RevistaBrasileiradeFarmacognosia25(2015)522–525
w w w. s b f g n o s i a . o r g . b r / r e v i s t a
Original
Article
Identification
of
terpenes
and
phytosterols
in
Dipteryx
alata
(baru)
oil
seeds
obtained
through
pressing
Fernanda
G.
Marques,
Jerônimo
R.
de
Oliveira
Neto,
Luiz
C.
da
Cunha,
José
Realino
de
Paula,
Maria
Teresa
F.
Bara
∗FaculdadedeFarmácia,UniversidadeFederaldeGoiás,Goiânia,GO,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received18March2015 Accepted15July2015
Availableonline3September2015
Keywords: Dipteryxalata
Vegetableoil Pressingextraction GC–MS
-Sitosterol
a
b
s
t
r
a
c
t
TheoilfromseedsofDipteryxalataVogel,Fabaceae,popularlyknownasbaru,wasextractedbyhydraulic andcontinuousscrewpressing.Atotalofelevenchemicalconstituentsobtainedbyhydraulic press-ing,includingsteroids,monoandsesquiterpenes andtocopherolderivativeswereidentifiedbygas chromatography–tandemmassspectrometry(GC–MS).Compoundslimonene,-elemene,␥-elemene, ␣-caryophyllene,-caryophyllene,campesterol,stigmasterol,-sitosterolandcycloartenolarebeing describedforthefirsttimeinthebaruoil.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Baru(Dipteryx alata Vogel, Fabaceae) is a vegetal species of
thecerrado whose seeds(nuts) present great nutritionalvalue (Takemotoetal.,2001),aconsiderablecontentofphenolic com-pounds(568.9mg/100g)(Lemosetal.,2012),antioxidantactivity (DPPHmethod)(Lemosetal.,2012),apreventiveeffecton iron-induced oxidative stress in rats (Siqueira et al., 2012) and the capacitytoreducecholesterol,triacylglyceridesandlipid peroxida-tioninrats(Fernandesetal.,2012).Oilextractedfromtheseseeds ispopularlyusedasananti-rheumaticagentandpresentssudorific, tonicandmenstrualregulatoryproperties(Sanoetal.,2004).The baru oilalsocontains tocopherols and highcontent of unsatu-ratedfattyacids(81.2%)(Takemotoetal.,2001).Interestinedible vegetableoils,especiallythosewithhighunsaturatedfattyacid contentshasincreasedduetoitsbeneficialhealtheffects,suchas cholesterolreductionandatherosclerosisprevention(Gromadzka andWardencki,2011;PlatandMensink,2000;Ausmanetal.,2005). Amongthemostcommoncompoundsinvegetableoilsthere arefattyacids,hydrocarbons,tocopherols,tocotrienols,phenolic compounds,terpenesandphytosterols.Thepresenceandamount of these substances are related to the quality, nutritional and functional values of such oils, and can vary depending onthe species,cultivationclimateconditions,oilextractionsystemand
∗ Correspondingauthor.
E-mail:mbara@ufg.br(M.T.F.Bara).
refiningprocesses(Certetal.,2000;GromadzkaandWardencki, 2011).Theextractionof vegetableoils is commonlyperformed usinghydraulicpressing,mechanicalscrewpressingorsolvents. Extractionthroughuseofsolventisnotrecommendedsinceitcan generatetoxicresiduesintheproduct.Althoughmechanicalscrew pressingisthemostcommonmethodusedinoilseedindustries, hydraulicpressingisstillusedintheproductionofcertain special-izedoils(Kemper,2005;Savoireetal.,2013).
However,despitethestudiesfocusingonthefunctional poten-tial of baru seeds, it has not been given enough attention to theknowledgeofthechemical compositionof theoilfromthe seed of baru.Given this background,thepresent study wasto evaluate the chemical composition of the baru oil using gas chromatography–tandem mass spectrometry (GC–MS) and to analyzetheinfluenceofextractionprocesses(hydraulicand con-tinuousscrewpressing)onoilcomposition.
Materialsandmethods
Plantmaterialandoilextraction
Baruseeds(DipteryxalataVogel,Fabaceae)werecollectedin August2012fromdifferenttreesinJussara,Goiás,Brazil(15◦51′
South;50◦52′West;317maltitude),identifiedbyDr.JoséRealino
de Paula, and stored at freezer. The seeds were joined, stored forthree monthsina coolplace andthentheoilwasobtained usingboth manual hydraulic pressing (manufacturer:Ribeiro®
) andmechanicalcontinuouspressing(MPE-40,Ecirtec®).Hydraulic
http://dx.doi.org/10.1016/j.bjp.2015.07.019
F.G.Marquesetal./RevistaBrasileiradeFarmacognosia25(2015)522–525 523
pressingwasperformedwith50gofseedsoverwhichwasapplied apressureof1.2×107to1.4×107Paforthreeconsecutivetimes.
Forcontinuouspressing,afrequencyinverterwasusedoperating at50cyclesperminute,with0.4mmspacers.Afterpressing,using bothmethods,oilwassubmittedtocentrifugation(5000×gfor 10min).
Theextractionyieldwascalculatedastheratiobetweentheoil massobtainedandseedmasssubmittedtothisprocess.Extraction efficiencywascalculatedastheratiobetweentheyieldandthe percentageoilpresentintheseeds,calculatedbytheBligh&Dyer technique(BlighandDyer,1959).
Oilcharacterizationandquality
Specificgravity(AssociationofOfficialAnalyticalChemists–AOAC OfficialMethod920.212–PycnometermethodAOAC,2000), refrac-tiveindex(AOAC,2000),iodineabsorptionnumber(AOACOfficial Method920.158–HanusmethodAOAC,2000)andsaponification number(AOACOfficialMethod920.160AOAC,2000)wereapplied toobtainoilcharacterization,whileoilqualitywasanalyzedby peroxidevalue(AOAC OfficialMethod 965.33AOAC,2000)and freefattyacidvalue(modifiedAOACOfficialMethod940.28AOAC, 2000–alcoholwassubstitutedbyanether:alcohol(2:1)solution toimproveoilsolubilization).Allanalyseswereperformedin trip-licate.
ChemicalcompositionofbaruoilanalyzedbyGas
chromatography–tandemmassspectrometry(GC–MS)
A baru oil solution (20% in hexane) was analyzed by a GCMS-Q2010Plussystem(Shimadzu®)withaRTX-5MScapillary
column(5%diphenyl/95%dimethylpolysiloxane,0.25mm×15m, Restek®).Anelectronimpactionizationof70eV.Helium(White
Martins®6.0)wasusedasacarriergasataflowrateof0.66ml/min.
Injections(3.0l)wereperformedbyanautomaticinjector (AOC-5000,Shimadzu®).
Twodifferentmethods, basedonprevious studies(Kadioglu etal.,2009;Lin etal.,2012),wereappliedtoidentifythe com-pounds.A:Injection,interfaceanddetectortemperaturesof220◦C,
270◦C and 270◦C, respectively, and column oven temperature
startingat40◦C(holdingfor1min),risingto220◦Cat10◦C/min
(heldfor30min).B:Injection,interfaceanddetectortemperatures of300◦Candcolumnoventemperaturestartingat150◦C(holding
for1min),risingto310◦Cat10◦C/min(heldfor30min).
Equipmentcontrol,aswellaspeakidentificationandarea inte-gration,wasperformedbyGCMSsolutionsoftware.
Compounds were identified by mass spectra analysis (35–500m/z range) using the NIST 05 library and GCMS solu-tionsoftwaretocalculatethesimilarityindex.Similarityindexes between90and100%wereconsideredacceptable(Toraneetal., 2011).Relativearea(%)ofeachidentifiedcompoundwas calcu-latedbytheratiobetweenthecompoundpeakareaandthesum ofallpeakareasofidentifiedcompounds.
Statisticalanalysis
Comparativedataanalysiswasperformedbyat-testusinga sig-nificancelevelof0.05.Thep-valueswerecalculatedusingMicrosoft OfficeExcel2007software.
Resultsanddiscussion
Oilobtainment
Theextractionyieldscalculatedforhydraulicandcontinuous screwpressingwere7.99and25.0%,respectively.Thelipidcontent
Table1
Physical–chemicalpropertiesandqualitycharacterizationofbaruseedoilobtained byhydraulicandcontinuousscrewpressing.
Results Hydraulicpressing Continuousscrew pressing
Iodinevalue(g/100g) 89.88±4.42a 89.44± 2.59a
Saponificationvalue(mgKOH/g) 159.92± 3.00a 156.41± 1.32a
Refractiveindex 1.468±0.000a 1.469±0.000a
Relativedensity 0.917±0.038a 0.917±0.014a
Acidvalue(mgKOH/g) 0.41±0.01b 0.30±0.01b
Peroxidevalue(meq/kg) 1.61±0.05b 1.36±0.05b
aNostatisticallysignificantdifferenceforthisparameter(p>0.05). bStatisticallysignificantdifferenceforthisparameter(p≤ 0.05).
foundinbaruseedswas36.01±1.40%(similartoearlierpublished dataof38.2±0.4%(Takemotoetal.,2001))resultinginextraction efficienciesof22.19and69.43%forhydraulicandcontinuousscrew pressing,respectively.MacielJúnior(2010)obtainedanextraction efficiencyhigherthan89%tobaruoilseedsusingcontinuousscrew pressing.Nostudydescribingthehydraulicpressingofbaruseeds hasbeenfoundyet.
Duetothehigherextractionefficiencyprovidedby continu-ousscrewpressing,hydraulicpressinginoilseedindustrieshas graduallybeenreplacedovertheyearsbyscrewpressing(Savoire etal.,2013),althoughhydraulicpressisstillusedintheindustrial productionofoliveoilwhichisobtainedintheabsenceofhigh temperatures,whichincreasesthecommercialvalueofthe prod-uct(Kemper,2005).Itshouldbenotedthatthecontinuousscrew pressinghasothersadvantagesassimplicityofoperation,quickly andeasilyadapttovarioustypesofoilseeds(Silva,2009). More-overcontinuouspressinghaveahighenergyconsumption,which isdissipatedinfrictionandcansubstantiallyincreasetheproduct temperature,whichincreasestheriskofthermal degradationof heat-sensitivesubstances(Guedes,2006).
Oilcharacterizationandquality
Table1summarizesthephysicalchemicalpropertiesand qual-itycharacterizationresultsofbaruseedoil.Nostatisticaldifference wasobservedbetweeniodinevalues,saponificationvalues, refrac-tiveindexandrelativedensityofoilsobtainedbythetwoextraction methods(p>0.05).
Acidandperoxidevalueshavebeenestablishedasquality char-acteristics by Codex Alimentarius (2011). For cold pressed and virginoils, themaximum acceptablelevelsfor acidand perox-ideare4.0mgKOH/gand15meq/kg,respectively.Thelevelsfor oilsobtainedbybothextractionmethodswereapproximatelyten
Table2
Similarityindexandrelativeareaofcompoundsidentifiedinbaruseedoilobtained byhydraulicandcontinuousscrewpressing.
Compound Similarityindex Relativearea(%)
Hydraulic pressing
Continuousscrew pressing
-Sitosterol 89% 63.89 55.30 Stigmasterol 92% 14.21 17.74 ␣-Tocopherol 96% 7.43 7.72
Campesterol 92% 5.50 7.31
Cycloartenol 90% 4.61 6.43
␥-Tocopherol 91% 3.61 5.44 -Caryophyllene 94% 0.25 NDa
␥-Elemene 90% 0.12 NDa
-Elemene 95% 0.09 NDa
␣-Caryophyllene 94% 0.08 NDa
Limonene 90% 0.03 NDa
Total 100% 100%
524 F.G.Marquesetal./RevistaBrasileiradeFarmacognosia25(2015)522–525
timeslowerthantheacceptancecriteria(Table1),whichsuggests considerablehydrolyticandoxidativestabilityforbaruoil.
ChemicalcompositionofbaruoilbyGC–MS
TheanalysisofbaruseedoilbyGC–MSresultedinthe iden-tificationofelevenchemicalconstituents,nineofthemhavenot beendescribedyetforthisoil,suchaslimonene(1),-elemene(2),
␥-elemene(3),␣-caryophyllene(4),-caryophyllene(5), campes-terol(6),stigmasterol,-sitosterolandcycloartenol(7).
Thesimilarityindexandrelativeareaofthesecompoundsinthe twoextractionmethods(Table2)showsthat-sitosterolpresented asimilarityindexlowerthan90%intheNIST05libraryandits iden-titywasconfirmedbycomparingitwithacommercialstandard (purity≥95%,Sigma),whichresultedinasimilarityindexof97%.
Ofthecompoundsidentified,there aremono and sesquiter-peneswithwell-knownbiologicalactivities.Limonene(1)presents antioxidant activity(Yang et al., 2010) and exerts a protective effectongastricmucosa(Moraesetal.,2009).-Caryophyllene(5) isknownforitsanti-inflammatory(AwadandFink,2000;Sousa etal.,2008),antibiotic,antioxidant,andanti-carcinogenicactivities (Adorjan and Buchbauer, 2010). Elemene hasanti-tumor activ-ity(Yangetal.,1996).Tocopherols,recognizedantioxidants,had alreadybeenidentifiedinbaruoilinpreviousstudies(Takemoto et al., 2001). In addition, phytosterols, including campesterol, stigmasterol,-sitosterolandcycloartenol(7)foundinbaru oil, present antioxidant (Yoshida and Niki, 2003; Ju et al., 2004), hypocholesterolemic(Spositoet al.,2007; Bartnikowska, 2009), anti-carcinogenic(AwadandFink,2000;Moreauetal.,2002), anti-inflammatory(Garcíaetal.,1999;HänninenandSem,2008)and estrogenic activities(Ju et al., 2004, Maliniand Vanithakumari, 1993). -Sitosterol,stigmasterol and campesterol are themost commonphytosterolsinvegetables(Moreauetal.,2002)andin herbalmedicines(Yeetal.,2010).
It was observed that -sitosterol was the major compound foundinthebaruoilobtainedbybothmethods,whilethe essen-tialoilfractionwasfoundinconsiderablyloweramounts,andwas notdetectedinthebaruoilobtainedbycontinuousscrew extrac-tion.Thetemperatureofthepressincreasesinthecrushingprocess ofseedsreachingaround60◦C.Thismaybeduetheincreasein
oiltemperatureduring continuousscrewextraction, which can degradeessentialoilcompoundsorfacilitatetheirvolatilization (Savoireetal.,2013).
Thepopularuseofbaruoilasa menstruationregulatorand ananti-rheumatic agent(Sanoetal., 2004)canberelated with theestrogenic effectof phytosterolsand theanti-inflammatory effectsof caryophylleneand phytosterols.In addition,the asso-ciationofphytosterolswithhighunsaturatedfattyacidcontents (81.2%,Takemotoetal.,2001)isanindicatorthattheoilcouldhave ahypocholesterolemiceffect(GromadzkaandWardencki,2011; HänninenandSem,2008).Thistheoryissupportedbythefactthat theconsumptionofbarunutsledtoacholesterol,triacylglyceride andlipidperoxidationreductioninrats(Fernandesetal.,2012). Thesefindingssuggestthatbaruoilcouldbeusedbothasa func-tionalfoodandformedicinalpurposes.
Authors’contributions
FGMcontributedtocollectingtheplantsample,carryingout lab-oratorywork,conductingchromatographicanalysis,dataanalysis andinitialpaperdrafting.JRONcontributedtochromatographic analysis. LCC contributed to the analysis and interpretation of theGC–MSdata.JRPcontributedtocollectingandidentifyingthe plants,andfinalapprovalofthearticle.MTFBcontributedtothe conceptionanddesign,supervisionoflaboratorywork,provision ofstudy materials,draftingofthepaper andcritical reading of themanuscript.Allauthorshave read thefinal manuscript and approveditssubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
ThisresearchwassupportedbyFAPEGandCNPq.
References
Adorjan,B.,Buchbauer,G.,2010.Biologicalpropertiesofessentialoils:anupdated review.FlavourFrag.J.25,407–426.
Ausman,L.M.,Rong,N.,Nicolosi,R.J.,2005.Hypocholesterolemiceffectofphysically refinedricebranoil:studiesofcholesterolmetabolismandearlyatherosclerosis inhypercholesterolemichamsters.J.Nutr.Biochem.16,521–529.
Awad,A.B.,Fink,C.S.,2000.Phytosterolsasanticancerdietarycomponents:evidence andmechanismofaction.J.Nutr.130,2127–2130.
Bartnikowska,E.,2009.Biologicalactivitiesofphytosterolswithparticularattention totheireffectsonlipidmetabolism.Pol.J.FoodNutr.Sci.59,105–112.
Bligh,E.G.,Dyer,W.J.,1959.Arapidmethodoftotallipidextractionandpurification. Can.J.Biochem.Physiol.37,911–917.
Cert,A.,Moreda,W.,Pérez-Camino,M.C.,2000.Chromatographicanalysisofminor constituentsinvegetableoils.J.Chromatogr.A881,131–148.
CodexAlimentariusCodexStandardforNamedVegetableOils,2011.CodexStan 210-1999(Amendment2005,2011).WorldHealthOrganization,Foodand Agri-cultureOrganizationoftheUnitedNations,Rome,pp.1–16.
Fernandes,D.C.,Alves,A.M.,Sousa,A.G.O.,Castro,G.S.F.,Junior,A.A.,Naves,M.M.V., 2012.Effectofbarualmond,peanutandBrazilnutonserumlipidprofileof ratstreatedwithhigh-fatdiets.In:16thWorldCongressofFoodScienceand Technology,FozdoIguac¸u,Brazil.
García,M.D.,Sáenz,M.T.,Gómez,M.A.,Fernández,M.A.,1999.Topical antiinflam-matoryactivityofphytosterolsisolatedfromEryngiumfoetidumonchronicand acuteinflammationmodels.Phytother.Res.13,78–80.
F.G.Marquesetal./RevistaBrasileiradeFarmacognosia25(2015)522–525 525
Guedes,A.M.M.,Dissertac¸ãodeMestrado2006.Estudodaextrac¸ãodeóleodapolpa detucumãporCO2supercrítico.ProgramadePós-graduac¸ãoemCiênciae Tec-nologiadeAlimentos,UniversidadeFederaldoPará,Belém,78p.
Hänninen,O.,Sem,C.K.,2008.Nutritionalsupplementsandfunctionalfoods: func-tionalsignificanceandglobalregulations.In:Bagchi,D.(org.),Nutraceuticaland FunctionalFoodRegulationsintheUnitedStatesandAroundtheWorld.Elsevier Inc.,Houston,pp.11–35.
Ju,Y.H.,Clausen,L.M.,Allred,K.F.,Almada,A.L.,2004.-Sitosterol,-sitosterol glu-coside,andamixtureof-sitosteroland-sitosterolglucosidemodulatethe growthofestrogen-responsivebreastcancercellsinvitroandinovariectomized athymicmice.J.Nutr.134,1145–1151.
Kadioglu,Y.,Demirkaya,F.,Demirkaya,A.K.,2009.Quantitativedeterminationof underivatized˛-tocopherolincowmilk,vitaminandmultivitamindrugsby GC-FID.Chromatographia70,665–670.
Kemper,T.,2005.Oilextraction.In:Bailey,A.E.,Shahidi,F.(org.),Bailey’sIndustrial OilandFatProducts.JohnWiley&SonsInc.,NewJersey,pp.57–98.
Lemos,M.R.B.,Siqueira,E.M.A.,Arruda,S.F.,Zambiazi,R.C.,2012.Theeffectof roast-ingonthephenoliccompoundsandantioxidantpotentialofbarunuts(Dipteryx alataVog.).FoodRes.Int.48,592–597.
Lin,Q.,Li,M.,Zhou,R.,Liu,Y.,2012.Chemicalcompositionandanti-bacterialactivity ofessentialoilfromCidrelasinensis(A.Juss.)Roem.seed.Afr.J.Biotechnol.11, 1789–1795.
MacielJúnior,S.,Dissertac¸ãodeMestrado 2010.Caracterizac¸ãofísico-química, qualidade, e estabilidade oxidativas do óleo de Dipteryx alata Vog. (baru). Programa de Pós-graduac¸ão em Tecnologia de Processos Quími-coseBioquímicos,Universidade FederaldoRiodeJaneiro,RiodeJaneiro, 105p.
Malini,T.,Vanithakumari,G.,1993.Effectofbeta-sitosterolonuterinebiochemistry: acomparativestudywithestradiolandprogesterone.Biochem.Mol.Biol.Int. 31,659–668.
Moraes,T.M.,Kushima,H.,Moleiro,F.C.,Santos,R.C.,Rocha,L.R.M.,Marques,M.O., Vilegas,W.,Hiruma-Lima,C.A.,2009.Effectsoflimoneneandessentialoilfrom Citrusaurantiumongastricmucosa:roleofprostaglandinsandgastricmucus secretion.Chem.-Biol.Interact.180,499–505.
Moreau,R.A.,Whitaker,B.D.,Hicks, K.B.,2002.Phytosterols, phytostanols,and theirconjugatesinfoods:structuraldiversity,quantitativeanalysis,and health-promotinguses.Prog.LipidRes.41,457–500.
.OfficialMethodsofAnalysisofAOACInternational,vol.II.AOACInternational, Gaithersburg,MD.
Plat, J., Mensink, R., 2000. Vegetable oil based versus wood based stanol ester mixtures: effects on serum lipids and hemostatic factors in non-hypercholesterolemicsubjects.Atherosclerosis148,101–112.
Sano,S.M.,Ribeiro,J.F.,Brito,M.A.,2004.Baru:biologiaeuso.In:Documentos116. EMBRAPA,Planaltina,Brasil,pp.11–20.
Savoire,R.,Lanoisellé,J.L.,Vorobiev,E.,2013.Mechanicalcontinuousoilexpression fromoilseeds:areview.FoodBioprocess.Technol.6,1–16.
Silva,I.C.C.,Dissertac¸ãodeMestrado 2009.Usodeprocessoscombinadospara aumentodorendimentodaextrac¸ãoedaqualidadedoóleodemacaúba. Pro-gramadePós-graduac¸ãoemTecnologiadeProcessosQuímicoseBioquímicos, UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,99p.
Siqueira,E.M.A.,Marin,A.M.F.,Cunha,M.S.B.,Fustinoni,A.M.,Sant’ana,L.P.,Arruda, S.F.,2012.Consumptionofbaruseeds(DipteryxalataVog.),aBraziliansavanna nut,preventsiron-inducedoxidativestressinrats.FoodRes.Int.45,427–433.
Sousa,O.V.,Silvério,M.S.,Del-Vechio-Vieira,G.,Matheus,F.C.,Yamamoto,C.H., Alves,M.S.,2008.Antinociceptiveandanti-inflammatoryeffectsoftheessential oilfromEremanthuserythropappusleaves.J.Pharm.Pharmacol.60,771–777.
Sposito, A.C.,Caramelli, B., Fonseca,F.A.H., Bertolami, M.C., 2007. IVDiretriz BrasileirasobreDislipidemiasePrevenc¸ãodaAterosclerose.Arq.Bras.Cardiol. 88,1–19.
Takemoto,E.,Okada,I.A.,Garbelotti,M.L.,Tavares,M.,Aued-Pimentel,S.,2001.
Chemicalcompositionofseedsandoilofbaru(DipteryxalataVog.)nativefrom Pirenópolis,StateofGoiás,Brazil.Rev.Inst.AdolfoLutz60,113–117.
Torane,R.C.,Kamble,G.S.,Gadkari,T.V.,Tambe,A.S.,Deshpande,N.R.,2011.GC–MS study of nutritiousleaves ofEhretia laevis. Int. J. Chem. Technol.Res. 3, 1589–1591.
Yang,H.,Wang,X.,Yu,L.,1996.Theantitumoractivityofelemeneisassociatedwith apoptosis.Chin.J.Oncol.18,169–172.
Yang,S.,Jeon,S.,Leeb,E.,Shim,C.,Lee,I.,2010.Comparativestudyofthechemical compositionandantioxidantactivityofsixessentialoilsandtheircomponents. Nat.Prod.Res.24,140–151.
Ye,J.,Chang,W.,Hsieh,D.J.,Hsiao,M.,2010.Extractionandanalysisof-sitosterol inherbalmedicines.J.Med.PlantsRes.4,522–527.
