Dual Inhibition of AChE and BChE with the C-5 Substituted Derivative of Meldrum's Acid

13 Pages·2017·0.75 MB·English

marine drugs
Article
Cytotoxic Effects of Sarcophyton sp. Soft Corals—Is
There a Correlation to Their NMR Fingerprints?
MohamedA.Farag1,*,MostafaI.Fekry1,MontasserA.Al-Hammady2,MohamedN.Khalil1,
HeshamR.El-Seedi5,6,AchimMeyer3,AndreaPorzel4 ,HildegardWestphal3
andLudgerA.Wessjohann4,*
1 PharmacognosyDepartment,CollegeofPharmacy,CairoUniversity,KasrelAinist.,P.B.11562Cairo,Egypt;
[email protected](M.I.F.);[email protected](M.N.K.)
2 NationalInstituteofOceanographyandFisheries,RedSeaBranch,84511Hurghada,Egypt;
[email protected]
3 LeibnizCentreforTropicalMarineResearch,FahrenheitStr.6,D-28359Bremen,Germany;
[email protected](A.M.);[email protected](H.W.)
4 DepartmentBioorganicChemistry,LeibnizInstituteofPlantBiochemistry,Weinberg3,
D06120Halle(Saale),Germany;[email protected]
5 DepartmentofMedicinalChemistry,DivisionofPharmacognosy,UppsalaUniversity,Box574,SE-75123
Uppsala,Sweden;[email protected]
6 DepartmentofChemistry,FacultyofScience,El-MenoufiaUniversity,32512ShebinEl-Kom,Egypt
* Correspondence:[email protected](M.A.F.),[email protected](L.A.W.);
Tel.:[phone](M.A.F.),[phone](L.A.W.)
AcademicEditor:VassiliosRoussis
Received:30May2017;Accepted:27June2017;Published:4July2017
Abstract: Sarcophytonsp. softcoralsarerichincembranoidditerpenes,whichrepresentthemain
chemicaldefenseofcoralsagainsttheirnaturalpredatorsinadditiontotheirmyriadbiologicaleffects
inhumans. QuantitativeNMR(qNMR)wasappliedforassessingthediterpenevariationin16soft
coralspecimensinthecontextoftheirgenotype,origin,andgrowinghabitat. qNMRrevealedhigh
diterpenelevelsinSarcophytonsp. comparedtoSinulariaandLobophyton,with(ent)sarcophinesas
majorcomponents(17–100µg/mg)ofthecoraltissues. Multivariatedataanalysiswasemployed
to classify samples based on the quantified level of diterpenes, and compared to the untargeted
NMRapproach. ResultsrevealedthatqNMRprovidedastrongerclassificationmodelofSarcophyton
sp. thanuntargetedNMRfingerprinting. Additionally,cytotoxicityofsoftcoralcrudeextractswas
assessedagainstandrogen-dependentprostatecancercelllines(PC3)andandrogen-independent
coloncancercelllines(HT-29),withIC valuesrangingfrom10–60µg/mL.Noobviouscorrelation
50
betweentheextracts’IC valuesandtheirditerpenelevelswasfoundusingeitherSpearmanor
50
Pearsoncorrelations. ThissuggeststhatthistypeofbioactivitymaynotbeeasilypredictedbyNMR
metabolomicsinsoftcorals,orisnotstronglycorrelatedtomeasuredditerpenelevels.
Keywords: cembranoids; Sarcophyton; metabolomics; quantitative nuclear magnetic resonance
(qNMR);terpenoids
1. Introduction
In spite of marine organisms’ taxonomic biodiversity amounting up to 30 × 106 species with
a wide habitat covering more than 70% of the planet surface, the number of described bioactive
naturalmarineproductsamounttomerelyafewthousand[1,2]. Thusitcanbeexpectedthatmarine
organismsrepresentavastsourcefornovelbioactivecompoundswithpotentialfordrugdevelopment.
Thegrowinginterestinnaturalmarineproducts,particularlyintheareaofanticancercompounds,
is attributed to the urgent therapeutic need for novel cytotoxic agents [3,4]. Roughly 40% of the
Mar.Drugs2017,15,211;doi:10.3390/md15070211 www.mdpi.com/journal/marinedrugs
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180softcoralspeciesidentifiedworldwidearenativetotheRedSea[5]. ThegenusSarcophytonis
rich in cembrane terpenoid natural products [6–8]. In addition to the unique ecological function
of cembranoids, acting to enhance the corals’ fitness to their own marine environment, they also
exhibitrelevantbioactivitiesinhumanssuchasneuro-protective,anti-inflammatory,antimicrobial,
andanti-tumorproperties[9,10].
It is of interest to develop analytical tools for soft coral metabolite-based classification in the
context of genotype, geographical origin, or growth environment, similar to that well developed
forterrestrialplants—especiallymedicinalplants[11–13]. RecentlyNMRhasbeenappliedforthe
qualitativeandquantitativecharacterizationofmetabolitesincrudebiologicalextracts. Forinstance,
quantitative 1H-NMR (qNMR) was employed to determine the amount of the anti-diabetic agent
“trigonelline”inBalanitesaegyptiaca(datefruit)extract,andthehepatoprotectiveagent“cynaropicrin”
inartichokeleafextract[14,15]. ComparedtoHPLC,NMRallowsfortheabsolutequantificationof
metaboliteswithouttheneedforpeakseparationortheuseofspecificinternalstandardsforeach
metabolite[16]. Aphenomenonthatisuniquefor1H-NMRsignalintensityliesinitsproportionality
tothenumberofnuclei[17]. ComparedtoMS,NMRanalysisrequiresminimalsamplepreparation
andisnon-destructive. WehaverecentlyreportedmetabolitefingerprintingofSarcophytonspecies
from the Red Sea area by NMR and LC-MS. This comparative metabolomics approach revealed
relativecompositionaldifferencesinlipidsandcembranoidsamongcorals,thoughwithnoabsolute
quantification[18].
Ourobjectiveinthecurrentstudywasto(1)employqNMRtostandardizecoralextractswith
absolutemeasurementsoftheirmajorditerpenelevelsand(2)investigatewhetheracorrelationexists
betweenthediterpenecompositionandcytotoxiceffectsofsoftcoralextracts. Fourditerpenesand
gorgosterol(asterol)werequantifiedvia1H-NMRin16differentsoftcoralspecimens(Suppl.TableS1).
Finally,multivariatedataanalysiswasappliedtoclassifycoralsamplesbasedonmeasuredditerpene
levelsasatargetedmetabolomicsapproachtobecomparedwiththepreviouslyuntargetedNMR
fingerprintingclassificationmodel[18].
2. Results
2.1. SelectionandQuantificationofTerpene/SterolNMRSignals
Full assignment of the major NMR signals in coral secondary metabolites described herein
was performed as detailed in our previous work using 1D (one-dimensional) NMR and 2D
(two-dimensional) NMR experiments [18]. Nevertheless, for the quantification described herein
using 1D NMR to be optimal, full relaxation of the protons of diterpene signals and the internal
standardHMDShadtobeachieved. Forthat,aratherlargesumofrelaxationdelayandacquisition
time of 23 s was employed for NMR acquisition, as the longest relaxation times were 4.5 s for the
HMDS protons. For quantification, NMR signals unique to each metabolite, and also sufficiently
separatedfromneighboringsignals,wereselected. Methylsignalswereofpremiumpreferenceas
theirlargeintegrationdemarcatesthemreadilyfromthenoisybackgroundsetoffbythemanysingle
protonsderivedfromother,lessabundantcompounds(Figure1). Figure2showsarepresentative
1H-NMRspectrumofSarcophytonconvolutum(SC1),withassignedNMRsignalsusedformetabolite
quantification as summarized in Suppl. Table S2. Quantified NMR signals belonged to one
sesquiterpene viz. guaiacophine, four diterpenes viz. sarcophine, and one sterol viz. gorgosterol
(Figure 1). For N1 guaiacophine, the signal of H -14 (δ 1.13 ppm, d, J = 6.9 Hz) was used for
3 H
quantification,beingmoreintensethanitsolefinicprotonH-6(δ 6.26ppm)andalsobetterseparated
H
than H -12 (δ 1.81 ppm) and H -13 (δ 1.82 ppm) signals. The latter ones slightly overlapped
3 H 3 H
withH -17(δ 1.79ppm)presentinditerpenesN2–N5(Figure2). AbsenceofN1guaiacophinein
3 H
somesoftcoralspecimenswasaffirmedbytheabsenceofitsH -14andolefinicH-6NMRsignals
3
asevidentinFigure2. ThecembranoidditerpenesN2-N5exhibitalmostsimilarstructuralfeatures
(Figure 1), including a 14-membered macrocyclic sarcophine skeleton fused to a five-membered
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α,β-unsaturated-γ-lactonering. Withregardtostructuraldifferences,thediterpeneenantiomersN2/3
eenxhanibtiitomaneresp oNx2y/3b reixdhgiebibt eatnw eeepnoxCy7 /brCid8,gwe hbeertweaesetnh eCd7/iCas8t,e wrehoemreearss Nth4e/ dNia5saterereeopmimeresr sNw4/iNth5 twaroe 
vepiciimnaelrsh ywdirtohx tywlgor ovuicpinsaalt hCy7d/rCo8x.yCl gornosueqpus eantt lCy,7t/hCe8.s iCgnonalseoqfuHe-n7tliyn, Nth2e/ 3s,igNn4a,l aonfd HN‐75 wina Nsu2/s3e,d Nfo4r, 
qanudan Nti5fi cwataiso nusaepdp feoarr iqnugaanttiδfi2ca.6t3io,n3 .a4p4,paenadrin3g.5 5atp δp m2.6,3r,e s3p.4e4c,t iavnedly 3..I5n5 dpeptmail,, rienstpegecrtaitvioelny.o Ifnt hdeeHtai-l7, 
isnigtengarlaattioδnH o2f. 6th3ep Hpm‐7 wsiagsnauls eadt δtHo 2q.u6a3n ptpifmyN w2a/s3 uasnedd ttoo dqiusatinntgifuyi sNh2t/h3e amndfr toom dtihsteindgiausistehr ethoeismom freormic 
pthaei rdNia4s/te5r.eoAinsoamloegroicu splya,irt hNe4s/i5g. nAanlsaolofgHou-7slayp, ptheea rsiinggnaaltsδ oHf 3H.4‐74 papppmeaarnindgδ aHt 3δ.H5 53.p4p4 mppimn Nan4da nδdH 
3N.555w peprmeu isne dNf4o arnthde Nir5r ewspeerec tuivseedq ufoarn ttihfieciar trioesnp.eTchtievset eqruoaindtgifoicragtoiosnte.r Tolh(eN s6te)rboeidar gsoarcghoasrtearcotel r(iNst6ic) 
bcyecalrosp aro cphaanreacrtienrgisitnici tcsyscildoepcrhoapiann(ea rgionrgg oins taitnse s-tiydpe ecshiadien c(haa igno)ragnodstwanaes‐tqyupaen tsiifideed cfhroamin)i tasnudn iwquaes 
qHu-3a0natif(iδeHd 0f.r4o8mp pitms u&nidqdu,eJ H=‐93,04a.2 (δHHz 0)..4I8n pcopnmtr &as td,ds,i gJ n=a 9l,f o4r.2H H-3z0).b Iwn acsonfotruansdt, tsoigbneailn ftoerr fHer‐i3n0gb wwiaths 
footuhenrdu tno kbneo iwntnerpferoritnogn sw.iAthl tohtohuerg hunHk3n-o2w7nsi gpnroalto(nδHs. 0A.8lt0hopupgmh &H3d‐2,7J s=ig6nHalz ()δiHs 0q.8u0it eppdmis t&an dt, fJr o=m 6 
HHz3-)1 i5s qinuiNte1 d(iδsHta0n.t8 f9ropmpm H3&‐1d5, iJn =N61 H(δzH) ,0i.8t9w papsmno &t uds, eJ d= 6fo Hrzq)u, aitn wtifiasc antoiot nusaesdi tfoshr oqwuaendtimficoarteiotnh aans 
iotn sehcorwosesdp meaokrec otrhraenla otinoen cirnosHspSQeaCk (cdoarrtaelnatoiotnsh ionw HnS)Q. CCo (ndsaetqau neontt lsyh,oswignn)a. lCoofnHse-3q0uaenwtlays, suisgendalf oorf 
qHu‐a3n0ati fiwcaatsio nu,sdedes pfoitre iqtsulaonwtifsiicgantiaoln/,n odiesespriattei oiwts hleonwc osmigpnaarle/dnotoiseit srcaotuion twerhpeanrt Hco3m-2p7a(rFeidgu  rtoe Si1ts). 
Acolulonttherepramrte Hth3y‐2l7g r(oFuigpusrieg nS1a)ls. AofllH o3t-h1e8r, Hm3e-t1h9y,lH g3r-o2u1p, Hsi3g-n26a,lsH o3f- 2H8,3‐a1n8d, HH33‐1-299, Hin3‐N216, wHe3r‐e26n, oHtw3‐2e8ll, 
arensdo lHve3‐d29fr ionm Nt6h owseerbee nloont gwinelgl rtoesootlhveerdd firtoemrp ethnoessef obuenlodnigninegx ttroa cott.her diterpenes found in extract.  

Figure1.StructuresofthemajorC15-andC20-terpenoidsandasterolquantifiedinSarcophytonextracts
Figure 1. Structures of the major C15‐ and C20‐terpenoids and a sterol quantified in Sarcophyton 
usingqNMR,anddiscussedinthemanuscript.Notethatthesamecarbonnumberingsystemisused
extracts using qNMR, and discussed in the manuscript. Note that the same carbon numbering 
foreachcompoundthroughoutthemanuscriptforNMRassignmentandthusisbasedonanalogy
system is used for each compound throughout the manuscript for NMR assignment and thus is 
ratherthantheInternationalUnionofPureandAppliedChemistry(IUPAC)rules.Signalshighlighted
based on analogy rather than the International Union of Pure and Applied Chemistry (IUPAC) rules. 
inredrepresentthoseusedforqNMR.
Signals highlighted in red represent those used for qNMR.
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Figure 2. Representative 1H‐NMR spectrum (0–6.5 ppm) of S. convolutum (SC1). For complete 
Figure 2. Representative 1H-NMR spectrum (0–6.5 ppm) of S. convolutum (SC1). For complete
assignment of quantifiable NMR signals refer to Suppl. Table S2. Signals used in quantification are 
assignment of quantifiable NMR signals refer to Suppl. Table S2. Signals used in quantification
marked in blue boxes.  
aremarkedinblueboxes.
Sesquiterpenes are represented by guaiacophine (N1), which was found to occur at its highest 
Sesquiterpenesarerepresentedbyguaiacophine(N1),whichwasfoundtooccuratitshighest
levels of ca. 10 μg/mg mostly in S. convolutum (SC) and in one of the S. glaucum specimens (SG1) 
levels of ca. 10 µg/mg mostly in S. convolutum (SC) and in one of the S. glaucum specimens
(Figure 3). Compared to the sesquiterpene “guaiacophine”, diterpenes (N2–5) form the major class 
(SG1) (Figure 3). Compared to the sesquiterpene “guaiacophine”, diterpenes (N2–5) form the
of secondary metabolites in corals. The enantiomers sarcophine/ent‐sarcophine (N2/3) were the most 
major class of secondary metabolites in corals. The enantiomers sarcophine/ent-sarcophine
abundant diterpenes in samples of S. acutum (SA), S. convolutum (SC2), S. ehrenbergi (SE2‐4), S. 
(N2/3) were the most abundant diterpenes in samples of S. acutum (SA), S. convolutum (SC2),
glaucum  (SG),  and  S.  regulare  (SR1‐3).  Among  the  monitored  metabolites, 
S. ehrenbergi (SE2-4), S.glaucum(SG), and S. regulare (SR1-3). Among the monitored metabolites,
dihydroxydeepoxysarcophine  diastereoisomers  (N4/N5)  and  gorgosterol  (N6)  were  the  ones 
dihydroxydeepoxysarcophinediastereoisomers(N4/N5)andgorgosterol(N6)weretheonesdetected
detected in all species examined (Figure 3). The highest levels of diterpene N4 were recorded in S. 
inallspeciesexamined(Figure3). ThehighestlevelsofditerpeneN4wererecordedinS.convolutum
convolutum (SC1) and in an unidentified Sarcophyton sp. (S) at ca. 25.6 and 31 μg/mg, respectively, 
(SC1)andinanunidentifiedSarcophytonsp. (S)atca. 25.6and31µg/mg, respectively, versusthe
versus the lowest levels detected in the Lobophyton (LP) species (ca. 5.5 μg/mg). Interestingly, N5, a 
lowestlevelsdetectedintheLobophyton(LP)species(ca. 5.5µg/mg). Interestingly,N5,adiastereomer
diastereomer of N4, showed a slightly different accumulation pattern to N4 with maximum levels 
ofN4,showedaslightlydifferentaccumulationpatterntoN4withmaximumlevelsobservedinboth
observed in both S. glaucum (SG1) and S. convolutum (SC2) at ca. 33 μg/mg, and minimal abundance 
S.glaucum(SG1)andS.convolutum(SC2)atca. 33µg/mg,andminimalabundanceinS.ehrenbergi
in S. ehrenbergi (SE4) at 4.6 μg/mg. Gorgosterol, a common sterol, was found at levels ranging from 
(SE4)at4.6µg/mg. Gorgosterol,acommonsterol,wasfoundatlevelsrangingfromca. 26µg/mgin
ca. 26 μg/mg in S. acutum (SR1) to almost being absent in Sinularia species (SP).  
S.acutum(SR1)toalmostbeingabsentinSinulariaspecies(SP).
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FFiigguurree 33.. SStatcakckeded cocloulmumn ngrgarpahp hshsohwowinign gN1N–1N–6N m6emtaebtaoblioteli tleevleelvse ilns sinofst ocfotrcaol rsaplescpieesc. ieVsa.luVeasl uarees 
areexpressedasmean(µg/mg)±SE.(n=3). Forcompleteinformationonsamplecodesreferto
expressed as mean (μg/mg) ± SE. (n = 3). For complete information on sample codes refer to Suppl. 
Suppl.TableS1.
Table S1. 
22..22.. MMuullttiivvaarriiaattee DDaattaa AAnnaallyyssiiss ooff tthhee TTaarrggeetteedd MMeettaabboolliittee PPrroofifilliinngg bbyy qqNNMMRR ooff SSoofftt CCoorraallss 
TTaarrggeetteedd mmeettaabboolliittee aannaallyysseess ooff tthhee 1166 ssoofftt ccoorraall ssppeecciimmeennss ((SSuuppppll.. TTaabbllee SS11)) wweerree bbaasseedd oonn tthhee 
qquuaannttiiffiiaabbllee ccoommppoouunnddss( F(igFuigruer1e) .1T)h. isTahlilso waleldowusedto uasss etsos tahsesehsest etrhoeg ehneetiteyroagmeonnegityc oaramlospnegc imcoernasl 
sapnedcitmoecnosm apnadr etoi tctoomthpearuen itta trog ethteed uNntMarRgeatepdp rNoaMchR raeppporrotaecdhe raerplioerrte[1d8 ]e.arTlhieer t[w18o].m Tahjeo rtwpori nmcaipjoarl 
pcorimncpiponale nctosm(PpCon1e/nPtCs 2()PaCc1c/oPuCn2t)e dacfcoorucnat.e8d5 .f8o%r coaf. t8h5e.8t%ot aolfv athriea ntoctea.l Tvhaerilaonacdei.n Tghpel oltoa(Fdiignugr epl4oat) 
(rFeivgeuarlee d4tah)a trdeivheyadlerodx ytdheaet podxihyysadrrcooxpyhdieneepaonxdysgaorcrgoposhtienreo l(aNnd4- 6g)owregroestmerooslt r(eNsp4o‐6n)s ibwleerfeo rmcoorsatl 
rdeisspcroinmsiinbaleti ofonrv ciaorPaCl 1d.iDscirhimydirnoaxtyiodne evpioa xPyCsa1r.c oDpihhiynderaonxdydgeoerpgooxstyesraorlcwoperheinbeo tahnpdo gsiotirvgeolsytecororrl ewlaeterde 
btootPhC p1o.sIniticvoenlytr acsotr,rgeulaatieadc otpoh PinCe1(.N In1) caonndtrsaasrtc, ogpuhainiaec/oepnhti-nsaer c(Nop1h) inaned(N s2a/rc3o)pwheinree/aesnsto‐csaiartceodphwiinteh 
(sNam2/p3)le wseerger eagsastoicoinataeldo nwgitPhC s2a.mSpinlue lsaerigaraengadtiLoonb oaplhoyntgo nPwC2e.r eSicnluulsatreirae adntdo gLeotbhoeprhoyntonth wenereeg acltuivsetesriedde 
toofgPeCth1e,rs uogng tehset inneggtahtaivtea staidrgee otef dPCpr1o, fisluingggeasptipnrgo atchha,t laim taitregdetaesdi tpirso,fcioliunlgd arpeapdriolaycdhi,s lcirmimitienda taes tiht eims, 
cforoumld Sraeracdoiplhyy dtoisncrgiemniunsa.teA tqhueamri ufrmomg rSoawrcnopshoyfttocno graenlsu(sS.E A4qaunadriSuGm3 g)rwoewrne csloufst tceorreadlst o(SgEe4th aenrdo nSGth3e) 
wneegrea tcivluessteidreedo tfoPgCe1th.eTrh oenp tohsei tniveegastiidvee osfidPeC o1f sPhCo1w. eTdhec lpuostseitriivneg soidfSe .orfe gPuCl1ar sehsopw. (eSdR c1l-u3s)tienrdinivgi odfu Sa.l 
rsepgeucliamree snps. s(uSgRg1e‐s3t)i ningdthivaitdtuhael gsepoegcrimapehnisc asluigmgpeastcitnogn tmhaett atbhoe ligteeocgormapphoisciatilo inmispancott ovne rymsettraobnoglitine 
cthomespeospsieticoiens .isI nnocto nvterrays ts,tirnoncags ieno tfheeisteh esrpSec.igelsa.u Icnu mcosnptr.a(sStG, i1n-3 c)aosre So.f eehirtehnebre rSg. igslpau.c(uSEm1 s-4p). s(pSGec1im‐3)e nosr, 
Ssa. mehprelnesbefragiil esdp.t o(ScElu1‐s4t)e rsptoegciemtheenr.s, samples failed to cluster together.
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(a) 

(b) 

FigureF4ig.u(ar)e T4a. r(ga)e Tteadrg1eHte-dN 1MHR‐NsMigRn asligbnipall obtipplroitn pcripinaclipcoaml cpomonpeonnteannt aalnyasliyss(iPs C(PAC)A(b) ()ba)n adndh iheirearracrhcihciaclal 
cluster analysis (HCA) of soft coral species showing two clear clusters of soft coral species described 
clusteranalysis(HCA)ofsoftcoralspeciesshowingtwoclearclustersofsoftcoralspeciesdescribedby
by two principal component vectors accounting for 84.6% of the total variance. For complete 
twoprincipalcomponentvectorsaccountingfor84.6%ofthetotalvariance.Forcompleteinformation
information on sample codes refer to Suppl. Table S1.
onsamplecodesrefertoSuppl.TableS1.
Hierarchical cluster analysis (HCA) is an additional tool for revealing interrelationships among 
Hierarchicalclusteranalysis(HCA)isanadditionaltoolforrevealinginterrelationshipsamong
coral species. It also offers a more intuitive graphical way for result interpretation. HCA showed 
coral stwpeoc cielesa.rI ctlualsstoeros:f fgerrosuap mA ocoremipnrtiuseitdiv oef gsirxa sppheicciamlewnas,y wfhoerrreeassu gltroiunpte Brp irnectlautdioend .thHe CreAmsahinoiwnge d10 
twoclseoafrt ccloursatle rssp:egcirmouenpsA (Fciogmurpe ri4sbe)d. oGfrosiuxps pAe csihmoewnesd, wa hdeirsetainscgt rsouubpclBusitnerc lu(1dAe)d otfh eSirneumlaariian ianngd 
10 sofLtocboorpahlytsopne scpimeceienss t(hFaitg aurree c4hbem).icGalrloyu dpiffAereshnto wfroemd athde irsetsint cotf Ssuarbccolpuhsytteorn (s1pA., )proofvSidininugla erviaidaenndce 
Lobophfoytro tnhesp veacliiedsittyh aotf athree cehmepmloicyaeldly tdarigffeetreedn tpfrroofimlintgh eaprepsrtooacfhS afrocro pcohryatlo nclsapss.,ifpicraotvioind.i nSgineuvlairdiae nacned 
for theLovbaoplihdyittoyn osfptehcieese mweprleo yinecdlutdaerdge itne dthpe rsotufidliyn gasa dpipstraonatc ohuftolirercso troa lascsleassss iwfichaettihoenr .thSei nmuulalrtiivaaarniadte 
Lobophdyatotan sapneaclyiessisw meroedinelc lcuadne defifnecthtievesltyu ddyisatsindgiustiashn tboeuttwlieeersn tovaarsisoeusss wSahrectohpehryttohne mspu. lGtivroauripa teAd aaltsao 
analysdiisspmlaoydeedl caa dnisetfifnecctt isvuebl‐yclduisstteirn g(2uAis) hcobnesttwiteuetendv oafr itohue saSqauracroipuhmy tgornoswpn. Gsorfot ucporAalsa (lsSoE4d iasnpdla SyGe3d). 
adistiSnicmtsiluabr -tcol uPsCteAr r(2esAu)ltcso (nFsitgiuturtee 4dao),f atlhl espaeqcuimareiunms fogrr oewithnesr oSf.t gcloauraclusm(S oEr4 Sa. nehdreSnGbe3r)g.iS fiamileilda rtoto clPuCstAer 
resultsin( Foingeu grreo4uap) ,(Failgluspree 4cibm). e nsforeitherS.glaucumorS.ehrenbergifailedtoclusterinonegroup
(Figure4b).
2.3. Cytotoxic Activity of Soft Corals  
2.3. CytotoxicActivityofSoftCorals
Soft coral ethyl acetate extracts subjected to NMR fingerprinting were further assessed for their 
in vitro cytotoxicity against human colon epithelium adenocarcinoma cell lines (HT29) and human 
SoftcoralethylacetateextractssubjectedtoNMRfingerprintingwerefurtherassessedfortheir
prostate cancer cell lines (PC3). Both cell lines showed a comparable response towards S. ehrenbergi 
invitrocytotoxicityagainsthumancolonepitheliumadenocarcinomacelllines(HT29)andhuman
Mar.Drugs2017,15,211 7of13
Mar. Drugs 2017, 15, 211   7 of 13 
prostatecancercelllines(PC3).BothcelllinesshowedacomparableresponsetowardsS.ehrenbergi(SE2
&S(ES4E)2, S&. gSlEau4c),u Sm. (gSlaGu1cu&mS (GSG3)1, S&. rSeGgu3l)a, rSe.( SreRg3u)l,araen d(SRSi3n)u, laanrdia SainndulLaoribao pahnydt oLnobsoppehcyietosn, wspitehcireess,p wecitthiv e
IC5r0evspaelucteisveo fICc5a0. v2a2lu,3es3 ,o3f 6c,a6. 02,22, 13,38, ,3a6n, d609, 2µ1g, /8m, aLnda s9s μhog/wmnL inasF sihgouwren5 i.n SF.iagcuurteu m5., SS.. accountvuomlu, tSu.m  ,
S. gcloanuvcoulmutu(mSG, S2.) ,galanudcuSm. r(eSgGu2la),r ean(SdR S1.) rdegisuplalarey e(SdRa1)s dtriospnlgaeyredcy ato sttorxoincgaecr tcivyittoytoaxgica iancsttivHityT 2a9g,awinhsti le
S.rHegTu2la9r,e w(ShRile2 )Sw. raesgumlaorer e(SeRff2e)c wtivaes magoarien esftfPecCti3v.e against PC3.  

FigFuirgeu5r.e C5.y Ctoyttooxtoicxiitcyitdya dtaatoaf osfo sfotfct ocroarlals pspeeccieiessa aggaaiinnsstt PPCC33 aanndd HHTT2299 cceellll lliinneess. .RReseusultlst saraer eexepxrpersessesde d
asmase amneaICn 5I0C(5µ0 g(μ/gm/mL,Ll,o lwowererv avlaulueses= =h higighheerr aaccttiivviittyy)) ±± SSEE.. ((nn == 33)).. FFoorr ccoommpplelteet eininfofromrmataiotino nono n
samspamlepcloed ceosdreesf reerfetor tSou Spuppl.pTl.a TbalbelSe 1S.1. 
2.4.2M.4.e Mtabetoalbitoel/iMte/eMtaebtoalbiotelitaen adnMd Meteatbaobloitliet/eC/CyytottootoxxicicitityyC Coorrrreellaattiioonn AAnnaallyyssiiss 
WeWaett eamttepmtepdtetdo tfou rftuhretrheerx aemxaimneinew hwehtehtehrera nani ninnneerr cocorrrreellaattiioonn eexxiissttss aammoonngg mmeetatbaobloitleitse soro r
between metabolites and cytotoxicity using two different algorithms: Spearman rank order correlation 
betweenmetabolitesandcytotoxicityusingtwodifferentalgorithms: Spearmanrankordercorrelation
and Pearson linear correlation [19]. Pearson’s and Spearman’s correlation coefficient results are 
and Pearson linear correlation [19]. Pearson’s and Spearman’s correlation coefficient results are
summarized in Table 1. The strongest positive metabolite inner correlation was observed among 
summarized in Table 1. The strongest positive metabolite inner correlation was observed among
dihydroxydeepoxysarcophine (N4/5) and gorgosterol (6), with a Pearson’s and Spearmean’s R2 
dihydroxydeepoxysarcophine (N4/5) and gorgosterol (6), with a Pearson’s and Spearmean’s R2
ranging from 0.86 to 0.89 (Table 1). N4‐6 were tightly linked to the major principal component (PC1) 
ranging from 0.86 to 0.89 (Table 1). N4-6 were tightly linked to the major principal component
that  differentiated  soft  corals,  as  revealed  from  the  loading  plot  (Figure  4a).  The  weakest 
(PC1) that differentiated soft corals, as revealed from the loading plot (Figure 4a). The weakest
inter‐metabolite correlation was observed among guaiacophine (N1) and sarcophine/ent‐sarcophine 
inter-metabolitecorrelationwasobservedamongguaiacophine(N1)andsarcophine/ent-sarcophine
(N2/3). With regard to a correlation between the targeted metabolite levels and cytotoxicity data, the 
(N2/3). Withregardtoacorrelationbetweenthetargetedmetabolitelevelsandcytotoxicitydata,the
IC50 for HT29 did not show any obvious correlation with any of the quantified metabolites using 
IC forHT29didnotshowanyobviouscorrelationwithanyofthequantifiedmetabolitesusingeither
5e0ither algorithm. The strongest positive correlation is found between PC3 cytotoxicity data and 
algorithm. ThestrongestpositivecorrelationisfoundbetweenPC3cytotoxicitydataandgorgosterol
gorgosterol content (N6, Spearman’s R2 = 0.55), and next with dihydroxydeepoxysarcophine (N4, R2 = 
con0t.e5n).t  (N6,Spearman’sR2=0.55),andnextwithdihydroxydeepoxysarcophine(N4,R2=0.5).
TabTleab1l.eC 1o. rCroerlaretiloatniocno ecoffiecffiiecnietnstasm amonogngm meteatbabooliltietea abbuunnddaannccee aanndd ttooxxiicciittyy ddaattaa. .PPeeaarsrosonn cocrorrerlealtaiotino n
coecffioceifefinctisenatrse agriev egniviennt hine ltohwe elorwleefrt pleafrtt poafrtth oeft athbele twabhleil ewShpileea rSmpeaanrmcoarnre claotriroenlactoioenffi ccoieefnfticsieanretsg airvee n
atthgieveunp apte trhrei guphptepra rritgohtf pthaert toafb tlhee. tVaabllue.e Vsailnuebso ilnd baorled sairgen siifigcnaifnictaantt pat< p 0<. 00.50.5. 
Variables  N1  N2‐3 N4 N5 N6 PC3 HT29 
Variables N1 N2-3 N4 N5 N6 PC3 HT29
N1  0.064 0.246 0.393 0.236 0.061 0.031 
N1 0.064 0.246 0.393 0.236 0.061 0.031
N2‐3  0.167  0.599  0.512  0.735  0.297 0.235 
N2-3 0.167 0.599 0.512 0.735 0.297 0.235
N4  0.411  0.532  0.878  0.884  0.501  0.434 
N4 0.411 0.532 0.878 0.884 0.501 0.434
N5 N5  0.572 0.572 0.4810.481 0.8640.864  00.8.82626  0.403.5435 0.224 0.224
N6 N6  0.294 0.294 0.7210.721 0.8390.839  0.7604.764  0.408.4585 0.359 0.359
PC3 PC3  0.268 0.268 0.1280.128 0.3400.340 0.3106.316 00.4.04404  0.638 0.638
HT29 HT29 −0.079−0.079− 0.07−90.0790.2790.279 0.0206.026 00.2.22525 0.506.55 65
Mar.Drugs2017,15,211 8of13
3. Discussion
Theabundanceofcembranoidditerpenesincoralsisascribedtoadefensiveroleagainstmarine
predators[20]. Thehighestlevelsofsarcophine/ent-sarcophine(N2/3)wererecordedinspecimens
collectedinSafaga(SC2,SG2,andSR3)withca. 106.9,94.3,and74.8µg/mgdryweight,respectively,
providingevidencethatgeographicalorigincanimpactsecondarymetabolitecontentanddistribution,
asobservedinterrestrialplants[21].Inagreementwithourresults,Bilasyetal.revealedthatS.glaucum
fromHurghadacontains82.8µg/mgsarcophine[22]whereasintheVietnamesesoftcoralS.mililatensis,
levelsdroppedto0.025µg/mg[23]. However,suchahypothesisneedstobefurthervalidatedthrough
analyzingalargesamplepoolofcoralsrepresentingmorediverselocalitiesand/orwithdifferent
ecological environments. It should be noted that sarcophine/ent-sarcophine was not observed in
Sinulariapolydactelaspecies,althoughthegenusisknowntoproducecembranoidditerpenes.Itisworth
mentioningthatS.convolutumfromSafaga(SC2)wasthespeciesrichestinallmonitoredmetabolites,
suggestingthatfocusoughttobedirectedonthepropagationofthisspeciesandgenotypeifaraw
material for marine diterpene production is selected. Conversely, both Sinularia and Lobophytum
specieswerethepoorestinditerpenes,asrevealedfromqNMRanalysis(Figure3). Withregardto
comparingmetabolitelevelsfromwildsoftcoralsversusaquariumgrownones,forbothS.ehrenbergi
andS.glaucum, tankculturedsoftcoralsappearedtocontainlessbioactivecompoundscompared
totheircorrespondingwild-keptones(Figure3), whichisinagreementwithourpreviousresults
usingLC-MS[18]. Thediscrepancybetweenwildandaquariumgrowncoralsmightbeattributableto
differencesinecologicalbackgrounds,asaquariumcoralsmighthavelostorinterruptedtheirfitness
toproducethesechemicalsfunctioningasdefensesagainstpredatorsnottobefoundinthetanks[24].
However,finalproofcanonlybemadeifthesamecloneisusedinbothenvironments,something
thatwasnotavailableyetforourstudy. Anotherexplanationcouldbeattributedtodifferencesin
harboredorganismssuchaszooxanthellae,fungi,andbacteriainsidecorals,whichmightbecritical
fortheproductionofsecondarymetabolitesinholobiontmarinesoftcorals[25]. Lessthan1%oftotal
microbialdiversitycanbesuccessfullyculturedincoraltanks,whichmaynolongerberepresentative
oftheenvironmentwherethesoftcoralsoriginatedfrom[26,27]. Ourresultsalsorevealthatthesea
depthwhereacoralgrowscanimpactitsditerpenelevelsinanegativerelationship. Atgreatersea
depthlevels,adecreaseinditerpenelevelswasfound,asinthecaseofinS.ehrenbergi(SE1vs. SE3)
and S. convolutum (SC1 vs. SC2), shown in Figure 3. This result indicates the importance of light
(mostlikely)orwavemovement(lesslikely)tothephotosymbioticunicellulardinoflagellatesliving
insidethecorals’polyps. Theyareessentialtofulfillthesecorals’energeticrequirementsviaproviding
photosynthesisproducts(i.e.,sugarsandmaybealsoenhancedprecursorslikeprenyldiphosphates)
thatmayultimatelyaffecttheyieldofthebioactivemetabolites[28,29].
ComparedtoPCAderivedfromthefull1H-NMRspectra[18],qNMR-derivedPCAresultedin
astrongerclassificationmodel(Figure4a).ThisobservationcanbeattributedtothefactthatPCAbased
onall1H-containingcompoundsincludesdatafromprimarymetabolites(e.g.,sugarsor,especially,
fattyacids)appearingatδ 0.84–1.36ppm(Figure2). Suchprimarymetabolitecontentsaretightly
H
linkedtothegrowthconditionsandareoftenoflittleormostlynotaxonomicalvalue,thusdiluting
orthwartingclassificationmodelsasobservedin[30]. Interestingly,aquariumgrownS.glaucumsp.
(SE4)andS.ehrenbergisp. (SG3)wereclusteredtogetheralongthenegativesideofPC1,beingless
enrichedincembranoids(N1-5)comparedtowildcollectedmarinecoralsofthesamespecies. Overall
itcanbestatedthatthedifferencesingrowingconditionssupercedethoseofspeciationinthisclade
ofcorals. Thus,theclusteringbasedonthequantificationofalimitedsetofisoprenoids,common
toalloratleastmostofthespecies,islessvaluableforchemotaxonomybutmightgiveinsightinto
environmentalandgeographicalfactorsaffectingsecondarymetaboliteproduction.
In general, wild soft corals demonstrated stronger cytotoxicity activity against both cell lines
comparedtotheircorrespondingaquariumgrownones,whichisinagreementwithqNMRresults
(Figure 3). No difference in IC values was observed for extracts prepared from reef flat corals
50
comparedto2−3mseadepthcorals, suggestingthatseadepthinthisrangedoesnothavemuch
Mar.Drugs2017,15,211 9of13
impactonthecorals’cytotoxiceffects. Unexpectedly,SinulariaandLobophytonspeciesdisplayedthe
strongestcytotoxicactivityagainstbothcelllines,althoughtheyhavelowercontentsofthequantified
isoprenoidsandthusthemostdistinctqNMRfingerprintfromotherSarcophytonsp.[18,31]. Therefore,
itcanbeconcludedthatthecompoundsresponsibleforthehighercytotoxicityaremostlikelynot
coveredbytheqNMRanalysis.
ThesignificantpositivecorrelationbetweencembranoidsN4/5andthesterolN6(Table1)has
beenpreviouslyobservedinS.glaucum,wherecembranoidswerefoundasmajorcomponents[32,33]
along with sterols [33,34]. The relative slight positive correlation between PC3 cytotoxicity data
andgorgosterolcontentsuggeststhatthePC3celllineis,ingeneral,lessresponsivetogorgosterol,
inagreementwithCarvalhoetal.whoreportedalowersensitivityofPC3towardsoxysterolscompared
totheHT29cellline[35].Additionally,PC3cellshavepreviouslybeendemonstratedtohavearelatively
lowsensitivitytowardsterolsversuspolyphenols[36,37].
4. MaterialsandMethods
4.1. SoftCoralMaterialCollectionandIdentification
Sarcophyton ehrenbergi, S. regulare, S. glaucum, S. convolutum, S. acutum, along with Lobophyton
pauciliforum and Sinularia polydactela samples from Red Sea coastal regions were collected from
two different diving sites, namely Al-Guna and Makadi bay, along the Egyptian Red Sea coastal
areaatwaterdepthsrangingfrom2–5mbelowsea-levelusingSCUBAdiving. Scleriteidentification
followingtheprotocolof[38]wascarriedouttoconfirmthespeciestype. Samplecollection,handling,
identification,andaquariumculturingconditionsofS.ehrenbergiandS.glaucumgrowninaquariums
arepreviouslyreported[18].
4.2. ChemicalsandReagents
Hexamethyldisiloxane(HMDS)andacetone-D6(99.80%D,99%purity)werepurchasedfrom
DeuteroGmbH(Kastellaun,Germany). HMDS(0.94mMasafinalconcentration)wasaddedforboth
chemicalshiftadjustmentandabsolutemetabolitequantification. Sarcophinestandardwaspurchased
fromAGScientific, SanDiego, CA(St. Louis, MO,USA).Allotherchemicalsandstandardswere
availablefromSigmaAldrich(St. Louis,MO,USA).
4.3. SoftCoralExtraction,SamplePreparation,andNMRAnalyses
100 mg of dried soft coral umbrella tissue was cut using a scalpel under liquid nitrogen, and
furthergroundusingamortarandpestle. Thepowderedsoftcoraltissuewassubsequentlyextracted
by 5.0 mL of 100% ethyl acetate using an ultrasonic bath for 20 min. The debris was removed by
centrifugation at 12,000× g for 5 min. 3 mL of the ethyl acetate extract was aliquoted and left to
evaporateunderanitrogengasstreamuntilcompletedryness. Thedrypelletwasre-suspendedin
800µLofacetone-D6containingHMDS(0.94mmol/Lfinalconcentration)andaftercentrifugationat
13,000×gfor1min,transferredtoa5mmNMRtube. 1HNMRspectrawererecordedonanAgilent
VNMRS600NMRspectrometer(Agilent,SantaClara,CA,USA)usingaprotonNMRfrequencyof
599.83MHz. NMRspectralparameterswereidenticaltothosedescribedbyFaragetal.[18].
4.4. NMRQuantification
FormetabolitequantificationusingNMRspectroscopy,thepeakareasofselectedprotonsignals
belongingtothetargetcompoundsandtheinternalstandard(HMDS)wereintegratedmanuallyfor
allsamples. Thefollowingequationwasappliedforabsolutemetabolitelevelcalculations:
I x
m = M × T × St ×c ×v
T T St St
I x
St T
Mar.Drugs2017,15,211 10of13
m : massofthetargetcompound[µg]inthesolutionusedfor1HNMRmeasurement
T
M : molecularweightofthetargetcompound[g/mol]
T
I : relativeintegralvalueofthe1HNMRsignalofthetargetcompound
T
I : relativeintegralvalueofthe1HNMRsignalofthestandardcompound
St
x : numberofprotonsbelongingtothe1HNMRsignalofthestandardcompound
St
x : numberofprotonsbelongingtothe1HNMRsignalofthetargetcompound
T
c : concentration of internal standard (HMDS) in the solution used for 1H NMR measurement
St
[mmol/L]
v : volumeofsolutionusedfor1HNMRmeasurement[mL]
St
4.5. CytotoxicityAssay
Cytotoxicity assays against human prostate PC3 and colon cancer HT29 cell lines were
conducted following the protocol described by Farag et al. [39]. The cells were cultured in RPMI
(Roswell Park Memorial Institute) 1640 containing 1% L-glutamine and 10% heat-inactivated
fetal bovine serum (FBS) at 37 ◦C, within a 5% CO humidified atmosphere. Cells were left
2
to attach in 96-well plates at a density of 1 × 104/well for 24 h. After 24 h, the media was
replaced with RPMI media containing the dried ethyl acetate extracts dissolved in DMSO (at a
concentration of 2 mg/mL). Each extract was tested at the following concentrations: 5, 10, 50,
and 100 µg/mL. The maximum DMSO concentration was 0.1%, which was not cytotoxic to the
cell lines. After 72 h, the medium was replaced by 100 µL of 0.3 mg/mL XTT-solution (2,3-bis
(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide) (Roche
AppliedScience,Mannheim,Germany)followedbyincubationat37◦Cfor4hours. Digitoninwas
used as a positive drug control in DMSO, with an IC value of 1.7 µg/mL. A microplate reader
50
(Beckman Coulter, DTX 880 Multimode Reader) was used to measure the absorbance at 490 nm
againstareferencewavelengthof650nm. Theexperimentswererepeatedintriplicateandfortwo
consequent passages for each cancer cell line. IC values were calculated with GraphPad Prism
50
version5software,usingthesigmoidaldose-responsefunction.
4.6. StatisticalAnalysis
SpearmanrankordercorrelationandPearsonlinearcorrelationwerecarriedoutusingXLSTAT
statisticalsoftware(Addinsoft,NewYork,NY,USA).
5. Conclusions
ThisstudyemployedqNMRtoinvestigateSarcophytontaxaheterogeneityaccordingtospecies
level, geographicalorigin, seadepth, andgrowinghabitat. qNMRrevealedditerpeneenrichment
in Sarcophyton sp. compared to Sinularia and Lobophyton species, with sarcophine enantiomers as
major components (17–100 µg/mg). It remains to be examined whether such different metabolite
accumulationpatternsamongsoftcoralsareduetoprecursorlimitation(i.e.,isoprenylatedbuilding
blocks) or, more likely, to differences in regulation or specific activities of enzymes in Sarcophyton
spp. versus other species. Probing enzymatic activity or gene expression levels could provide
amoreconclusiveunderstandingofsuchmetabolomicresultsincorals. Theresultsfurtherrevealed
that qNMR provided a stronger classification model of Sarcophyton sp. than untargeted NMR
fingerprinting. Among the selected corals studied, more focus should be directed towards the
propagationofS.convolutumasarawmaterialforditerpeneproduction,beingthespeciesrichestinall
monitoredmetabolites. However,growingconditionsobviouslyhaveaverystrongeffecton(targeted)
isoprenoidproduction,evensupercedingmostspecies’differences. Thelackofcorrelationbetween
thecompositionandabundanceofthetargetedisoprenoidsandtherespectiveextract’santicancer
effectssuggeststhatatleastthistypeofbioactivitymaynotbeeasilypredictedbyNMRprofiling.
Anotherhypothesisisthatotherditerpenesandbiscembranoidsofhigherspecificactivitypresentin