w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Antiproliferative
effect
of
Momordica
cochinchinensis
seeds
on
human
lung
cancer
cells
and
isolation
of
the
major
constituents
Jae
Sik
Yu
a,1,
Hyun-Soo
Roh
b,1,
Seul
Lee
b,
Kiwon
Jung
c,
Kwan-Hyuck
Baek
b,∗,
Ki
Hyun
Kim
a,∗aSchoolofPharmacy,SungkyunkwanUniversity,Suwon,RepublicofKorea
bDepartmentofMolecularandCellularBiology,SamsungBiomedicalResearchInstitute,SungkyunkwanUniversitySchoolofMedicine,Suwon,RepublicofKorea cInstituteofPharmaceuticalSciences,CollegeofPharmacy,CHAUniversity,Seongnam,RepublicofKorea
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received3November2016
Accepted21February2017
Availableonline9March2017
Keywords:
Gac Saponin
Lungcancercells
Endothelialcells
a
b
s
t
r
a
c
t
Gac,Momordicacochinchinensis(Lour.)Spreng.,Cucurbitaceae,isanindigenousSouthAsianediblefruit andhasbeenusedtherapeuticallyinTraditionalChineseMedicine.PreviousstudieshaveshownthatM. cochinchinensisseed(MomordicaeSemen)hasvariouspharmaceuticalpropertiessuchasantioxidantand anti-ulcereffectsaswellascontainssecondarymetaboliteswithpotentialanticanceractivitiessuchas triterpenoidsandsaponins.However,itsbiologicalactivitiesincancerhavenotyetbeeninvestigated. Inthisstudy,wefoundthatitsethanolextractreducedcellproliferationinfourhumanlungcancercell lines,A549,H1264,H1299andCalu-6.Phytochemicalinvestigationoftheethanolextractwascarried out,andresultedinisolationoftwomajorsaponins,whichwereidentifiedasgypsogenin3-O--d
-galactopyranosyl(1→2)-[␣-l-rhamnopyranosyl(1→3)]--d-glucuronopyranoside(1)andquillaicacid
3-O--d-galactopyranosyl(1→2)-[˛-l-rhamnopyranosyl(1→3)]--d-glucuronopyranoside (2).
Treat-mentwiththeseisolatedcompounds(1and2)decreasedcel1proliferationinallhumanlungcancercell linestested.Inaddition,thecompoundsattenuatedprimarylungendothelialcellproliferation.Taken together,thesefindingssuggestM.cochinchinensisseedshaveantiproliferativeactivityonhumanlung cancercellsaswellasangiostaticeffectonlungendothelialcells.
©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Momordicacochinchinensis(Lour.)Spreng.,Cucurbitaceae,isa dioecious vinethat growsthroughout SoutheastAsia andsmall partsof NorthernAustralia.M.cochinchinensisfruitsareknown asbabyjackfruitor“gac”inVietnam.Outershellofthefruitis notconsidered edible,but theseedsand thesoftpulp are edi-bleandtheyhavebeenusedinTraditionalChineseMedicine.M. cochinchinensisseeds(MomordicaeSemen)havebeenappliedfor many therapeutic purposes including relieving muscle spasms, rheumaticpain,hemorrhoids,andbruises(Perry,1980;Gao,2005; Tranetal.,2016).PreviousstudieshaveshownthatMomordicae Semenhavepharmaceuticalproperties,suchasantioxidanteffects (Tsoi et al., 2005), immune response enhancement (Xiaoet al., 2007),andanti-ulcereffects(Kangetal.,2010).Inaddition,the
∗ Correspondingauthors.
E-mails:khbaek@skku.edu(K.Baek),khkim83@skku.edu(K.H.Kim).
1Theseauthorscontributedequallytothiswork.
seedsaremixedwithglutinousricetomakeapopularVietnamese dishservedatweddingsandNewYearcelebrations.Ripegacfruits containhighamountsoflycopeneand-caroteneandare associ-atedwithvarioushealthbenefitsincludingstrokereductionand VitaminAdeficiencyprevention(Vuong,2000).Previous chemi-calinvestigationsonthisfruithavedemonstratedthepresenceof majorsecondarymetabolitesincludingtriterpenoidsandsaponins (Iwamotoetal.,1985;DeShanetal.,2001;Kanetal.,2006;Jung etal.,2013,2016).OurrecentstudyrevealedthatsaponinsfromM. cochinchinensisseedhaverenoprotectiveeffectsagainst cisplatin-induceddamagetoLLC-PK1kidneycells(Jungetal.,2016).
As an ongoing investigation of biological activities of M. cochinchinensisseeds, weexploredeffectsofitsEtOHextracton cell proliferationin humanlung adenocarcinoma celllines and found that the EtOH extract treatment decreased proliferation of all human lung cancer cell lines tested. To the best of our knowledge,theseantiproliferativeactivitiesofM.cochinchinensis
seedonhumancancercells havenot beenreportedelsewhere. Additionally, phytochemical investigation was conducted to identifyconstituentsofM.cochinchinensisseedcontributingtoits
http://dx.doi.org/10.1016/j.bjp.2017.02.002
0102-695X/©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
anti-proliferative activityagainst humanlung cancercells. This resultedinsuccessfulisolationoftwomajorsaponins.Thesewere furtherevaluatedfortheirantiproliferativeactivityagainsthuman lungcancercellsaswellasprimarylungendothelialcells.
Materialsandmethods
Generalexperimentalprocedures
JASCODIP-1000digitalpolarimeter(Jasco,Tokyo,Japan)was usedtomeasureopticalrotations.IRspectrawerecollectedusing aJASCOFT/IR 300Espectrophotometer (Jasco).High resolution-FAB-MSwasperformedwithaJEOLJMS-700massspectrometer (Akishima,Tokyo,Japan).WiththeBuchiB-540apparatus(Buchi, Flawil, Switzerland) the melting point was recorded. Nuclear magneticresonance(NMR)spectrasuchas,DEPT,HSQC,HMBC, TOCSY,andNOESYexperiments,wereperformedunderaBruker AVANCEIII700NMRspectrometeroperatingat700MHz(1H)and 175MHz(13C)(Bruker),withchemicalshiftslabeledasppm(ı). Preparativehighperformanceliquidchromatography(HPLC)was performedusingaWaters1525BinaryHPLCpumpwithWaters996 PhotodiodeArrayDetector(WatersCorporation,Milford,CT,USA). Semi-preparativeHPLCwasconductedwithaShimadzu Promi-nenceHPLCSystemwithSPD-20A/20AVSeriesProminenceHPLC UV–visDetectors(Shimadzu,Tokyo,Japan).Silicagel60(Merck, 230–400mesh)andRP-C18silicagel(Merck,40–63m)wereused forcolumnchromatography.Merckpre-coatedSilicagelF254plates andRP-18F254splates(Merck,Darmstadt,Germany)wereusedfor TLC.SpotsweredetectedonTLCunderUVlightorbyheatingafter sprayingwithanisaldehyde-sulfuricacid.
Plantmaterial
Mormodicacochinchinensis(Lour.)Spreng.,Cucurbitaceae,seeds usedinthisstudywereobtainedfromalocalmedicinalherbstore locatedneartheYunnanprovince,People’sRepublicofChinain May2010.Theseedswereclassifiedbyoneoftheauthors(K.H. Kim).Avoucher(MBJ-2010-05)wasdepositedintheherbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.
Extractionandisolation
Momordicacochincinensisseeds(1.2kg)weregroundintosoft powderandextractedwith50%ethanolunderrefluxtwicefor4h. Theextractwasfollowed byfiltrationand thenconcentratedin vacuo.Theresultedresidue(41g)wasstirredwithdistilledwater thenpartitionedwithhexane, ethylacetate(EtOAc),and BuOH, yielding2.1,3.1,and11.6g,respectively.Beforefurtherisolation process,allofthepartitionedfractionswereprocessedwithLC/MS analysis,and BuOHsoluble fractionresultedas a mainfraction andselectedforisolationofchemicalconstituents.TheBuOH sol-ublefraction(4g)waspurifiedusingpreparativeHPLCof10–70% MeOHgradientsolventsystemwithEclipseDBX-C18column, Agi-lent (250mm×21.2mm i.d., 7m, at a 5ml/min flow rate) to producesevenfractions(C1–C7).Thesefollowingfractionswere performedbyLC/MSanalysisforthoroughselectionprocess. Frac-tionC7(150mg)wasselectedforfurtherpurification.FractionC7 (150mg)wasseparatedbyasemi-preparativereversedphaseHPLC usingacolumnofLunaphenyl-hexyl(250mm×10mmi.d.,10m, ata2ml/minflowrate)with30%MeCNisocraticsystemtoyield compounds1(18mg)and2(20mg).
H
CO
2H
CHO
R
1R=H 2R=OH
O
O
O
HO
2C
HO
O
O
OH
OH
HO
OH
O
OH
OH
OH
Cellculture
Human non-small cell lung cancer cell lines, A549, H1264, H1299andCalu-6,weremaintainedinRPMI-1640medium (Wel-GENE,Seoul,Korea)supplementedwith10%FBS(Hyclone,Logan, UT,USA),2mMl-glutamine(GibcoBRL,GrandIsland,NY,USA), 50U/mlpenicillin,and50g/mlstreptomycin(WelGENE).Primary lungendothelialcells(LEC)wereisolatedfromwild-typeC57BL/6 miceaspreviouslydescribed(Shinet al.,2014)and culturedin AdvancedDMEM(GibcoBRL)supplementedwith15mMHEPES (Sigma,St.Louis,MO,USA),100g/mlheparin(Sigma),100g/ml endothelialcellgrowthsupplement(ECGS;Biomedical Technolo-gies, Stoughton,MA,USA),20%FBS, 2mMl-glutamine,50U/ml penicillinand50g/mlstreptomycin(GibcoBRL).ThepurityofLEC wasassessedbystainingforCD31,anendothelialcellmarker,using anti-CD31antibody(Abcam,Cambridge,UK)followedby exami-nationunderaconfocalmicroscope(CarlZeiss,Jena,Germany)as previouslydescribed(Shinetal.,2014).
Cellproliferationanalysis
Humanlungcancercells(5×103)and3×103LECwereplated in triplicatein 96 welltissue cultureplates (Thermo Scientific, Waltham,MA,USA)overnightandthentreatedwithEtOHextract ofM.cochinchinensisseedsorisolatedcompounds(1and2)for 48h.Cellproliferationwasthenassessedbyincubatingwith1/10 volume of WST-1 reagent (Daeil Lab Service, Seoul, Korea) for 2hfollowedbymeasuringabsorbanceat450nmwithascanning multi-wellspectrophotometer(MolecularDevice,Sunnyvale,CA, USA).Cellproliferationwasthendeterminedbypercentoftreated tountreatedcellabsorbance.
Statisticalanalysis
Unpairedstudent’s two-tailed t test wasusedto assess sta-tistical differencesbetween cells treatedwiththe EtOHextract ofM.cochinchinensisseedsorisolatedcompounds(1and2)and untreatedcontrols.Dataarepresentedasmean±SEMandpvalues lessthan0.05wereconsideredtobestatisticallysignificant.
Resultsanddiscussion
120
100
80
60
40
20
0
A549
% of viab
le cells
H1264 H1299
Untreated Doxo MSEX
Calu-6
Fig.1.TheethanolicextractofMomordicacochinchinensisseedreduceshumanlung
cancercellproliferation.Fourhumanlungcancercelllines,A549,H1264,H1299and
Calu-6,weretreatedwith60g/mlofM.cochinchinensisseedEtOHextract(MSEX)
or1Mdoxorubicin(Doxo)asapositivecontrolfor48h.Cellproliferationwasthen
assessedbyWST-1assay.Dataarepresentedasmean±SEM.*p<0.05,**p<0.01.
toinvestigatenovelbiologicalactivityofM.cochinchinensisseeds, wefirstpreparedEtOHextractfromM.cochinchinensisseedsand thenexaminedtheeffectsofitstreatmentoncellproliferationin fourhumanlungcancercelllines,A549,H1264,H1299andCalu-6, usingtheWST-1cellproliferationassay(Fig.1).Treatmentwith EtOHextractat60g/mlfor48hdecreasedA549,H1264,H1299 andCalu-6cellproliferationupto25,15,13and11%,respectively,
comparedwithuntreatedcontrols,indicatingthatEtOHextractof
M.cochinchinensisseedpossessedanti-proliferativeactivityagainst humanlungcancercellsinvitro.
TheEtOHextractofM.cochinchinensisseedswassubjectedto fractionationwithhexane,EtOAc,andBuOHtoyieldthreefractions. ThroughthoroughprocessofLC/MSanalysis,BuOHsoluble frac-tionwassubjectedtophytochemicalinvestigationwhichresulted inisolation andidentificationoftwo triterpenoidalsaponinsby HPLCpurification.Isolatedcompounds1and2werestructurally resolvedbyspectroscopicmethodsincluding1Hand13CNMR,and ESI-MSanalysis.Thepurifiedcompoundswereclassifiedas gyp-sogenin 3-O--d-galactopyranosyl(1→2)-[␣-l-rhamnopyranosyl (1→3)]--d-glucuronopyranoside (1) (Jung et al., 2013, 2016) and quillaic acid 3-O--D-galactopyranosyl(1→2)-[␣ -l-rhamnopyranosyl(1→3)]--d-glucuronopyranoside (2) (Jung etal.,2013,2016)aftercomparisontopreviouslyreported1Hand 13CNMRandMSdata.
Todeterminewhethertheanti-proliferativeactivityof EtOH extract againsthuman cancercells isattributed totheisolated compounds 1 and 2, we evaluated theireffects onin the pro-liferationofA549,H1264,H1299andCalu-6cells (Fig.2).After treatmentwithcompounds1and2at20and60g/mlfor48h, WST-1assayrevealedadecreaseincellproliferation inallfour humanlung cancercelllinesinadose-dependentmanner, sug-gestingthatanti-proliferativeactivityoftheEtOHextractwasdue tothesecompounds.
Previouslypublished studieshave shown that triterpenoidal saponinsattenuatetumorangiogenesis,thegrowthofnew cap-illarybloodvesselswhichisessentialfortumorstobecomerapidly growingmalignanttumors(Folkman,2007).Thisisaccomplished throughinhibitionofendothelialproliferation,leadingtotumor growthsuppressionintumorxenograftmodelsofmouse(Araietal.,
120
A
B
D
C
100
80
60
40
20
0
Untreated 20 60 20
Conc. (µg/ml)
H1299 Calu-6
H1264 A549
% of viab
le cells
60
120
100
80
60
40
20
0
Untreated 20 60 20
Conc. (µg/ml)
% of viab
le cells
60
120
100
80
60
40
20
0
Untreated 20 60 20
Conc. (µg/ml)
% of viab
le cells
60 120
100
80
60
40
20
0
Untreated 20 60 20
Conc. (µg/ml)
Compound 1 Compound 2
% of viab
le cells
60
Fig.2. Compounds1and2isolatedfromtheethanolicextractofMomordicacochinchinensisdecreasehumanlungcancercellproliferation.Antiproliferativeactivityof
compounds1and2isolatedfromtheEtOHextract(MSEX)inA549(A),H1264(B),H1299(C)andCalu-6(D)cellswasassessedbyWST-1assay48haftertreatmentatthe
120 LEC
A
B
Br
ight-field
CD31
Hoechst
+
100
80
60
40
20
0
Untreated
% of viab
le cells
MSEX
Compound 1 Compound 2
Fig.3.Compounds1and2attenuateprimarymouselungendothelialcellproliferation.(A)Bright-field(upper)andimmunofluorescence(lower)imagesofprimarylung
endothelialcellsisolatedfrommiceshowingthetypicalculturedendothelialcellcobblestonemorphologyandCD31expression,respectively.Scalebars:50mM.(B)
Antipro-liferativeeffectsoftheethanolicextractofMormodicacochinchinensisseeds(MSEX)anditsmajorconstituents,compounds1and2,inprimarymouselungendothelialcells
wereassessedbyWST-1assayat48haftertreatmentwith60g/mlofMSEXand20g/mlofcompounds1and2.Dataarepresentedasmean±SEM.**p<0.01.
2011;Guanetal.,2015).Therefore,wealsoexaminedwhetherthe EtOHextractofM.cochinchinensisseedanditsmajorconstituents, compounds1and2,affectedendothelialcellproliferation(Fig.3). SincewefocusedontheinhibitoryactivityofM.cochinchinensis
seedsagainstlungcancercells,weusedprimaryLECisolatedfrom mice(Fig.3A).AlthoughtheEtOHextractfailedtoexhibitprofound effectsonendothelialproliferation,treatmentwithcompounds1 and2at20g/mldecreasedcellproliferationinprimaryLECupto 12and24%,respectively(Fig.3B).
Takentogether,ourobservationsindicatethatcompounds1and 2alsopossessedanti-proliferativeactivityinLEC,aswellasinlung cancercells.
Conclusions
MormodicacochinchinensisisanindigenousSouthAsian fruit thathaslongbeenusedasfoodandinTraditionalChinesemedicine formanytherapeuticpurposes.ThistraditionalAsianfruitisknown tocontainhighlevelsofcarotenoids,whichpartiallyexplainsits seeds’therapeuticeffects.Yet,therehasbeennoinvestigationof biologicalactivitiesofM.cochinchinensisfruitincancerandits com-ponentswhichmayberesponsibleforthebiologicalactivities.This studyidentifiedtheantiproliferativeeffectsofEtOHextractofM. cochinchinensisseedsagainsthumanlungcancercells.Inaddition, compounds1and2,identifiedasitsmajorconstituents,inhibited cellproliferationinhumanlungcancercellsaswellasprimaryLEC, implyingthattheisolatedcompoundscontributeto antiprolifera-tiveactivityoftheextractagainsthumanlungcancercellsaswell ashaveangiostaticactivityinvitro.Thesenovelobservationsraise thepossibilityofitspotentialuseasa therapeuticfoodforlung cancerintervention.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisinvestigation.
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearsinthisarticle.
Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearsinthisarticle.
Authors’contribution
JSY,HSR,andSLcontributedtheexperiments.JSY,KHB,andKHK preparedandwrotethemanuscript.KJ,KHB,andKHKcontributed analysisanddiscussion.KHBandKHKcontributedthedesignofthe projectandreviewedthemanuscript.Alltheauthorshavereadthe finalmanuscriptandapprovedthesubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
This research was supportedby theBasic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Plan-ning(2015R1C1A1A02037383)andbytheMinistryofEducation (NRF-2012R1A5A2A28671860).S.Leewassupportedbythe NRF-2015-Global PhD Fellowship Program of the National Research FoundationofKorea(NRF)grantfundedbytheKoreanGovernment (NRF-2015H1A2A1034135).
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