Anti-angiogenic activity of iridoids from Galium tunetanum

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RevistaBrasileiradeFarmacognosia28(2018)374–377

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

Short

communication

Anti-angiogenic

activity

of

iridoids

from

Galium

tunetanum

César

Mu ˜

noz

Camero

a

_,

_Maria

_Paola

_Germanò

b

_,

_Antonio

_Rapisarda

b

_,

_Valeria

_D’Angelo

b

_,

Smain

Amira

c

_,

_Fatima

_Benchikh

c

_,

_Alessandra

_Braca

a,∗

_,

_Marinella

_De

_Leo

a

a_Dipartimento_di_Farmacia,_Università_di_Pisa,_Pisa,_Italy

b_Dipartimento_di_Scienze_Chimiche,_Biologiche,_{Farmaceutiche}_e_Ambientali,_Università_degli_Studi_di_Messina,_Polo_{Universitario}_SS,_Annunziata,_Messina,_Italy c_Department_of_Animal_Biology_and_Physiology,_University_of_Setif,_Setif,_Algeria

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29January2018 Accepted29March2018 Availableonline25May2018 Keywords:

Anti-angiogenicactivity Asperuloside

Chickchorioallantoicmembrane Geniposidicacid

IridoidV1

a

b

s

t

r

a

c

t

ThephytochemicalstudyofGaliumtunetanumLam.,Rubiaceae,leavesledtotheisolationof13 com-poundsfromthechloroform–methanolandthemethanolextracts,including sixiridoidglycosides, onenon-glycosideiridoid,twop-coumaroyliridoidglycosides,twophenolicacids,andtwoﬂavonoid glycosides.Thestructuraldeterminationoftheisolatedcompoundswasperformedbymono-and bidi-mensionalNMRspectroscopicdata,aswellasESI-MSexperiments.Allcompoundswereisolatedfrom thisspeciesfortheﬁrsttime.Theanti-angiogeniceffectsoftheisolatediridoidswerealsoreportedon newbloodvesselsformationusingthechickembryochorioallantoicmembraneasinvivomodel.Results showedthatamongtheisolatediridoidstestedatthedoseof2␮g/egg,asperuloside(1),geniposidicacid (2),andiridoidV1(3)reducedmicrovesselformationofthechorioallantoicmembraneonmorphological observationsusingastereomicroscope.Theanti-angiogeniceffectsoftheactivecompounds,expressed aspercentagesofinhibitionversuscontrol,were67%(1),59%(2),and54%(3),respectively.Inaddition, theactivecompoundswereabletoinhibitangiogenesisinthechorioallantoicmembraneassay,ina dose-dependentmanner(0.5–2␮g/egg)ascomparedtothestandardretinoicacid.

Introduction

Plants belonging to Galium genus, Rubiaceae, comprising

approximately1300species,areknowninethnobotanicalﬁeldfor

thetreatmentofavarietyofpathologicalconditions,suchas

psori-asis,skininfections(Oumeish,1999),hepatitis(Bolivaretal.,2011),

kidneydisorders,andassedative,diuretic,andtotreattheepilepsy

andhysteria(Shahetal.,2006).G.tunetanumLam.isaperennial

herb,nativetoTunisia,Algeria,Marocco,Spain,andSicily(Casimiro

etal.,2012).Tothebestofourknowledge,intheliteraturethere

isonlyonereportabouttheantioxidantactivityofthemethanol

extractofitsleaves(Gaamouneetal.,2014)butnophytochemical

studieshavebeencarriedoutsofar.

Galium genus is well-known for producing several classes

of secondary metabolites such as iridoid glycosides, saponins,

triterpenes, anthraquinones, and ﬂavonoid glycosides (Mocan

et al., 2016). Iridoids are a large class of natural products,

exhibitingawiderangeofpharmacologicalactivitiessuchas

anti-inﬂammatory,anticancer,cardioprotective,andneuroprotective.

Interestingly,theiridoidglycosidegeniposidewasfoundtohave

∗ Correspondingauthor.

E-mail:alessandra.braca@unipi.it(A.Braca).

apotentanti-angiogenicactivityinthechickembryo

chorioallan-toicmembrane(CAM)assay(Kooetal.,2004).Angiogenesisisthe

growthofnewbloodvesselstoensurewoundhealing,

reproduc-tion,anddevelopmentsofcells.Thisphysiologicalprocessplaysan

importantroleintheexpansionofveinsandbloodcapillariesand

inthenutritionoftumorcells.Thus,angiogenesisinhibitionmight

beapromisingapproachforanticancertherapies.

Inthecourseofourinvestigationonplantsbelongingtothe

African ﬂora (Beladjila et al., 2017), the chemical study of G.

tunetanum leaves was performed, and the isolation and

struc-tural characterization of 13 compounds, including nine iridoid

glycosides(1–9),twophenolic acids(10–11),andtwo ﬂavonoid

glycosides(12–13)washereinreported.Theanti-angiogeniceffect

ofiridoids1–8onnewbloodvesselsformation,usingtheCAMassay

asinvivomodel,wasalsoexplored.

Materialsandmethods

Oneandtwo-dimensionalNMRexperimentswereperformed

on a Bruker DRX-600 spectrometer at 300K (Bruker BioSpin,

Rheinstetten,Germany) equippedwitha Bruker5mm TCI

Cry-oProbe, acquiring the spectra in methanol-d4. Pulse sequences

and phase cycling wereused for DQF-COSY,TOCSY, HSQC, and

HMBC,experiments.NMRdatawereprocessedusingXWinNMR

https://doi.org/10.1016/j.bjp.2018.03.010

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C.M.Cameroetal./RevistaBrasileiradeFarmacognosia28(2018)374–377 375

software (De Leo et al., 2017). ESI-MSwere obtained from an

LCQAdvantageThermoFinniganspectrometer(ThermoFinnigan,

USA), equipped with Xcalibur software. Column

chromatogra-phies (CC) were performed over Sephadex LH-20 (40–70␮m,

AmershamPharmaciaBiotechAB,Uppsala,Sweden)andIsolera®

Biotage® puriﬁcationsystem(ﬂashSilicagel60SNAP340g

car-tridge,ﬂowrate90ml/min)(Milellaetal.,2016).Reversephase–

highperformance liquidchromatography(RP-HPLC)separations

were conducted on a Shimadzu LC-8A series pumping system

equippedwithaShimadzuRID-10Arefractiveindexdetectorand

ShimadzuinjectoronaC18␮-Bondapakcolumn(30cm×7.8mm,

10_␮mWaters,ﬂowrate2ml/min,Milford,MA,USA).ThinLayer

Chromatography(TLC)analyseswerecarriedoutusingprecoated

Kieselgel60F254(0.20mmthickness)plates(Merck,Darmstadt,

Germany);compoundsweredetectedbyceriumdisulfate/sulfuric

acid(Sigma–Aldrich, Milan,Italy). Allthesolvents used forthe

extractionandseparationprocessesandretinoicacidusedforthe

CAMassayasantiangiogenicreferencecompoundwerepurchased

fromSigma-Aldrich(Milan,Italy).

GaliumtunetanumLam.,Rubiaceae,leaveswerecollectedand

identiﬁedbyauthorsSmainAmiraandFatimaBenchikhinDjilma,

45kmawayfromJijel,NortheastAlgeria,inJune2013.Avoucher

specimen has been deposited at the Herbarium Horti Botanici

Pisani,Pisa,Italy(n.8486Galiumtunetanum/1,NuoveAcquisizioni).

Brieﬂy, dried leaves of the plant (1kg) were extracted

with solvents of increasing polarity: n-hexane, chloroform,

chloroform–methanol(9:1),andmethanolbyexhaustive

macera-tiontogive4.0,13.3,11.9,and48goftherespectiveresidues.The

methanolextractwaspartitionedbetweenn-butanolandwaterto

affordan-butanolresidue(10.8g),thatwassubmittedtoSephadex

LH-20 column chromatography (5×75cm, ﬂow rate 1ml/min)

usingmethanolaseluentandcollectingninemajorfractions(A–I)

groupedbyTLC.PartofthefractionB(1.5g)wassubjectedto

RP-HPLCwithmethanol–water(3:7)aseluent,togivecompounds2

(0.7mg,tR7min)and7(1.4mg,tR14min).FractionsE(273.3mg),

F(707.3mg),G(724.0mg),andI(818.2mg)weresubmittedto

RP-HPLCusingmethanol–water(35:65)aseluent,togivecompounds

3(5.0mg, tR 14min) and 8(1.7mg, tR 55min) fromfractionE;

compounds10(1.5mg,tR9min)and 9(0.5mg,tR22min)from

fractionF;compounds 11 (6.0mg, tR 6min) and 12 (1.3mg, tR

32min) fromfractionG;compound13 (2.6mg,tR 39min)from

fractionI,respectively.TheremainingfractionsB(874.2mg)andC

(922.3mg)weresubjectedtoRP-HPLCwithmethanol–water(1:4)

aseluent,togivecompound6(1.3mg,tR5min)fromfractionBand

compound2(1.3mg,tR8min)fromfractionC,respectively.Partof

thechloroform–methanolresidue(5.6g)wassubjectedtoIsolera

Biotagecolumnchromatography(340gsilicaSNAPcartridge,ﬂow

rate90ml/min),elutingwithchloroformfollowedbyincreasing

concentrationsofmethanolinchloroform(between1%and100%).

Fractionsof27mlwerecollected,analyzedbyTLCandgroupedinto

ﬁvemajorfractions(A–E).FractionsB(331.4mg)andC(1481.8mg)

weresubjectedtoRP-HPLCwithmethanol–water(3:7)aseluent,

togivecompounds5(1.3mg, tR 6min)and 7(3mg, tR 15min)

from fractionB; compound 1 (23.6mg, tR 8min) from fraction

C,respectively.FractionE(509.7mg)wassubmittedtoRP-HPLC

withmethanol–water(1:4)aseluent,togivecompound4(6.6mg,

tR7min).

The CAM assay was performed following the method of

Germanòetal.(2015)modiﬁed(Certoetal.,2017).Fertilizedeggs

ofGallusgalluswerepreviouslymaintainedinahumidiﬁed

incu-batorat37◦Cand,afterfourdaysofincubation,asmallwindow

wascreatedonthebroadsideoftheeggstoapplydifferentdoses

ofpurecompounds(0.5–2␮g/egg) directlyontheCAMsurface,

previously suspended in albumen. Retinoicacid(2␮g/egg) was

used as antiangiogenic reference compound. After treatment,

theeggswerereincubatedfor24h,thentheywereobservedby

meansofasteromicroscope(ZeissStemi2000-c)equippedwith

a digitalcamera(Axiocam MRc5 Zeiss)andphotographed.The

antiangiogeniceffectsontheCAMwerequantiﬁedbycountingthe

numberofbloodvesselbranchpointsinastandardizedareausing

aZeisssoftwareformicromorphometricanalysisandexpressedas

%ofinhibitionrespecttocontrol.Eachexperimentwasrepeated

threetimes.Thesigniﬁcanceof thedifferenceswasassessedon

thebasisofthet-test,consideringthedifferencesforp<0.05,and

ﬁnallycalculatedwithrespecttothelotofcontroleggstreatedonly

withalbumen.

Resultsanddiscussion

The phytochemical study of chloroform–methanol and

methanol extracts of G. tunetanum leaves afforded the

isola-tionofthirteencompounds1–13.Theirstructuraldetermination

was performed by 1D and 2D NMR spectroscopic techniques,

massspectrometryanalyses, andcomparisonofthesedatawith

those reported in the literature. Isolated compounds included

six iridoid glycosides identiﬁed as asperuloside (1) (Otsuka

etal.,1991),geniposidicacid(2)(Güvenalpetal.,2006),iridoid

V1 (3) (Mitova et al., 1999), deacetylasperuloside (4) (Otsuka

etal.,1991),monotropein(6)(Tzakouetal.,2007),and

daphyl-loside (7) (Demirezer et al., 2006); one non-glycoside iridoid

macedonine (5) (Mitova et al., 1996); two p-coumaroyl

iri-doid derivatives, 10-O-p-coumaroyl-10-deacetyldaphylloside

(8) (Ahn and Kim, 2012) and

10-O-p-coumaroyl-10-deacetylasperuloside (9) (Bai and Hu, 2006); two phenolic

acids characterized as p-hydroxyhydrocinnamic acid (10) and

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376 C.M.Cameroetal./RevistaBrasileiradeFarmacognosia28(2018)374–377 1 mm 1 mm 1 mm 1 mm

A

B

C

D

Fig.1. Chickembryochorioallantoicmembrane(CAM)treatedwithatadoseof2␮g/egg.(A)Control;(B)asperuloside(1);(C)geniposidicacid(2);(D)iridoidV1(3).

80% 0.5 µg 1 µg 2 µg 70% 60% 40% 50% 30% 20% 10% 0% INHIBITION Geniposidic acid Asperuloside Iridoid V1 Retinoic acid

Fig.2.Dose-dependentanti-angiogenicactivityofasperuloside(1),geniposidicacid(2),andiridoidV1(3)inthechickembryochorioallantoicmembrane(CAM)assay. Retinoicacidwasusedasapositivecontrol.CAMsweretreatedwithcompoundsatdosesof0.5–2␮g/egg.Eachgroupcontainedatleast10eggs.Eachvaluerepresentsthe mean±SDofthreeexperiments.

glycosidesrutin (12)andapigenin-7-O-glucoside(13)(Agrawal,

1989).

Isolatediridoids,except9thatwasobtainedintoosmall

quan-tity, were subjected to CAM assay in order to evaluate their

anti-angiogeniceffects.

Theanti-angiogeniceffectsofisolatediridoids(2␮g/egg)inthe

CAMassayshowedthatcompounds1,2,and3wereabletoreduce

CAMmicrovesselformationwithinhibitionsof67%,59%,and54%,

respectively.Besides,compounds4–8demonstratedthefollowing

inhibitionvalues:43%,31%,23%,19%,and16%.Noteworthy,1has

ahigheranti-angiogenicactivityinrespecttothestandardretinoic

acid(62%).RepresentativemicroscopicimagesoftheCAMafter

treatmentwiththeactivecompounds1–3arereportedinFig.1.

Controleggsshowedthepresenceofaclearvascularnet-workwith

largevesselsconvergingtowardtheembryo(Fig.1A).Conversely,

avisiblereductionofbloodvesselbranchpointsisevidencedin

theCAM treatedwith1,2,and 3(Fig.1B–D).In addition,these

activecompoundsdemonstratedtoinhibitCAMangiogenesisina

dose-dependentmanner(0.5–2␮g/egg)(Fig.2).

Itisknownthatinhibitionofangiogenesishasbeenrecognized

tobeadvantageousforthepreventionofinﬂammationand

neo-plasticgrowth.Forthisreasonnowadaysthereisagrowinginterest

todiscovernewinhibitorsofangiogenesisfromnaturalsources.

TheCAMmodeloffersadvantagesthat includethecomparative

easeofculture,lowcost,andeasyobservationofthe

neovasculari-sation(Koutsavitietal.,2017).Amongtheisolatediridoidstested,

asperuloside(1),geniposidicacid(2),andiridoidV1(3)exhibited

highinhibitoryactivityonCAMangiogenesis.Theseresultsarein

accordancewiththestudyofKooetal.(2004)wheretheiridoid

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C.M.Cameroetal./RevistaBrasileiradeFarmacognosia28(2018)374–377 377

activityofGardeniajasminoidesfruitsethanolextract.Insummary,

theresultsobtainedmaybethestartingpointforconsideringG.

tunetanumanewsourceofanti-angiogeniccompounds.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thatnoexperimentswereperformedonhumansoranimalsfor

thisstudy.

Conﬁdentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authors’contributions

ABplannedtheexperiments.CMCcarriedouttheextractionand

puriﬁcationofcompounds.MDLperformedtheNMRandESI-MS

experiments.SAandFBcollected,identiﬁedtheplantmaterialand

contributedtotheinterpretationofresults.MPG,AP,VDperformed

thebiologicalassays.Allauthorscontributedtothecriticalrevision

ofthemanuscript.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

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