Rev. bras. farmacogn. vol.27 número1

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RevistaBrasileiradeFarmacognosia27(2017)67–69

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

Original

Article

Constituents

from

the

bark

resin

of

Schinus

molle

Gonzalo

Rodolfo

Malca-García

a,∗

_,

_Lothar

_Hennig

a

_,

_Mayar

_Luis

_{Ganoza-Yupanqui}

b,∗

_,

Alejandro

Pi ˜na-Iturbe

c

_,

_Rainer

_W.

_Bussmann

d

a_Institut_für_Organische_Chemie,_Fakultät_für_Chemie_und_Mineralogie,_Universität_Leipzig,_Leipzig,_Germany

b_Departamento_de_{Farmacología,}_Facultad_de_Farmacia_y_Bioquímica,_Universidad_Nacional_de_Trujillo,_Trujillo,_Peru

c_Escuela_de_{Microbiología}_y_{Parasitología,}_Facultad_de_Ciencias_Biológicas,_Universidad_Nacional_de_Trujillo,_Trujillo,_Peru

d_William_L._Brown_Center,_Missouri_Botanical_Garden,_St._Louis,_United_States

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13June2016

Accepted21July2016

Availableonline15September2016

DedicatedtoProf.Dr.LotharBeyeronthe

occasionofhis80thbirthday.

Keywords:

Schinusmolle

Resin Terpenes

Cytotoxicactivity

MIC

a

b

s

t

r

a

c

t

AtotaloffiveterpeneswasisolatedfromthebarkresinofSchinusmolleL.,Anacardiaceae,andtheir structuresweredeterminedbyspectroscopictechniques.Amongthesecompoundsthesesquiterpene hydrocarbonterebintheneshowedsignificantgrowthinhibitoryactivityagainsthumancoloncarcinoma HCT-116cells.Furthermore,terebintheneandpinicolicacid(5)alsoshowedantibacterialactivityagainst StaphylococcusaureusATCC25923andBacillussubtilisATCC6633.

Introduction

The family Anacardiaceae is of pantropical occurrence and includesafewrepresentativesintemperate regions.Thefamily comprisesabout70generaand600species.Manyofthemareused traditionallyashealing,stomachicand antidiarrhealagents,due tothepresenceoftanninsandoilresins(Duarteetal.,2006).In thisfamily,SchinusmolleL.(alsoknownasCaliforniapepperand pinkpepper)wasintroducedfromSouthAmericatomosttropical andsubtropicalareasoftheworld,aswellastheMediterranean (Rhoumaet al.,2009).InPeruviantraditional medicineS.molle

isusedasantibacterial,topicalantiseptic,digestiveandpurgative diuretic(Duke,2002),fortoothache,woundhealing,rheumatism, and menstrual disorders, as well as stimulant and antidepres-sant(Machadoetal.,2007),forrespiratoryandurinaryinfections (PerezandAnesini,1994),andasanalgesicandcentraldepressant (Barrachinaetal.,1997).ExtractsofS.molleshowedpromising anti-tumoraleffectsandcytotoxicactivity(IC5050±7␮g/ml)againsta

humanhepatocellularcarcinomaCellLine,HepGwasalsoreported (Ruffaetal.,2002).RecentstudiesrevealedthatS.molleessential

∗ Correspondingauthors.

E-mails:gonzalo.malca@uni-leipzig.de

(G.R.Malca-García),mganoza@unitru.edu.pe(M.L.Ganoza-Yupanqui).

oilwascytotoxicinseveralcelllines,anditwasmoreeffectiveon breastcarcinomaandleukemiccelllines(Díazetal.,2008).

ExtensiveinvestigationsofthefruitandleafessentialoilsofS. molleidentifiedabroadvarietyofconstituents(Abdel-Sattaretal., 2010;Bendaoudetal.,2010),showingthattheingredientsare sim-ilar, butwith differencesin theirpercentage depending onthe regioninwhichtheyaregrown.Maincomponentsare monoter-penehydrocarbonsandoxygenatedmonoterpenehydrocarbons. Noreportswerefoundconcerningisolationandcharacterizationof secondarymetabolitesfromthebarkresin.Therefore,wedecided toconcentrateoureffortinS.molleresin.

Materialandmethods

NMRandMSinfrastructureandmethods

NMRspectra(1_H,13_C,_APT,_NOESY1D,_H_,_H_-COSY,_edited_HSQC,

andHMBC)wererecordedonaVarianMercury400plus(400MHz for1_H,₁₀₀_MHz_for13_C)_and_a_Varian_Mercury₃₀₀_plus₍₃₀₀_MHz

for1_H,₇₅_MHz _for13_C) _{spectrometer,}_{respectively,} _at₂₆◦_C _and

withCDCl3asasolvent.Thechemicalshiftswerereported

rela-tivetotheresidualsolventpeak,usedasaninternalreference(1_H

7.26ppm,13_C _77.16_ppm)._Chemical_shifts_are_given _in_ı_values,

couplingconstantsJinHz.

http://dx.doi.org/10.1016/j.bjp.2016.07.004

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68 G.R.Malca-Garcíaetal./RevistaBrasileiradeFarmacognosia27(2017)67–69

Plantmaterial

Theresinbarkof“molle” wascollectedin March2009from Trujillo-Peruand thespecies wasidentified asSchinusmolle L., Anacardiaceae,byEricF.RodríguezRodríguezatHerbarium Trux-illense (HUT), National University of Trujillo, Peru. A voucher specimenunderNo.58335(HUT)documentingthecollectionwas depositedatHerbariumTruxillense(HUT)inPeru.

Extractionandisolation

Spottestswereusedforthequalitativedeterminationof sec-ondarymetabolitespresent intheresin ofS.molle(Dominguez, 1973;Harborne,1984).Weidentifiedsmallamountsofsteroidsand triterpenoids(darkgreen)bytheLiebermann-Burchard’stest,and flavonoids(red)bytheShinoda’stest.Noalkaloidsweredetected usingDragendorff’stest.

Theresin ofS.molle(10.14g;stillcontainingpiecesofwood andinsolublematerials)wasextractedwithCH2Cl2 andhexane

(250mleach).Bothextractswerecombinedyielding2.63gafter removal of the solvents. This residue was fractionated by col-umnchromatography on silica gel eluting with hexane, called fraction1(0.92g)andbyusingCH2Cl2systemtogivefraction2

(1.50g).Fraction1wassubjectedtocolumnchromatographywith hexane/CH2Cl2 (100:0→99.5:0.5→99:1)systemtogive

germa-creneD(1)(264mg;Rf:0.81)andterebinthene(2)(18.4mg;Rf:

0.91).

Fraction2wassubjectedtocolumnchromatographywiththe eluentsystemCH2Cl2/EtOAc(1:0→2:0.1→3:0.1→4:0.2→5:0.3)

tofurnishisomasticadienonalicacid(4)(120mg;Rf0.65)andto

CH2Cl2/EtOAc(5:0.3→3:1→2:1.5→1:2)resultingintheisolation

ofisomasticadienoicacid(3)(336mg;Rf:0.6)andpinicolicacid(5)

(18.6mg;Rf:0.2).

Bioactivityassays

Cytotoxicityscreen

ThehumanHCT-116coloncarcinomacellline(ACC-581)was obtainedfromtheGermanCollectionofMicroorganismsandCell Cultures(DeutscheSammlungfürMikroorganismenund Zellkul-turen,DSMZ)and wascultured underconditionsrecommended bythedepositor. Cellswereseeded at6×103 _cells_per _well_of

96-wellplatesin180␮lcompletemedium(90%McCoy’s5A+10%

FBS)andtreatedwithsesquiterpenehydrocarbonterebinthene(2) inserialdilutionafter2hofequilibration.Terebinthene(2)was testedinduplicate aswellastheinternal solventcontrol.After 5dincubation, 20␮lof 5mg/mlMTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)inPBS(phosphate-buffered saline;pH7.4)wasaddedperwellanditwasfurtherincubated for2hat37◦_C. _The_medium_was_then_discarded_and_cells_were

washedwith100␮lPBSbeforeadding100␮l2-propanol/10NHCl

(250:1)inordertodissolveformazangranules.Theabsorbanceat 570nmwasmeasuredusingamicroplatereader(TecanInfinite M200Pro),andcellviabilitywasexpressedaspercentagerelative totherespectivesolventcontrol.IC50valuesweredeterminedby

sigmoidalcurvefittingandvaluesrepresenttheaverage±SD of twoindependentmeasurements.

Bacteriastrainsassay

ThestrainsusedwereStaphylococcusaureusATCC25923, Bacil-lussubtilisATCC6633,EscherichiacoliATCC25922andPseudomonas aeruginosa(isolatedattheHospitalRegionalDocentedeTrujillo, Peru).ThebacterialculturesweregrowninMueller-Hintonagar mediaincubatedat37◦_C_for₂₄_h_following_literature₍_CLSI,₂₀₁₂_).

Agar-well diffusion methods: The agar-well with diameter of 5.5mm were inoculated to a concentration equivalent to

0.5McFarland. Compounds2 and5wereinoculated at40␮lin

DMSO,atconcentrations of5and 10mg/ml.Afterbacterial dif-fusionfor10min,thosepetri plateswereincubatedat37◦_C_for

22h.

BrothmacrodilutionMIC(minimalinhibitionconcentration)

DryextractCH2Cl2 (3–5)wasdilutedinDMSO,tween80and

water in ratio2:1:3 according tothe following concentrations: 0.062,0.125,0.25,0.5,1.0,2.0,4.0,8.0and16.0mg/ml.EachMICtest wasrepeatedtwiceandalltubeswereincubatedfor22hat37◦_C.

AfterincubationMICresultsweredeterminedasminimum con-centrationwhichshowednovisiblebacterialgrowth.Then50␮lof

resazurinsodiumsalt(Sarkeretal.,2007)wereaddedtoeachtube toafinalconcentrationof0.02%,andthesampleswereincubatedat 37◦_C_for₁_h._MIC_was_confirmed_as_minimum_{concentration}_when

nocolorchangeoftheresazurinsodiumsalt(frombluetopink) happened.

Resultsanddiscussion

The structure of all five already known compounds germacrene-D (1), terebinthene [(6R*_,8_R*

)-9-spiro(cyclopropa)-2,4,4,8-tetramethylbicyclo[4.3.0]non-1-ene(2)],isomasticadienoic acid (3), isomasticadienonalic acid (4), and pinicolic acid [3-oxo-5␣-lanosta-8,24-diene-21-oicacid(5)]wasdeterminedafter

carefulpurificationofthecompoundsusingspectroscopicmethods andcomparisonofthedatawithliteratureresults.

1

2

5

3

4 R=CHO

R=CH

3

CO2H

HO2C H

H R

H O O

H H

GermacreneDisabiogeneticprecursorofmanyother sesquiter-penestructures(BülowandKönig,2000),terebinthenewasisolated fromS.terebinthifolius(Richteretal.,2010),thetriterpenoidacids

3and4werefoundintheberriesorthestemexudateofS.molle

(Pozzo-Balbietal.,1978;Abdel-Sattaretal.,2007)and5was iden-tifiedasanti-inflammatorytriterpenoidfromGanodermagenus(Ko et al.,2008).Surprisingly, there areno reports inliterature for sesquiterpenehydrocarbon 2asaningredient ofS.molle, but2

wasisolatedforthefirsttimefromS.terebinthifolius(Richteretal., 2010).Compound5wasnotfoundtobeactiveagainstanytypeof cancer,butinducedplateletaggregation(Mosaetal.,2011).

Terebinthene (2): (6R*_,8_R*

)-9-spiro(cyclopropa)-2,4,4,8-tetramethylbicyclo[4.3.0]non-1-ene:1_H_NMR_(400.1_MHz,_CDCl 3):

ı=0.49(1H,m,H-10a),0.53(1H,m,H-11a),0.60(1H,m,H-10b), 0.69(3H,s,CH3-15),0.71(1H,s,H-11b),1.00(1H,m,H-7a),1.06

(3H,s,CH3-13),1.08(1H,H-5a),1.10(3H,s,CH3-14),1.28(3H,m,

CH3-12),1.63(1H,m,J=18.5Hz,H-5b),1.83(1H,m,H-7b),2.00

(1H,m,H-8),2.12(1H,m,H-3a),2.12(1H,m,H-3b),2.65(1H,br, H-6);13_C_NMR₍₁₀₀_MHz,_CDCl

3):ı=6.33(C-10),7.36(C-11),13.00

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G.R.Malca-Garcíaetal./RevistaBrasileiradeFarmacognosia27(2017)67–69 69

Table1

Diametersofinhibitionzonesatconcentrationsof5and10mg/ml.

Compounds Concentration(mg/ml) Diameter(mm)

S.aureusATCC25923 B.subtilisATCC6633 E.coliATCC25922 P.aeruginosa

2 10₅ 9.3_8.0 9.3_8.6 0₀ 0₀

5 10₅ 7.1_6.8 9.0_8.8 0₀ 0₀

Table2

MICfromthebarkresinofSchinusmolle.

Resin MIC

S.aureusATCC25923 B.subtilisATCC6633

CH2Cl2extract 8.0mg/ml 0.125mg/ml

CytotoxicityonHCT-116cells

The in vitro cytotoxicity of 2 was evaluated in a tetra-zoliumsalt-based assay(MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) against human HCT-116 colon

carcinomacells.Compound2displayedamoderateinhibitionof HCT-116cellswithIC5014.23±1.3␮g/ml.

Antibacterialactivity

Additionally,wedeterminedtheantibacterialactivityof com-pounds2and5byscreeningagainstStaphylococcusaureusATCC 25923,BacillussubtilisATCC6633,EscherichiacoliATCC25922and

Pseudomonasaeruginosa(Table1).Theotherconstituents(1,3and

4)arewellknownfortheirantibacterialactivityand pharmacolog-icalproperties(Duarteetal.,2006;Rhoumaetal.,2009;Chantraine etal.,1998).TheMICofCH2Cl2extractwasreportedinTable2.

Conclusions

Herewereporttheisolationandcharacterizationoffive con-stituents form the bark resin of S. molle: germacrene D (1), terebinthene(2),isomasticadienoicacid(3),isomasticadienonalic acid(4),andpinicolicacid(5).TheresinofS.mollecontained10%of germancreneD(1),whichisveryhighcomparedwithothernatural sources(Kapooretal.,2009).Terebinthene(2)wasidentifiedasa constituentofS.molleforthefirsttimeanditshowedsignificant cytotoxicactivityagainstahumancoloncarcinomacellline.

Authors’contributions

GRMGcontributedrunningthelaboratorywork,and drafted thepaper;LHdidtheNMRinvestigations;RWBcontributedin col-lectingplantsamplesandrevisedthepaper;MLGYandAPIcarried outbiologicalactivitytestsandwroteonepartofthemanuscript.

Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsinterest.

Acknowledgements

WewouldliketothankProf.R.Müller,J.HerrmannandMrs. ViktoriaSchmitt(HIPS,DepartmentofMicrobialNaturalProducts, Saarbrücken,Germany)fortechnicalassistancewiththecytotoxic assay.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.07.004.

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