotectiveeffectsofnatural antioxidantsagainstoxidativestressrelateddisorderslikeageing, degenerativediseases andcancer(Cozzietal.,1997).According

toBidcholetal.(2011),phenolic compoundsmayhavea direct

contributionintheantioxidantactivity.Theantioxidantpotential invariousmedicinalplantshasbeenshowntobemainlydueto phenoliccompounds(Jayasingheetal.,2003;Alietal.,2006;Kim

etal.,2006;Alietal.,2007).Althoughartemisininhasbeenreported

tobethemajorcompound againstmalariain Artemisiaspecies, reportsareavailableonthesynergisticeffectsofdietaryflavonoids aloneandincombinationsagainstthisdisease(LehaneandSaliba,

2008;Ferreiraetal.,2010).Inthepresentstudy,apositive

cor-relationwasobservedamongTPC(3.57mgGAE/gDW),gallicacid accumulation(104␮gg−1)andantioxidantactivity(82.7%)in30 dayoldsuspension cultures.Our resultsarein agreementwith apreviousreportwhereapositivecorrelationbetweenhighTPC andTFCandantiradicalandantioxidantactivitiesinA.absinthium L.wasobserved(Canadanovic-Brunetet al.,2005).Senguletal.

(2009)havealsoreportedapositivecorrelationbetweentotal

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Table3

Comparativeanalysisoftotalphenoliccontent,totalflavonoidcontentandantioxidantactivityinseedderivedplantlets,calluscultureandcellsuspensionculture.Values aremean±standarderrorofthreereplicates.

Tissuetype Totalphenoliccontent(mgGAEg−1₎ _Total_flavonoid_content_(mg_QE_g−1₎ _Antioxidant_activity_(%)

Seedderivedplantlets 2.75±0.077 1.200±0.121 72.3±1.068

Callus 1.48±0.101 0.48±0.011 63.3±1.760

Cellsuspension 3.57±0.088 1.77±0.168 82.7±3.530

antioxidant activity than callus and seed germinated plantlets

(Table3).

4. Conclusion

Thepresentreportdescribesestablishmentofcellsuspension culturesofA.absinthiumL.fortheenhancedproductionof pheno-licsandflavonoids.Cellsuspensioncultureswerefoundtoproduce significantlyhigherlevelsoftotalphenolics,totalflavonoidsand gallicacidandshowedhigherantioxidantactivitythantheseed derived plantlets. Furthermore, a positive correlation between phenolicsandantioxidantactivitywasfoundinsuspension cul-tures.Itcanbeconcludedthattheinvitrocultures, particularly cellsuspensionculture ofA. absinthiumL. hasthepotentialfor scaleupstudiesoncommerciallevelbypharmaceuticalindustries, inorder tofurtherenhancethemedicinallyimportant phenolic compounds.

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