Rev. bras. farmacogn. vol.26 número2

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

Original

Article

Comparative

HPTLC

analysis

of

bioactive

marker

barbaloin

from

in

vitro

and

naturally

grown

Aloe

vera

Devendra

Kumar

Pandey

a

_,

_Sidharth

_Parida

b

_,

_Abhijit

_Dey

c,∗

a_Department_of_{Biotechnology,}_Lovely_Faculty_of_Technology_and_Sciences,_Lovely_Professional_University,_Phagwara,_Punjab,_India

b_Department_of_{Biotechnology,}_MITS_School_of_{Biotechnology,}_Affiliated_to_Utkal_University,_Bhubaneswar,_Odisa,_India

c_Department_of_Biological_Sciences,_Presidency_University_(Erstwhile_Presidency_College),_Kolkata,_India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13May2015 Accepted18August2015 Availableonline31December2015

Keywords:

Aloevera

Barbaloin

Invitro

HPTLC Densitometry

a

b

s

t

r

a

c

t

Aloevera(L.)Burm.f.,Xanthorrhoeaceae,asucculent,producesbarbaloin,abioactivecompoundsused invariouspharmaceuticalproducts.Extractspreparedfromtheleaveshavebeenwidelyusedas bitter-ingagents,tastemodifiersandalsoascatharticagentagainstsevereconstipation.Barbaloinisreported foritsanti-inflammatory,anticancer,antiviralandanticanceractivitiesandthesepropertiesaremostly mediatedbyitsantioxidativecapacity.Presently,astudyhasbeenconductedonthecomparativeHigh PerformanceThinLayerChromatographyanalysisofbarbaloinfromthedriedleafskinpowderofinvivo andinvitrogrownA.vera.ShoottipsofA.verawereculturedinMurashigeandSkoogmediasupplemented withdifferentcombinationof6-benzylaminopurineand1-naphthaleneaceticacid.[Bestmultiplication responsewasnotedinbenzylaminopurine(2.0mg/l)+1-naphthaleneaceticacid(0.1mg/l)supplemented MurashigeandSkoogmedia].Thequantitativedeterminationofbarbaloinwasperformedonsilicagel60 F254HPTLCplatesasstationaryphase.Thelinearascendingdevelopmentwascarriedoutinatwintrough

glasschambersaturatedwithamobilephaseconsistingofethylacetate:methanol:water(100:16.5:13.5) atroomtemperature(22±2◦_C)._CAMAG_Thin_Layer_{Chromatography}_scanner-3_equipped_with_CATS software(version:1.4.4.6337)wasusedforspectrodensitometricscanningandanalysisintheultraviolet regionat=366nm.Themethodwasvalidatedforlinearity,precisionandaccuracy.Correlation coeffi-cient,limitofdetection,limitofquantificationaswellasrecoveryvalueswerefoundtobesatisfactory. Outofthefivepopulationsstudied,theleafskinofA.veracollectedfromJodhpur(Rajasthan,India)and raisedinvitrowasfoundtocontainhigheramountofbarbaloin(2.78%)whencomparedtoitsnaturally growingcounterparts(2.46%)andotherplantpopulations.

Introduction

Aloevera(L.)Burm.f.,Xanthorrhoeaceae,isasucculentplant indigenoustoNorthernAfricaandMediterraneancountriesand hasbecome naturalized almost in all parts of India (Klein and Penneys, 1988). The original commercialuse of theplant was meantfortheproductionofalatexsubstancecalledbarbaloin(1) or aloin (molecularformula: C21H22O9)which is yellow-brown incolorwithandlingeringtasteandwasusedaslaxativeuntil worldwar-II(Saeedetal.,2004).A.verahasexhibitedanticancer, woundshealing,immunomodulatory,antiviral,anti-inflammatory, dentalprotective, laxative,antiseptic,gastroprotective, moistur-izingandanti-agingproperties(Surjusheetal.,2008;Parketal., 2011;Yoneharaetal.,2015;Hashemietal.,2015;Mangaiyarkarasi

∗ Correspondingauthor.

E-mail:[email protected](A.Dey).

etal.,2015).OraladministrationofA.veraforthetreatmentof dia-betesmellitusanddyslipidemiahasalsobeeninvestigated(Ngo etal.,2010).Besidesbarbaloin,theothermainingredientofA.vera

iscalledleafgel,aclear,colorlessandtastelesssubstance,which coversinnerportionsoftheleaves(ReynoldsandDweck,1999).The gelisusedforcommercialuseinpharmaceuticals,functionalsfoods andcosmetics(Hamman,2008).SterolspresentinA.veragel stim-ulatedcollagenandhyaluronicacidsynthesis byhumandermal fibroblasts(Tanakaetal.,2015).A.veragelalsopromotedcesarean woundhealinginwomen(Molazemetal.,2014).Barbaloin(also namedaloin),theC-glucosideofaloeemodinanthrone,localizes intheouterrindoftheplanthasbeenreportedtoconstituteupto 30%oftheplant’sdriedleafexudates(GroomandReynolds,1987) andproposedasapartofthedefensemechanismsagainst herbi-vores(GuttermanandChauser-Volfson,2000;Changetal.,2006). Barbaloinwasreportedforitshistaminereleaseinhibitory, anti-inflammatory,antiviral,antimicrobial,anticancer,antioxidantand catharticeffects(Pateletal.,2012)

http://dx.doi.org/10.1016/j.bjp.2015.08.016

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162 D.K.Pandeyetal./RevistaBrasileiradeFarmacognosia26(2016)161–167

In another Aloe species A. ferox, aloesin, aloeresin a and anthraquinone(asbarbaloin)wasestimatedbyareversed-phase high-performanceliquidchromatographic(HPLC)method(Zahn etal.,2008).InleafexudateofA.secundiflorathemajor compo-nentswereanalyzedbyHPLC-massspectroscopy(Rebeccaetal., 2003). Barbaloinin aloe capsule wasalsodetermined by HPLC methods (Chen et al., 2002). Aloin was selectively determined in different matrices by HPTLC densitometry in fluorescence mode (Coran et al., 2011). Earlier, barbaloin was estimated in

A.veraandinitscommercialproducts(Pandeyetal.,2012).An efficient micropropagation protocol was adopted in A. vera in Murashigeand Skoog(MS)mediumsupplemented withgrowth regulators (Aggarwal and Barna, 2004). Rapid propagation by theshootgenerationcalliwasachievedinpolyvinylpyrrolidone (PVP)andgrowthregulatorsupplementedMSmedium(Royand Sarkar,1991).However,noattempthasyetbeenmadetoquantify barbaloin(1)comparativelyinnaturalorinvitropopulationsof

A.vera.Sincebarbaloinisconsideredasanimportantbiologically activecompound in A. vera, thepresent investigationdepicts a validated HPTLC protocol for determination of barbaloin from

insituandinvitrogrownpopulationsofA.vera.

O

OH OH

HO

O H

HO OH

OH OH

1

₁

Materialsandmethods

Plantmaterialandchemicals

Different populations of Aloe vera (L.) Burm. f., Xanthor-rhoeaceae,werecollectedfromfivedistricts(fromfourdifferent states of India) viz. Jalandhar (Punjab), Jodhpur (Rajasthan), VaranasiandLucknow(UttarPradesh)andBhubaneswar(Odisha). Plantswith9–11leavesandofalmostsamesizeandagewere har-vestedintheirvegetativestageduringthemonthofJune2012to September2012andmaintainedattheherbalmedicinalgarden, LovelyProfessionalUniversity,Punjab.Thematerialswere identi-fiedandauthenticatedonthebasisofmorphologicalcharactersby abotanistintheDepartmentofBotany,BanarasHinduUniversity, Varanasi.Avoucherspecimen(VoucherNo.080912)wasdeposited attheDepartmentofBiotechnology,LovelyProfessional Univer-sityforfuturereference.Allsolvents(HPLCgrade)wereobtained fromE.Merck(Mumbai,India).Standard barbaloin(∼97%pure) waspurchasedfromSigma–Aldrich(USA).

MicropropagationofAloevera

MS (Murashige and Skoog) medium supplemented with 6-benzylaminopurine(BAP)(0.5–2.0mg/l)and1-naphthaleneacetic acid(NAA)(0.1–1.0mg/l)andagar(0.8%)wasusedforshoot pro-liferation.After4–5weeksofcultureperiod,theinvitrogrown plantletswithnewlyformshootsweretakenoutasepticallyandthe shootswereexcisedfromtheparentplantwiththehelpofsterile scalpelbladeandforcepsandinoculatedintonewbottles contain-ingsolidMSmediumwithdifferentsetofgrowthregulators(PGR) asmentionedearlier.Newlyformedshootsmeasuring3–4cmin lengthwereexcisedindividuallyfromtheparentplantandwere transferredintothetwotypesofrootingmedia:MSmedia sup-plementedwithNAA(0–2.0mg/l)andindole-3-butyricacid(IBA)

(0–2.0mg/l)individually.Allcultureswereincubatedunder16h photoperiod(cool,whitefluorescentlight(30␮M/s))and temper-atureof25±1◦_C_with_50–80%_relative_humidity._After₃₀_days_of cultureonrootingmedia,theplantletswereshiftedtoplasticpots forhardeningpriortofinaltransfertonaturalconditions.For hard-ening,plantswithnewlyformedroots weretakenoutfromthe culturebottleswithutmostcaretopreventanydamagetotheroots. Theplantswerethendippedinwarmwatertoremoveanytraces ofagarfollowingwhichtheplantsweredippedin1%(w/v)solution ofBavistintopreventanyfungalinfectioninthenewlydeveloped plants.Theplantletswerethenplantedinplasticpotscontaining 1:1mixtureofsoilandmanure.

Preparationofsampleandstandardsolution

ThesheddriedleafskinofA.verawerepowderedinamixer grinder(ChampEssentials,MorphyRichards,India).Leafskin(0.1g each from in situ and in vitro grown plants) were separately extractedwithmethanol(2×20ml)for15minunderrefluxona waterbathat70◦_C._The_extracts_were_filtered_through_Whatman no:1filterpaper(separatelyforinsituandinvitrosamples)and wereevaporatedundervacuumusingarotaryevaporator(Eyela, N-1100,China)tofurnishasolidmassofextract.Theextractwaskept infreezingtemperaturefreeofmethanolbecausebarbaloin(1)is convertedintoaloeemodinandanumberofunknowncompounds astheglucosepartisremovedfromthecompoundwhenkeptin methanol(Changetal.,2006).Theextract(0.1g/ml)obtainedfrom each sample wereprepared in HPLC-grade methanolfor quan-titativeanalysis. Stocksolutions of barbaloinwere preparedby dissolving10mgofthecompoundin10mlofmethanol.

Chromatographicconditions

The HPTLC system was composed of a CAMAG (Muttenz, Switzerland)Linomat-5automaticsampleapplicatorandCAMAG TLCscanner-3providedwithCATSsoftware(version:1.4.4.6337). Thestationaryphasewascomposedofpre-coatedsilicagel60F254 HPTLCplates(20cm x10cm;with0.25mmthickness).Samples wereadministeredtotheplatesas5mmwidebandsvia Linomat-5 automatic sample applicator (with nitrogen flow) equipped witha100␮lHamiltonsyringe.DeliveryratefromtheHamilton syringewasfixedat100nl/s.Linearascendingmodeof develop-mentuptoa distanceof80mm,withethylacetate:methanol: water(100:16.5:13.5)asmobilephase(WagnerandBladt,1996; GuttermanandChauser-Volfson,2000),wasimplementedatroom temperature(22±2◦_C) _and _50% _relative _humidity_in _a _CAMAG twintroughglasschamber(20cm×10cm)saturatedearlierwith mobilephase vapour for 20min.Afterdevelopment,the plates weredriedat100◦_C_for₁₀_min,_derivatized _with₁₀₀_ml_of_10% (v/v)alcoholicKOHsolution,followingwhichdensitometric scan-ningwasexecutedat366nm(WagnerandBladt,1996).Theslit dimensions were5mm×0.45mm and the scanningspeed was 100nm/s. For calibrationand toestimate thelinearity, marker stocks(0.3,0.6,0.9,1.2,1.5␮l)wereadministeredtotheplateto furnishamountsintherange300–1800ngperband.Peakareas wereplottedagainstthecorrespondingconcentrationsand regres-sionanalysiswasexecutedtogeneratethecalibrationequation.To analyzeplantsamples,1.0␮lextractofeachofthesampleswas administeredtotheplate.Afterdevelopment,derivatization, scan-ningandmeasurementofpeakareatheamountofbarbaloin(1) wasdetermined,assumingthepurityofthemarkertobe100%. ChromatogramsareshowninFigs.2and3.

Methodvalidation

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Fig.1.DifferentstagesoftissuecultureinAloeveraplants:a.explantsource,b.(1–4).shootproliferationafter8weeksofcultureonMSmedium,c.(1–4):invitroroot inductiononmicroshootsandacclimatizationofrootedplantlets.

1000 [AU] 800 700 600 500 400 300 200 100 0

0.00 0.10 0.20 0.30 0.40 0.50 0.60 [Rf] 0.80 100

80

70

60

50

40

30

20

10

0 [mm]

Fig.2.HPTLCfingerprintingofbarbaloinalongwiththemethanolicextractof dif-ferentpartsofinvitrogrownA.veraplants(1–5:standard;6:lightgreen-gel,7: yellow-root;8:red-leafskin)fromJodhpur.

repeatability,percentagerecovery,intra-dayandintermediate pre-cision.Eachof thestandard solutionsofbarbaloin (0.3,0.6,0.9, 1.2,1.5␮gperband)wasadministeredintriplicate.The calibra-tionplotwasdevelopedbyplottingpeakareaagainsttheamount ofbarbaloinandlinearityrangewasresolved.Instrument preci-sionwasexaminedbyscanningthesamebarbaloinband(1␮g) sixtimes.Themean,standarddeviation,andcoefficientof varia-tion(CV)(%)weredeterminedforpeakareaandretentionfactor (Rf).Repeatabilitywasdeterminedbyanalyzingthebandfor bar-baloinafterapplicationofstandardsolutiontotheplate(n=10),

100.0

[AU]

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0

200.0 250.0300.0 350.0 400.0450.0 500.0 550.0 600.0 [nm] 700.0 100.0

[AU]

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0

Fig.3.Overlayspetraofbarbaloin.

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Table1

InvitromultipleshootformationfromlateralshootsofAloeveraasinfluencedbythegrowthregulators(4replicatespertreatment).

MS+growthregulators(mg/l) %ofculturesshowingshoot proliferation

Averagenumberofshootper explant(after4weeks) (mean±SE)

Averagenumberofshootper explant(after8weeks) (mean±SE)

BAP NAA

0.5 0.5 50 1.32±0.22 3.3±0.2

0.5 1.0 44.2 1.3±0.31 2.21±0.6

1.0 0.5 70.2 2.00±0.32 5.2±0.3

1.0 0 65.34 2.33±0.36 4.1±0.3

2.0 0.5 83.2 2.8±0.52 5.6±0.2

2.0 0.1 100 4.1±0.25 8.12±0.3

Datarepresentthemeanof3replicatesforeachtreatment.

Determinationofbarbaloininsamples

Sampleextractsandstandardsolutionmixtures(0.3,0.6,0.9, 1.2,1.5␮l)wereadministeredto20cm×10cmsilicagel60HPTLC platesandanalyzedasdescribedabove.Peakareaswerenotedand acalibrationplotwasgeneratedbyplottingpeakareaagainstthe amountofstandardbarbaloin(1)administered.Thecalibrationplot

wasusedtodeterminetheamountsofstandardspresentinthe

samples.

Resultsanddiscussion

EffectofPGRonorganogenesis

Inordertoachieve multipleshootinduction,various combi-nationsofcytokininandauxinwereinvestigated.Appearanceof shootsfromlateralshootexplants(suckers)wasfoundaftertwo weeksofcultureinallthehormonecombinations(Table1).Shoot budsinitiated werelight green to yellowish in colorand were formedeitherassingleorin clusters.Themaximumnumberof shootbud(4)perexplantwasnotedinMSmediasupplemented with2.0mg/lBAPand0.1mg/lNAAwithinfourweeksofculture.

EffectofPGRonshootmultiplicationandelongation

Shoot buds proliferated in MS medium supplemented with 2.0mg/lBAPand0.1mg/lNAAweremultipliedintoclustersofsmall budsandwereelongatedwhensubculturedinthesame regenera-tionmedia.Maximumshootswereobtainedwithineightweeksof cultureinthesamemedium(Table1).However,MSmedia contain-ing0.5mg/lBAPand1.0mg/lNAAhasyieldedsignificantlylower numberofshoots.

Rootinginregeneratedshoots

Rootingwasobservedwithintwoweeksinallrootingmedia (Fig. 3).Maximum (87%) rooting wasfound in MS media sup-plementedseparatelywith0.5mg/lNAAand2.0mg/lIBAwithin two weeks of culture (Table 2). Significant difference in root

numberwasnotedaftertwoweeksofcultureintworootingmedia. Therootlengthwassignificantlydifferentandthemaximumroot elongationwasobservedintheaforesaidmedium(Table2).Roots regeneratedinthismediumwererelativelythickandstrong com-paredtoothermediumevenduringinitialstagesofdevelopment.

Acclimatizationofrootedplantlets

Welldevelopedrootedplantlets(3.0–5cm)wereobtainedafter twomonthsofcultureonMSbasalmediumwhichwerethen trans-ferredtopotscontainingsoilandsandof1:1ratioand100%of theexplantsweresurvivedduringandaftertheacclimatization process.ThestagesofmicropropagationarepresentedinFig.1.

MethodvalidationbyHPTLC

PreparatoryTLCstudiesshowedthatthesolventsystemethyl acetate:methanol:water(100:16.5:13.5)wasidealasmobilephase whichproducedasinglespotwithanRf0.4forbarbaloin(1)and wellresolvedspotswerefoundforthetestsamples.Thespotswere observedat366nmaftersprayingwith10%(v/v)ethanolicKOH. Thethreedimensionaldensitogrampatternsofthetestsamples andbarbaloin demonstratedthat thepeakscorrespondingtoRf 0.4weresuperimposableinallthesamples.Thespectrum char-acteristicscorrespondingtothispeakwerealsonotedtomatch exactlydenotingthecompoundscorrespondingtoRfofthe stan-dardsandthetestsamplestobeidentical.Thepeakpuritytestwas performedbycomparingtheabsorptionspectraofthestandard barbaloinandthetestsamples;clearsuperimpossibilityspecified thepurityofthepeak(Fig.2).Linearityofthecalibrationcurve wasfoundbetween0.3and1.5␮g.Thecorrelationcoefficientfor acalibrationcurvebetween0.3and1.5␮gwasfoundtobe0.998 forbarbaloin.Theregressionequationforthecalibrationplotfor barbaloinissummarizedinTable3.Percentageofbarbaloinwas determinedbyusingthepeakareaparameter.ThisHPTLCmethod wasvalidatedforprecision,accuracyandrepeatability.Themethod isspecificforbarbaloinbecauseitresolvedthecompound(Rf=0.4) wellinthepresenceofothercomponentsinA.vera(Fig.2).A lin-earrelationshipwasachievedbetweenresponse(peakarea)and amountof barbaloin in therange 0.3–1.5␮gper band(Fig.4).

Table2

EffectofdifferentconcentrationsofIBAandNAAonthenumberofrootandrootlengthinAloevera.

MS+growthregulators(mg/l) %ofshootsproducingroots (mean±SE)

Numberofrootspershoot (mean±SE)

Averagerootlengthin(cm) (mean±SE)

IBA NAA

0.5 0.5 43.21±1.5 1.17±0.18 1.76±0.3

0.0 1.0 16.7±2.1 1.98±0.37 1.13±0.2

1.5 2.0 64.22±1.3 4.19±0.15 2.11±0.2

2.0 0.5 87.56±2.0 7.14±0.16 3.47±0.4

1.0 0.0 42.19±1.3 4.01±0.21 1.96±0.2

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1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 2600

2400

2200

2000

1800

1600

1400

1200

1000

Conc

Area

S 84.3814

R-Sq 98.1% R-Sq(adj) 97.5%

Fitted line plot

Area=834.4 + 1115 conc

Fig.4. Standardcurveofbarbaloin.

Table3

Methodvalidationparametersforthequantificationofbarbaloin.

Parameters Results

Rf 0.4

Linearityrange(ng/spot) 300–1500ng Regressionequation(area) Y=834+1115×X

LOD 50ng

LOQ 150ng

Standarddeviation(SD) 5.36 Correlationcoefficient(R2₎ _0.99

Table4

Intra-andinter-dayprecisionstudy(intermediateprecision)thequantificationof barbaloin.

Markercompound Concentration(␮g) Intra-day Inter-day

Barbaloin 0.5 1.34 1.45

1.0 1.65 1.23

1.5 1.39 1.56

Instrumentprecisionwasobservedbyscanningthesamespotof barbalointentimes(%CV=0.52;n=10).Therepeatabilityofthe methodwasinvestigatedbyanalyzingtenapplicationsofthesame standardsolution(%CV=0.62;n=10).Theaccuracyofthemethod wasdemonstratedatfourlevels(0,50,100,and150%)via addi-tionofknownamountsofbarbalointoleafskinextractofA.vera. Recovery atthefourlevels wasnotedtobe100, 100.5,100.25 and99.25%respectively(Table5).Repeatabilityandprecisionwere estimatedbymeasuringintra-dayandinter-day variation.Little intra-dayandinter-dayvariationwasnoted(Table4).Specificityof themethodwascalculatedbyacquiringthespectrumofbarbaloin standardandthecorrespondingpeakinthetest samplesinthe range200–800nm.Thespectraobtainedfromthebarbaloinand thebarbaloinpresentinleafpowdermatchedexactly,indicating

Table6

Barbaloincontentinvariouspopulations(insituandinvitro)ofAloevera.

Populations Barbaloincontent (insitu

populations)(%)

Barbaloincontent (invitropopulations) (%)

Jalandhar(Punjab) 2.23 2.32 Jodhpur(Rajasthan) 2.46 2.78 Varanasi(UttarPradesh) 2.34 2.38 Lucknow(UttarPradesh) 2.15 2.27 Bhubaneswar(Odisha) 2.20 2.29

nointerferencefromotherplantconstituents(Fig.3).LODandLOQ weredetermined(Table3)byusingtheequationsLOD=3×N/Band LOQ=10×N/B,whereNisthestandarddeviationofthepeakarea ofthestandard(n=3),takenasameasureofthenoiseandBisthe slopeofthecorrespondingcalibrationplot.

Barbaloincontentinsamples

Thepresentmethodwasusedtodeterminetheamountof bar-baloin (1)in leafskin extracts of A. vera.The identificationof barbaloinwasdoneonthebasisofRfvalues(Fig.3)and compari-sonofspectra(max362nm)(Fig.3).Agoodseparationofbarbaloin wasachieved(Fig.2).Calibrationcurveforbarbaloin(Fig.4)was foundtobelinearasshowninTable3.Thechromatogramsof var-iouspartsofinvitroraisedA.verafromJodhpurdistrictareshown inFig.2.LeafskinofinvitroculturedA.verafromJodhpurdistrict wasfoundtocontainthehighestamountofbarbaloin(2.78%) fol-lowedbytheinsitugrownleafskinsamplesfromthesamedistrict (2.46%).TheinsitugrownleafskinsamplecollectedfromLucknow districtwasfoundtocontaintheminimumamountofbarbaloin (2.15%).Intrapopulationalvariationsinbarbaloincontentamong

Table5

RecoverystudyofbarbaloinbyproposedHPTLCmethod.

Markercompound Amountpresentinthesample(␮g/mg) Amountadded(␮g) Amountfounda₍_␮_g) _Recovery_(%) _Average_recovery_(%)

Barbaloin 27.8 15 42.8±0.34 98.64 98.9

27.8 30 60.86±0.45 98.36

27.8 45 72.9±0.32 99.72

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T1=methanolic leaf extract (100 µg) (tissue cultured) T2=standard (barbolin) (4%)

T3=methanolic leaf extract (100 µg) (naturally grown) T4=standard (barbolin) (8%)

T5=methanolic gel extract (200 µg)

Fig.5.Achromatogram.

insituandinvitrogrownplantsamplesaretabulatedinTable6.A chromatogram(photographofTLCplateisprovidedinFig.5).

Anumberoffactorssuchasgenotype,culturemedium (includ-ing PGR and their combinations), physical environment and developmentalstageoftheexplantaffecttheadventitiousshoot regeneration fromtissue cultured explants (Zhang et al., 1998; Baiand Qu,2001).Moreover,theinvitroregenerationof direct adventitiousshootsisanessentialcomponenttoproduceplants fromelitematerialsastoavoidformationofsomaclones.Inthis presentstudy,BAPandNAAwereselectedforshootregeneration andmultiplicationasthesearereportedlyamongthePGRsused mostoftenfortheshootorganogenesis(KantiaandKothari,2002). Shootamplificationoccurredinalmost allthehormone combi-nationsbutacombinationofBAPandNAAwasnotedcrucialfor directshootregeneration.AccordingtoChaudhuriandMukundan (2001),thebestmediumforshootinductionfromshoottipsofA. verawasMSmediumsupplementedwith10mg/lBAP,160mg/l adeninesulphateand0.1mg/lIBA.However,thisisincontrastto theresultsobtainedinthepresentinvestigationaswhenBAPata higherconcentrationandincombinationwithNAAwasadded,it reducedtheshootproduction.OtherstudiesindicatethatBAPis moreefficientthanNAAforshootproliferationinA.vera(Velcheva etal.,2005;Debiasietal.,2007).Accordingtotheliterature,BAP isbetterthanothercytokininsforshootinitiationand prolifera-tion.TheshootsshowedrootinginMSmediumsupplementedwith 2.0mg/lBAP+0.5mg/lNAA.Someresearchersreportedrootingin hormone-freemedium(Natalietal.,1990;AggarwalandBarna, 2004)whilesomeothersshowedthepresenceofPGR is neces-saryforrooting(MeyerandStaden,1991;AbrieandStaden,2001; Velchevaetal.,2005).

Micropropagationisbeingconsideredasanadvancedtoolfor theproductionofhighvaluephytochemicalswithmedicinal poten-tial(Debnathetal.,2006).Growthregulatorsusedinvitrowasfound toenhancesomebiologicalactivityandmodulatephytochemical contentintissuecultureplantletswhichareotherwisedifferentin

theiracclimatizedcounterparts.Inonestudy,variouscytokinines werefoundtoinfluencephenolicacidsandantioxidantactivityof tissueculturedandacclimatizedMerwillaplumbea(Aremuetal., 2013).Inourpresentinvestigation,wehavefoundenhanced bar-baloincontentfromtheleavesofmicropropagatedplantsgrowing undertheinfluenceoftwogrowthregulators.Similarlyplantcell culturesarealsoreportedasasourceofmicro-propagationof virus-freeplantsusefulfornovelsecondarymetaboliteproduction(Sato, 2013).Invitropropagationalsoservesasavaluabletoolfor prop-agationavoidingtheoverexploitationofnaturalpopulationsand toapplybiotechnology-basedapproaches(Gonc¸alvesandRomano, 2013).

Inthepresentstudy,asimpleandvalidatedHPTLCmethodfor theseparation and quantification ofbarbaloin in A. vera leaves frominvitroand naturallygrown plantswaspresented. Italso quantifiesbarbaloincontentfrominvitroandinsitugrownplant samples.Invitroderivedleaveswerefoundtocontainmore bar-baloin than in situ grown samples. Besides being useful as a reproducible methodfor quantification of barbaloin fromplant samples,theresultsalsoindicatethepossibleenhancementof sec-ondarymetaboliteproductionfromtissueculturedplant.Bioticand abioticstressesdevelopedduringtissueculturalconditionmaybe exploitedinordertomodulatesomeplantmetabolitewithpossible therapeuticvalue.

Authorcontributions

DKPmonitoredtheexperimentsandpreparedthemanuscript draft.SPperformedtheexperiments.ADdesignedthe experimen-tationandfinalizedthemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthoristhankfultotheDepartmentofMedicinal Chem-istry, Institute of Medical Sciences, Banaras Hindu University, VaranasiforprovidingHPTLCfacilityforanalysisoftheplant mate-rials.

References

Abrie,A.I.,Staden,J.V.,2001.MicropropagationoftheendangeredAloepolyphylla. PlantGrowthRegul.33,19–23.

Aggarwal,D.,Barna,K.S.,2004.TissueculturepropagationofeliteplantofAloevera L.J.PlantBiochem.Biotechnol.13,77–79.

Aremu,A.O.,Gruz,J., ˇSubrtová,M.,Szüˇcová,L.,Doleˇzal,K.,Bairu,M.W.,etal.,2013. Antioxidantandphenolicacidprofilesoftissueculturedandacclimatized Mer-willaplumbeaplantletsinrelationtotheappliedcytokinins.J.PlantPhysiol.170, 1303–1308.

Bai,Y.,Qu,R.,2001.Factorsinfluencingtissuecultureresponsesofmatureseedsand immatureembryosinturf-typetallfescue.PlantBreed.120,239–242. Chang,X.L.,Wang,C.,Feng,Y.,Liu,Z.,2006.Effectsofheattreatmentsonthe

sta-bilitiesofpolysaccharidessubstancesandbarbaloiningeljuicefromAloevera Miller.J.FoodEng.75,245–251.

Chaudhuri,S.,Mukundan,U.,2001.AloeveraL.micropropagationand characteriza-tionofitsgel.Phytomorphology51,155–157.

Chen,J.D.,Li,W.,Li,S.Y.,2002.Quantitativedeterminationofbarbaloininaloe capsulebyhighperformanceliquidchromatography.SePu20,367–368. Coran,S.A.,Bartolucci,G.,Bambagiotti-Alberti,M.,2011.Selectivedetermination

ofaloinindifferentmatricesbyHPTLCdensitometryinfluorescencemode.J. Pharm.Biomed.Anal.54,422–425.

Debiasi,C.,Silva,C.G.,Pescador,R.,2007.MicropropagationofAloeveraL.Rev.Bras. PlantMed.Botucatu9,36–43.

Debnath,M.,Malik,C.P.,Bisen,P.S.,2006.Micropropagation:atoolfortheproduction ofhighqualityplant-basedmedicines.Curr.Pharm.Biotechnol.7,33–49. Gonc¸alves,S.,Romano,A.,2013.Invitrocultureoflavenders(Lavandulaspp.)and

(7)

Gutterman, Y., Chauser-Volfson, E., 2000. The distribution of the phenolic metabolitesbarbaloin,aloeresinandaloeninasaperipheraldefense strat-egyinthesucculentleafpartsofAloearborescens.Biochem.Syst.Ecol.28, 825–838.

Hamman,J.H.,2008.CompositionandapplicationsofAloeveraleafgel.Molecules 13,1599–1616.

Hashemi,S.A.,Madani,S.A.,Abediankenari,S.,2015.ThereviewonpropertiesofAloe verainhealingofcutaneouswounds.Biomed.Res.Int.2015,714216. Kantia,A.,Kothari,S.L.,2002.Highefficiencyadventitiousshootbudformationand

plantregenerationfromleafexplantsofDianthuschinensisL.Sci.Hortic.96, 205–212.

Klein,A.D.,Penneys,N.S.,1988.Aloevera.J.Am.Acad.Dermatol.18,714–720. Mangaiyarkarasi,S.P.,Manigandan,T.,Elumalai,M.,Cholan,P.K.,Kaur,R.P.,2015.

BenefitsofAloeveraindentistry.J.Pharm.Bioallied.Sci.7,S255–S259. Meyer,H.J.,Staden,J.V.,1991.RapidinvitropropagationofAloebarbadensisMill.

PlantCellTissueOrgan.Cult.26,167–171.

Molazem,Z.,Mohseni,F.,Younesi,M.,Keshavarzi,S.,2014.Aloeveragelandcesarean woundhealing;arandomizedcontrolledclinicaltrial.Glob.J.HealthSci.7, 203–209.

Natali,L.,Sanchez,I.C.,Cavallini,A.,1990.InvitrocultureofAloebarbadensisMill.: micropropagationfromvegetativemeristems.PlantCellTissueOrgan.Cult.20, 71–74.

Ngo,M.Q.,Nguyen,N.N.,Shah,S.A.,2010.OralAloeverafortreatmentofdiabetes mellitusanddyslipidemia.Am.J.HealthSyst.Pharm.67,1806–1808. Pandey,D.K.,Malik,T.,Banik,R.M.,2012.Quantitativeestimationofbarbaloinin

AloeveraanditscommercialformulationsbyusingHPTLC.Int.J.Med.Aromatic Plants2,420–427.

Park,C.H.,Nam,D.Y.,Son,H.U.,Lee,S.R.,Lee,H.J.,Heo,J.C.,etal.,2011.Polymer fractionofAloeveraexhibitsaprotectiveactivityonethanol-inducedgastric lesions.Int.J.Mol.Med.27,511–518.

Patel,D.K.,Patel,K.,Tahilyani,V.,2012.Barbaloin:Aconcisereportofits pharmaco-logicalandanalyticalaspects.AsianPac.J.Trop.Biomed.2,835–838.

Rebecca,W.,Kayser,O.,Hagels,H.,Zessin,K.H.,Madundo,M.,Gamba,N.,2003.The phytochemicalprofileandidentificationofmainphenoliccompoundsfromthe leafexudateofAloesecundiflorabyhigh-performanceliquid chromatography-massspectroscopy.Phytochem.Anal.14,83–86.

Reynolds,T.,Dweck,A.C.,1999.Aloeveraleafgel:areviewupdate.J. Ethnopharma-col.68,3–37.

Roy,S.C.,Sarkar,A.,1991.InvitroregenerationandmicropropagationofAloeveraL. Sci.Hortic.47,107–113.

Saeed,M.A.,Ahmad,I.,Yaqub,U.,Akbar,S.,Waheed,A.,Saleem,M.,etal.,2004.Aloe vera:aplantofvitalsignificance.Q.Sci.Vis.9,1–13.

Sato,F.,2013.Characterizationofplantfunctionsusingculturedplantcells,and biotechnologicalapplications.Biosc.Biotechnol.Biochem.77,1–9.

Surjushe,A.,Vasani,R.,Saple,D.G.,2008.Aloevera:ashortreview.Ind.J.Dermatol. 53,163–166.

Tanaka,M.,Misawa,E.,Yamauchi,K.,Abe,F.,Ishizaki,C.,2015.Effectsofplantsterols derivedfromAloeveragelonhumandermalfibroblastsinvitroandonskin conditioninJapanesewomen.Clin.Cosmet.Investig.Dermatol.20,95–104. Velcheva,M.,Faltin,Z.,Vardi,A.,Eshdat,Y.,Perl,A.,2005.RegenerationofAloe

arborescensviasomaticorganogenesisfromyounginflorescences.PlantCell TissueOrgan.Cult.83,293–301.

Wagner,H.,Bladt,S.,1996.Plantdruganalysis:athinlayerchromatographyatlas. SpringerScience&BusinessMedia.

Yonehara,A.,Tanaka,Y.,Kulkeaw,K.,Era,T.,Nakanishi,Y.,Sugiyama,D.,2015.Aloe veraextractsuppressesproliferationofneuroblastomacellsinvitro.Anticancer Res.35,4479–4485.

Zahn,M., Trinh,T.,Jeong,M.L., Wang,D.,Abeysinghe, P.,Jia,Q.,etal.,2008. Areversed-phasehigh-performanceliquidchromatographicmethodforthe determinationofaloesin,aloeresinAandanthraquinoneinAloeferox. Phy-tochem.Anal.19,122–126.