Rev. bras. farmacogn. vol.26 número6

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

Original

Article

Bioassay

guided

purification

of

cytotoxic

natural

products

from

a

red

alga

Dichotomaria

obtusata

Amir

Reza

Jassbi

_,

_Younes

_Mirzaei

_,

_Omidreza

_Firuzi

_,

_Jima

_N.

_Chandran

_,

_Bernd

_Schneider

b_{ResearchGroupBiosynthesis/NMR,MaxPlanckInstituteforChemicalEcology,Jena,Germany}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21January2016

Accepted7June2016

Availableonline12August2016

Keywords:

Dichotomariaobtusata Redalgae

Trans-phytolpalmitate Tocopherol

Cytotoxicactivity

a

b

s

t

r

a

c

t

DifferentsolventextractsofDichotomariaobtusata(J.Ellis&Solander)Lamark,Galaxauraceae,ared algaecollectedfromthecoastofBushehrinthePersainGulf,wasinvestigatedforitscytotoxic prop-ertiesandchemicalconstituents.Thefreshalga,afterextractionwithmethanolanddichloromethane werecombinedandpartitionedbetweenwater,dichloromethaneandethylacetate.Theabovefractions werethentestedagainstMOLT-4(humanlymphoblasticleukemia)cancercellline.TheIC50valuesof thedichloromethaneandethylacetatelayersofthecrudeextractwere29.8±3.1and30.6±7.9␮g/ml againstMOLT-4cells,respectively,whilethewaterlayershowedaweekactivitywithIC50>50␮g/ml. Afterfractionationoftheactiveextractsusingopencolumnchromatographyoversilicageland prepar-ativethinlayerchromatographypurification,twoterpenoidderivedcompounds,trans-phytolpalmitate and␥-tocopherolwereisolatedfromthedichloromethaneandethylacetateextracts.Thestructuresof thecompoundswereelucidatedusingdifferentspectraldataincluding1_HNMR,13_{CNMR,HSQC,HMBC} andEI-MS.TheIC50valuesofcompoundstrans-phytolpalmitate,␥-tocopherolandanundetermined mix-tureofcompounds(F-13-14)weredeterminedas43.4±1.6,–and20.3±6.2␮g/mlagainstLS180(human colonadenocarcinoma);53.2 ± 9.3,>100and27.6± 6.9␮g/mlagainstMCF-7(humanbreast adenocarci-noma)and40.0±4.1,48.8±1.8and15.9±0.3␮g/mlagainstMOLT-4celllines,respectively,whichwere comparabletotheIC50valuesofstandardanticanceragent,cisplatinagainstthesamecelllines.Thered algaecollectedfromthePersianGulfcontainedsubstancesthatcouldinhibitthegrowthofhumancancer celllinesandmayrepresentanaturalsourceforthediscoveryofnovelanticanceragents.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

ThenortherncoastsofthePersianGulfandtheGulfofOman intheIranianterritoryaresuitednaturalhabitatsforthegrowth of differenttypesof marineorganisms suchasred, brownand green algae. Dichotomaria obtusata (J. Ellis & Solander) Lamark is a redalga from thefamily Galaxauraceae reportedin differ-entcoastalareafromIndianandpacificoceansinKenyan(Bolton etal.,2007)andCubancoastalarea(Vázquezetal.,2011;Delgado etal.,2013)andalsofromGavaterandJoudintheGulfofOman (SohrabipourandRabei,2008).Theredandbrownalgaeareknown asasourceofbiologicallyactivenaturalproductswithcytotoxic, and antimicrobial activity against cancer cells, and pathogenic bacteria and fungi, respectively. For instance, different solvent extractsofHypneaflagelliformis,CystoseiramyricaandSargassum

∗ _{Correspondingauthor.}

E-mail:jassbiar@sums.ac.ir(A.R.Jassbi).

boveanumshowedantimicrobialactivityagainstbothgram pos-itive andgramnegativebacteriaand alsoexhibitedantioxidant potentialinan1,1-diphenyl-2-picrylhydrazyl(DPPH)freeradical scavengingassay(Jassbietal.,2013).Thephytochemicalanalyses ofthealgalextractsresultedinidentificationandquantificationof theirfattyacidsandsteroids(Jassbietal.,2013).

An aqueous extract of a fresh collection of D. obtusata has shownantineoceptiveandantiinflammatoryactivitiesinamouse modelof12-O-tetradecanoylphorbolacetate-inducedearedema and aceticacidwrithingin adose-dependent manner(Vázquez etal.,2011).Thepresenceofdifferentkindsofnaturalproducts in thealgal water extract, liketerpenoids, coumarins, lactones, phenolics and polysaccharides has been explored by chemical tests(Vázquez etal.,2011).Furthermore,theanalgesiceffectof amethanolicextractofD.obtusatahasalsobeenexaminedinthe crotonoil-inducedearedemamodelanditsphospholipaseA2 inhi-bitionhasbeenstudied(Delgadoetal.,2013).Thephytochemical analysesofdifferentsolventextractsshowedthepresenceof triter-penoids, flavonoids,phenolics,steroidsand fats(Delgadoetal.,

http://dx.doi.org/10.1016/j.bjp.2016.06.008

0102-695X/©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

2013).However,tothebestofourknowledge,nofurther phyto-chemicalinvestigationhasbeenperformedonthisalgaspecies.

Wereportherethechemicalconstituentsandcytotoxicactivity ofaDCM-EtOAcextractofD.obtusataanditschemicalconstituents collectedfromthecoastsofBushehrintheNorthernpartsofthe PersianGulfofIran.

Materialsandmethods

Instrumentsandadsorbents

NMR spectra of the purified compounds were recorded on a Bruker Avance 500 spectrometer (Bruker Biospin, Karlsruhe, Germany),operatingatresonancefrequenciesof500.13MHzfor 1_{H and 125.75MHz for} 13_{C, respectively. The NMR}

spectrome-terwas equippedwitha 5mm TCI cryoprobe.Standard Bruker pulsesequenceswereusedformeasuring1_HNMR,13_CAPT,1_H–1_H COSY,1_H–13_CHSQCand1_H–13_{CHMBCspectra.Tetramethylsilane} (TMS)wasusedasaninternalstandardforreferencing1_Hand13_C NMRspectra.NMRtubes(5mmi.d.and2mmi.d.)wereusedfor measurements.Dataacquisitionandprocessingwasaccomplished usingBrukerTopspin2.1.EI-MSwasrecordedonanAgilent5975C inertGC/MSinstrument.The chromatography separationswere performedusingopenCCusingsilicagel60(0.2–0.04mmparticle size),FCCusingsilicagel60(0.040–0.063mmparticlesize)andTLC usingsilicagel60F254pre-coatedplates(0.25mmfilmthickness). TheadsorbentswerepurchasedfromMerck,Darmstadt,Germany.

Algalmaterialandextractionprocedure

AllpartsofDichotomariaobtusata(J.Ellis&Solander)Lamarck, Galaxauraceae,werecollectedinApril2011fromthecoastalarea ofBushehrCityatthecoordinate(28◦₅₈′₄₂′′_N;50◦₄₉′₅₃′′_E)in thewestpartofthecoastofBushehr)inthePersianGulf,Iran,and wasidentifiedbyDr.JelvehSohrabipour(taxonomist).Avoucher specimen(PC-94-6-7-1.1) hasbeendeposited attheherbarium ofMNCRC.Thefreshalga(9.5kg)wasmaceratedfortwodaysin methanol(MeOH;2×10l)followedbythesamevolumeofDCM successivelyatroomtemperature.TheDCMandMeOHextracts weremixedand afterremovaloftheirsolventsinvacuum sub-jectedtoliquidextractiontoaffordDCM(3.1g),EtOAc(18.6g)and watersoluble(180g)extracts.

Isolationandpurificationoftheplantextracts

ThebioactiveDCMandEtOAcextracts(21.4g)werepooledand subjectedtocolumn(60×3.5cm)chromatographyoversilicagel (100g,0.2–0.04mm).Thecolumnwaselutedusingn-hexanewith gradientofDCMupto100%andthenfollowedbyincreasingthe polarityofthemobilephase withMeOH toafford27 fractions. Thefractionseluted fromthecolumn werecheckedby TLCfor theirpurityandsimilarfractionswerepooled.Thefractionswere subjectedtoacytotoxicitybioassayonMOLT-4celllinesandthe followingfractionswerefoundtobeactive(F7-F9=2.164g), (F10-F12=10g),(F13-F15=0.942g)and(F16-F21=0.918g).F-7-F9was subjectedtosilicagelFCCusinghexane–EtOAcasthemobilephase toafford25fractions.Thepurecompound(1)wasobtainedasa gummymaterialfrom fractionsF7-12,104mg.The F13-15was analyzedbythesameFCCmethodasforF97-F9andthepurityof thetwosubstancesF13-13(2,52.8mg)andF-13-14(80mg)were checkedbysilicagelTLC.AlthoughF-13-14detectedasasingle spotonsilicagelTLC(CHCl3:MeOH;9:1),aftersparingand subse-quentheatingat110◦_{C,by1%vanillineinH}

2SO4/ethanolreagent, butboth13_Cand1_{HNMRspectraldata(dataarenotshown)and}

RP-18TLC(MeOH:H2O;6:4)analysesshowedthatthemixtureis consistedofatleastfour–fivecompounds.

Transesterficationofcompound1andGC-MSanalysesofthe resultingfattyacidmethylester

Compound1(1mg)wastransesterifiedusing250␮lofa10%BF3 inMeOHasdescribedpreviously(Jassbietal.,2013).TheGC–MS analysesoftheresultingesterwasperformedona7890AGC cou-pledtoHP-6890massspectrometeroperatinginEImodeat70eV. ThecolumnwasaDB-5MS(J&WScientificcolumn,30m×0.25mm i.d.,0.25␮mfilmthickness).Onemicroliterofthefattyacidmethyl estersinahexane solutionwasanalyzedinthesamecondition describedpreviously;theoventemperaturewassetat150◦_Cand after4min,roseto250◦_Cat4◦_{C/minandkeptfor10minat250}◦_C (Jassbietal.,2013).

Spectroscopicdata

Trans-phytylpalmitate(codioester,1):EI-MSm/z(rel.int.%): 534[C36H70O2]+(trace),296[M-palmitate]+(1),292(3),278(6), 256(45),227(5),213(18),185(11),157(10),123(32),111(20), 97(31),71(100),55(54).1_{HNMR(500MHz,CDC1}

3):ı5.40(1H,t,

J=7.1Hz,H-2),4.16(2H,d,J=7.0Hz,H-1,1′_{),2.33(2H,t,J=7.6Hz,} H-2′_{), 1.2–1.65 (20 H, m, CH}

2), 1.66(3H, s, H-3a),0.88 (3H, t,

J=7.0Hz,H-16′_{),0.84(12H,d,J=6.5Hz,H-7a,11a,15a,16).}13_C NMR(125MHz,CDC13):ı179.9(C-1′),140.4(C-3),122.9(C-2),59.4 (C-1),39.88(C-4),39.4(C-14),37.45(C-10),37.37(C-8),37.3(C-12), 36.75(C-6),34.12(C-2′_{),32.81(C-11),32.73(C-7),29.78–29.02} (C-3′_-13′_{),28.01(C-15),24.82(C-13),25.1(C-5),24.51(C-9),22.72} (C-15a),22.68(C-16),19.71(C-11a),19.76(C-7a),16.2(C-3a),14.14 (C-16′_).

-Tocopherol (2): EI-MS m/z (rel. int.): 416 [M]+ _{(100), 191} [C12H15O2]+ (20), and 151 [C9H11O2]+ (85).1H NMR (500MHz, CDCl3):ı6.94(1H,s,H-5),2.50(2H,m,H-4),2.09(s,C7-CH3),2.00 (s,C8-CH3),1.58(2H,m,H-3),1.54(m,H-12′),1.50(m,H-1′),1.4(m, 4H,H-9′_,8′_,7′_,3′_),1.33(m,H-2′_{),1.27(m,H-10}′_),1.22(m,H-6′_),1.15 (m,H-11′_{),1.08(2H,m,H-4}′_,5′_{),1.07(s,C-2-CH}

3),0.88(d,J=6.6Hz, H-13′_,C12′_-CH

3), 0.86(d,J=6.6Hz,C-8′-CH3), 0.85(d,J=6.6Hz, C-4′_-CH

3)(BakerandMyers,1991).13CNMR(125MHz,CDCl3):ı 149.3(C-9),142.3(C-6),128.8(C-7),125.3(C-8),119.7(C-5),118.3 (C-10),75.8(C-2),40.9(C-1′_),39.4(C-11′_),37.59(C-3′_),37.6(C-7′_), 37.5(C-5′_),37.3(C-9′_),32.8(C-4′_),32.8(C-8′_{),31.1(C-3),28.0} (C-12′_),24.8(C-10′_),24.5(C-6′_),23.5(C2-CH

3),22.7(C12′-CH3),22.6 (C-13′_{),21.9(C-4),21.0(C-2}′_),19.7(C8′_-CH

3),19.6(C4′-CH3),13.0 (C7-CH3),11.9(C8-CH3)(BakerandMyers,1991).

Statisticalanalyses

TheIC50 values inTable1 aretheaverageof 3–5replicates. One-wayanalysesofvariance(ANOVA)andposthocmultiple com-parisontestswereusedfordeterminationofsignificationp≤0.05 betweendifferentmeasurementsusingSPSS(version11.5for win-dows)software.

Cytotoxicityassay

Celllines

Table1

Cytotoxicactivityofthealgalextracts,fractionsfromcolumnchromatographyandtheisolatedpurecompoundsagainstselectedhumancancercelllines.

Algalextracts/fractions/purecompounds IC50LS180A IC50MCF-7 IC50MOLT-4

Aqueousfraction –B _{– >50}A

Ethylacetatefraction – – 30.6±7.9bcC

Dichloromethanefraction – – 29.8±3.1bc

F7-9 – – 21.8±2.2cd

F10-12 – – 25.1±3.4c

F13-15 – – 27.1±4.8c

F16-21 – – 28.4±4.1c

F7-12(1) 43.4±1.6a 53.2±9.3a 40.0±4.1b

F13-13(2) – >100 48.8±1.8a

F13-14 20.3±6.2b 27.6±6.9b 15.9±0.3d

Doxorubicin 0.017±0.006c 0.036±0.004c 0.013±0.002e

Cisplatin 1.7±0.7c 2.4±0.8c 0.7±0.4e

A_IC

50valuesareexpressedas␮g/mlandarethemean±S.D.of3–4experiments.

B _{Notdetermined.}

C _{Valuesthatdonotshareanyletterhaveastatisticallysignificantdifferenceatp<0.05.}

100units/mlpenicillin-Gand100␮g/mlstreptomycin.Thepassage numberafterthawingcellswaskeptbelowtwelve.

Cellviabilityfollowingexposuretoplantextractswasmeasured bytheMTTreductionassay(Firuzietal.,2010;Mosmann,1983). Briefly,5000cells wereseededin96-wellmicroplatesandafter overnightincubation,3–4differentconcentrationsoftheextracts orpurecompounds(5,10,25 and50␮g/ml)wereaddedtothe wellsinduplicate.Extractsorisolatedcompoundswerefirst dis-solvedindimethylsulfoxide(DMSO)andthendilutedinthegrowth mediumatthedesiredconcentration.DMSOconcentrationinthe wellswaskeptunder0.25%.Cellswerefurtherincubatedfor72h andtheirgrowthmediumwasreplacedwithfreshmedium con-taining0.5mg/mlMTTandincubatedfor4hat37◦_C.Themedia wasremovedandtheformazancrystalsweredissolvedbyadding 200␮lDMSOtoeachwell.Theabsorbancewasmeasuredat570nm withbackgroundcorrectionat655nmusingaBio-Radmicroplate reader(Model680).ForMOLT-4cellsthatgrowinsuspension,the plateswerecentrifugedat500×gfor10minbeforeeachstepthat totalorpartialremovalofthemediawasnecessary.Thepercent inhibitionofviabilityforeachconcentrationoftheextractorpure compoundwascalculatedwithreferencetotheuntreatedcellsand IC50valueswerecalculatedwiththesoftwareCurveExpertversion 1.34forWindows(Fig.1).Eachexperimentwasrepeated3–4times. Knowncytotoxicagents,doxorubicinandcisplatinweretestedas positivecontrols.

Resultanddiscussion

ThecytotoxicactivityofEtOAc,waterandDCMsolublepartsof theDCM-MeOHextractofD.obtusatawereassayedagainst MOLT-4celllineinMTTcolorimetricassay.TheEtOAcandDCMextracts could effectively lower the viability of cancer cells with IC50s of30.6±7.9 and29.8±3.1(mean±S.D.),respectively andwere moreeffectivethantheaqueousphase(IC50>50␮g/ml)(Table1). Therefore,wecombinedbothDCMandEtOAcextractsand sub-jectedthemtodifferentchromatographytechniquestoseparate thebioactiveconstituentsoftheextractsinbioassayguided purifi-cation(Table1).

Trans-phytyl-1-palmitate(Codioester)(1)and␥-tocopherol(2) andfractionF-13-14 exhibitedcytotoxicactivitiesagainstthree cancercell lines LS180 (human colon adenocarcinoma), MCF-7 (humanbreastadenocarcinoma)andMOLT-4cells(humanacute lymphoblasticleukemia)withIC50srangingfrom15.9tomorethan 100␮g/ml(Table1).Allof2compoundsandF-13-14showed activ-itiesthatweresignificantlylowerthanstandardcytotoxicagents, doxorubicinandcisplatin.

Compounds1and2werepurifiedusingsilicagelCC,flash col-umnchromatography (FCC) and preparative TLC. The chemical

structuresofthecompoundswereelucidatedusingdifferent spec-traldataincluding1Dand2DNMRandEI-MSandcomparingtheir spectraldatawiththosedescribedintheliterature(Alietal.,2001; BakerandMyers,1991).

Briefly, compound 1 (104mg) was purified as a white pre-cipitate and thensubjected toEIMS exhibiting ionsat m/z296 (M+_-C

16H32O2),278,71(100%)andm/z256suggestingtheterpene fragments(C20H38),(C5H11)andtheacylmoiety(C16H32O2)ofthe molecule.Howeverthemolecularion[C36H70O2]+atm/z534in thespectrumwasdetectedastracepeakduetoeasylossofthe esterfragmentfromthemolecule.Inthe1_{HNMRspectrumofthe} compound1,agroupofmultipletsatı0.84–0.88represented15 hydrogenatomsoffourisoprenoidmethylgroupsandoneterminal methylgroupofthelongchainacylgroup.Asingletatı1.66for theolefinicmethylgroup,atripletofH-2atı5.41(J=7.1Hz)and adoubletatı4.16(J=7.0Hz)ofthephytolhydroxymethylenepart ofmoleculecompletedtheassignments.Theconfigurationatthe phytoldoublebondwasdeterminedusingtheattachedprotontest (APT)13_{CNMRspectrumbyobservingsignalsatı59.4(C-1),122.9} (C-2)and140.4(C-3)recordedpreviouslyfortrans-phytol palmi-tate(Codioester)isolatedfromagreenalgaeCodiumiyengarii(Ali etal.,2001).Compound1wastransesterifiedtoitsmethylester using10%BF3 inmethanolasdescribedpreviously(Jassbietal.,

2013).TheGC–MSanalysisoftheresultingfattyacidmethylester ofthecompoundresultedindetectionofmethylpalmitate.

Concentration (µM)

60 50 40 30 20 10 0

%

V

ia

vil

it

y

-20 0 20 40 60 80 100 120

LS180 cells

60 50 40 30 20 10 0

% V

ia

b

ili

ty

0 20 40 60 80 100 120 140

60 50 40 30 20 10 0

% V

ia

b

ili

ty

0 20 40 60 80 100 120

MCF-7 cells

MOLT-4 cells

Fig.1.Assessmentofcytotoxicityofcompounds1,3andF-13-14againsthuman

cancercelllines.Cytotoxicactivityoftheisolatedcompoundsincludingcompound1

(),compound2(䊉)andF-13-14()weretestedatdifferentconcentrationsagainst

LS180,MCF-7andMOLT-4cancercelllinesbyMTTreductionassay.

singletat1.13(3H)suggestedasaturatedacyclicditerpenemoiety inthemolecule.TheAPT13_{CNMRspectrumwasmoreinformative} withrepresenting29signalsconsistingoffivequaternarycarbons, fourmethynes,fourteen methyleneand sixmethyl signals.The quaternarycarbons atı 149.3,142.3,128.6,125.3,118.3and a methyneat ı119.7suggested a fivecarbon substitutedphenyl groupinthemolecule.Adownfieldsignalatı75.8togetherwith itsHMBCcorrelationwiththe methylsignalatı 1.13and two methylenesignalsatı21.9and31.18wereapartofachromane system.AnHMBCcorrelationbetweentheH-5andC-4confirmed thestructureofthecompound2asgammaisomeroftocopherol (Fig.2).Theabovementionedspectraldatawerecompatiblewith the␥-tocopherol(␥T)structurebiosynthesizedbydifferentalgae (BakerandMyers,1991).Howeverthereweredifferencesbetween

HO

O

Fig.2. TheHMBCcorrelations(500MHz,CDCl3)forassignmentofthesubstitution

andchemicalshiftsof1_Hand13_{CNMRsignalsof␥-tocopherol(2).}

chemical shiftsof thearomaticringof compound2 withthose recordedford-␥T(BakerandMyers,1991).

Severalbiological activitiesincluding antioxidant,anticancer andanti-inflammatoryactivitieshavebeenreportedforvitamin Eincluding␣T,␤T,␥T,␦Tandalsotheirmetabolicproductssuch astocopherylquinines(Hensleyetal.,2004).Ourresultsthatshow theabundantpresenceof␥TinD.obtusatamayexplainthe anti-inflammatory and antinociceptive activity of themethanol and aqueousextractsofthisalgapreviouslyreportedforacollection fromCuban coasts(García Delgadoet al.,2013; Vázquez etal., 2011).

Inarecentstudy,phytolalcoholhasshownanticanceractivityin hepatocellularcarcinomacells(Kimetal.,2015).Phytol’s cytotox-icityhasalsobeenreportedagainstseveralothercancercelllines (Pejinetal.,2014).Thismaysuggestthatthecytotoxicactivityof compound1ismainlyduetothepresenceofitsditerpenoidpart ratherthanitsfattyacidmoiety.However,tothebestofour knowl-edge,thisisthefirstreportonthecytotoxicactivityofaphytolester againstcancercells.

Conclusion

TheredalgaecollectedfromthePersianGulfcontained com-pounds1and2andotherunidentifiedsubstances(F-13-14)that couldinhibitthegrowthofhumancancercelllinesandmay repre-sentanaturalsourceforthediscoveryofnovelanticanceragents. Our primary cytotoxic bioassays showed that we still need to characterizethestructuresofthephytochemicalsofotheractive fractions(F-13-14)inthefuture.

Authors’contribution

ARJ:supervisingtheresearch,analyzingdataanddetermining thestructuresofnaturalproducts,writingthepaper,YM:chemical analyzingofthealgalmaterial,OF:performingbiologicalactivity, preparationofthemanuscript;JNC:performingNMRspectroscopy andhelpinginstructuralelucidationofnaturalproducts,BS: help-inginstructuralelucidationofnaturalproductsandeditingthe manuscript.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Conflictsofinterest

Acknowledgements

WewouldliketoexpressouracknowledgmenttoIranNational ScienceFoundation(ResearchGrantNo.88000545)andVice Chan-cellorforResearch,ShirazUniversityofMedicalSciencesforthe financialsupportofthisproject. Theauthorsalsowish tothank ProfessorIrajNabipourandDr.JelvehSohrabipourforcollection andidentificationofthealgalmaterialrespectively.

References

Ali,M.S.,Saleem,M.,Ahmad,V.U.,Shameel,S.,2001.Phytolandglycerolderivatives

fromthemarinegreenalgaCodiumiyengariioftheKarachiCoast(ArabianSea). Z.Naturforsch.B56,837–841.

Baker,J.,Myers,C.,1991.One-dimensionalandtwo-dimensional1_H-and13

C-nuclearmagneticresonance(NMR)analysisofvitaminErawmaterialsor analyticalreferencestandards.Pharmacol.Res.8,763–770.

Bolton,J.J.,Oyieke,H.A.,Gwada,P.,2007.TheseaweedsofKenya:checklist,history

ofseaweedstudy,coastalenvironment,andanalysisofseaweeddiversityand biogeography.S.Afr.J.Bot.73,76–88.

Delgado,N.G.,FríasVázquez,A.I.,Sánchez,H.C.,delValle, R.M.S.,Gómez, Y.S.,

SuárezAlfonso,A.M.,2013.Anti-inflammatoryandantinociceptiveactivitiesof

methanolicextractfromredseaweedDichotomariaobtusata.Braz.J.Pharm.Sci. 49,65–74.

Firuzi,O.,Asadollahi,M.,Gholami,M.,Javidnia,K.,2010.Compositionand

biolog-icalactivitiesofessentialoilsfromfourHeracleumspecies.FoodChem.122, 117–122.

GarcíaDelgado,N.,FríasVázquez,A.I.,CabreraSánchez,H.,delValle,S.,Menéndez,

R.,SierraGómez,Y.,Alfonso,S.,María,A.,2013.Anti-inflammatoryand

antinoci-ceptiveactivitiesofmethanolicextractfromredseaweedDichotomariaobtusata. Braz.J.Pharm.Sci.49,65–74.

Hensley,K.,Benaksas,E.J.,Bolli,R.,Comp,P.,Grammas,P.,Hamdheydari,L.,Mou,

S.,Pye,Q.N.,Stoddard,M.F.,Wallis,G.,2004.Newperspectivesonvitamin

E:␥-tocopherolandcarboxyethylhydroxychromanmetabolitesinbiologyand medicine.FreeRadic.Biol.Med.36,1–15.

Jassbi,A.R.,Mohabati,M.,Eslami,S.,Sohrabipour,J.,Miri,R.,2013.Biologicalactivity

andchemicalconstituentsofredandbrownalgaefromthePersianGulf.IranJ. Pharm.Res.12,339–348.

Kim,C.W.,Lee,H.J.,Jung,J.H.,Kim,Y.H.,Jung,D.B.,Sohn,E.J.,Lee,J.H.,Woo,H.J.,

Baek,N.I.,Kim,Y.C.,Kim,S.H.,2015.Activationofcaspase-9/3andinhibitionof

epithelialmesenchymaltransitionarecriticallyinvolvedinantitumoreffectof phytolinhepatocellularcarcinomacells.Phytother.Res.29,1026–1031.

Mosmann,T.,1983.Rapidcolorimetricassayforcellulargrowthandsurvival:

appli-cationtoproliferationandcytotoxicityassays.J.Immunol.Methods65,55–63.

Pejin,B.,Kojic,V.,Bogdanovic,G.,2014.Aninsightintothecytotoxicactivityof

phytolatinvitroconditions.Nat.Prod.Res.28,2053–2056.

Sohrabipour,J.,Rabei,R.,2008.RhodophytaofOmanGulf(SouthEastofIran).Iran

J.Bot.14,70–74.

Vázquez, A.I.F., Sánchez, C.M.D., Delgado, N.G., Alfonso, A.M.S., Ortega, Y.S.,

Sánchez,H.C.,2011.Anti-inflammatoryandanalgesicactivitiesofredseaweed