LipidProfileofNutmeg(Myristicafragrans)
byJulioFrancisco
FloridaInstituteofTechnology
Abstract:
Thefollowingexperimentwasatwopartexperimentinwhichitwasundertakento
isolateanunknownlipidmixturefromtheseedofMyristicafragrans(nutmeg)throughboth
thinlayerchromatography(TLC)andchemicalextractionmethods.Approximately1.20gof
crudeproductwasextractedfromthenutmeggivinga24.0%recoveryyield.Therecovery
yieldwaslargerthanpreviousstudiessuggested.Itcanbepostulatethatcrudeproduct
containedimpuritywhichgaveitalargeryieldthanexpected.Also,itwasidentifiedthatthe
nutmegcontainedpalmiticacidandhighlevelofmyristicacid.
Furthermore,thevariouspropertiesoflipidwerestudiedthroughdifferentchemical
andphysicaltechniques.Thesetechniquesincludesolubilityoflipids,emulsificationoflipids,
greasespottestforfat,saponificationoffats,andiodineabsorptiontest.
Inthesolubilitytest,itwasdeterminedthatvegetableshortening,glycerol,andoliveoil
wereinsolubleinwaterduetothehydrophobicpropertyoftheselipids.However,itwas
observedthatthesesamelipidsweresolubleinetherduetothehydrophobiclikeinteraction.
Theemulsificationtestrevealedthatbotholiveoilandbileareseparableinwaterand
detergentduetodifferencesindensities.Thegreasespottestindicatedthepresenceoflipid
byformationofatranslucentappearanceonthepaper.Thesaponificationtestindicatedthe
presenceoffattyacid.Lastly,theiodineabsorptiontestindicatedthatbothvegetable
shorteningandoliveoilareunsaturatedfattyacid.
Introduction:
Ithavebeenknownthatadiethighintransfat,saturatedfatsandcholesterolalong
withlowlevelofpolyunsaturatedfatscanleadstothedevelopmentofatheromatousplaques.
Theaccumulationofatheromatousplaqueswouldnarrowthecoronaryarterycausingthe
reductionofbloodflowtomuscles.Eventually,myocardialinfarctionwoulddevelop(Willett,
2012).Also,ithasbeenreportedthattransfatincreasestheriskforischemicheartdisease
by25%.Thefoodsthatpossesstransfatarefastfood(e.g.KFC,McDonalds,etc)and
commercialfat(e.g.vegetable).Ithasbeenrecommendthatapersonsdietshouldconsistof
highpolyandmonounsaturatedfats(Stender,Dyerberg&Astrup,2006).Thisisduetothe
factthatbothpolyandmonounsaturatedunsaturatedfattyacidhavebeneficialeffectonthe
cardiovascularandcognitivefunction(Robertsetal.,2010).Also,polyunsaturatedfattyacid
wasreportedtodecreaseLDLandincrease(ormaintain)HDLreducingtheriskofcoronary
heartdisease(Willett,2012).
Polyandmonounsaturatedfatshavebeenextensivelystudiedincommercialgoods
suchasoliveoilandevennutmeg.First,oliveoilpossesslowlevelofsaturatedfattyacidand
highlevelofessentialunsaturatedfattyacids.Theseessentialunsaturatedfattyacids
providesantioxidantandantiatherogenicproperty(Frankel,2011).Second,nutmegsare
commerciallyimportantspicethatgrowninmanyareas,particularlycultivatedinthe
Caribbean,southIndia,SriLanka,Sumatra,andMalaysia.Thisspiceisusedforflavoringand
medicinalpurposes.Infact,theessentialoilhavebeenusedforitshealingpropertyfor
variousofailmentsinmanypartsoftheworld.Theoilcomponentsofnutmegvariousfrom
geographicallocation(AlJumaily&AlAmiry,2012).
Withthisinmind,theaimofthisstudyistoextract,purify,andidentifytheoil
compositionofcommercializenutmeg.Inaddition,propertiesofoliveoilandvegetable
shortening(alongwithglycerolandbile)weredeterminedusingvariousofchemicaland
physicalmethods.
Method:
IsolationofOilfromNutmeg
Nutmegwasgroundintoafinepowder.About5gramsofnutmegpowderwasplaced
intoaflaskalongwithapproximately50mLofhexane:isopropyl(3:2ratio).Theflaskwas
thencoveredwithaluminumfoilandplacedinto37Cofhotwaterbathfor15minute.
Afterward,thesolutionwasfilteredthroughavacuumfiltrationmethod.Additional20mlof
warmhexane:isopropanolwaspourthroughthesolidresidueinthefilter.Thesolutionwas
thentransferredintoapreweighedbeaker.Thesolventwasremovedfromtheextractionin
90bathwhileflushingtheinsideoftheflashwithN2gastoreceiveayellowoil.Afterward,
theflaskwasplacedinicefor5minutesinorderforthelipidtosolidify.Theweightheflask
wasdetermined.Theweightofthecrudeproductwasdeterminedbytakingthedifference
betweentheinitialandfinalweightoftheflasks.Also,theyieldpercentageofcrudeproduct
wasdeterminedusingtheformula:
Recoveryyield(%) =
gramsofpurifiedlipid
gramsofcrudelipid x100
(Equation1)
ThinLayerChromatography(TLC)oftheStandardandNutmegExtract
TheTLCwasusedtoseparatecompoundsfromnutmegextractionandcompareitto
knowncompounds.First,asilicagel60Athinlayerplateswasspottedwithdifferentsolutions
atapproximately2cmfromthebottomandtheplatewasallowedtodry.Afterdrying,the
platewastransferredintoachromatocabfilledsolvent.Thechromatocabwassealedwitha
glassplateandthesolventwasallowedtorise.Theplatewasthenremovefromthe
chromatocab.TheRfvaluewascalculatedusingthefollowingequation:
Rf = Distancefromtheoriginmigratedbyacompund
Distancefromoriginmigratedbysolvent
(Equation2)
TheRfvalueofthestandardlipidswascomparedtothenutmeg.BycomparingtheRfvalue,
theidentityofthegeneralclassoflipidcanbedetermine.
SolubilityofLipids
Thesolubilityofglycerol,oliveoil,andvegetableshorteningwasdetermined.Approximately1
dropofthelipid(glycerol,oliveoil,orvegetableshortening)and1mLofsolvent(eitherwater,
ether,ormethanol)wasplacedintoatesttubeandobservationwasrecorded.
EmulsificationofLipid
Intheemulsificationoflipid,threetesttubeswerefilledwith0.2mlofoliveoiland2.5mlof
water,soapsolution,andbilesaltsolutionwereindividuallyaddedtoseparatetesttube.
Then,eachtubewereshakenvigorouslyfor30seconds.Afterwards,1mlofetherwasadded
toeachtubeandshaken.
GreaseSpotTestforFats
Inthegreasespottest,dissolveasmallamountoffatsolidwith1mlofetherintoatesttube.
Place1dropofsolutionintothecenterofafilterpaperandallowdrying.Thegreasespotwas
closelyexamined.
SaponificationofFat
IntheSaponificationtest,0.5mlofoliveoil,5mlofKOH,andaglassbeadwereplacedintoa
testtube.Thetubeswereheatedinboilingwaterbathfor10minutes.Adropofsolutionwas
withdrawnandaddedtoatesttubeofwater.Observationwasrecorded
IodineAbsorptionTest
About0.2mlofoliveoil,0.2gofsolidfat,1mlofchloroform,and3to4dropsofiodinewere
placeinatubeandtheresultswererecorded.
Result:
Table1.ThinLayerChromatographyofStandardandNutmegLipid.Separationwas
performedonsilicagel60Athinlayerplate(20x20cm)usingasolventconsistingof
hexane:diethyl:aceticacid(80:20:1).TheRfvaluewasdeterminedbytakingthequotientof
thedistancefromtheoriginmigratedbyacompoundoverthedistancefromtheorigin
migratedbythesolvent.Theresultsshowedthattheboldnumberwasthedarkestbandand
thatalltheotherbandsrepresentothernonprominentlipids.
Compounds:
Rf
Nutmeg
0.058,0.18,0.24,0.31,0.60,0.75,0.89
Tripalmitin
0.56
Tristearin
0.96
Phosphatidylcholine
0.22
MyristicAcid
0.30
Triolein
0.60
PalmiticAcid
0.38
Table2.Thefollowingtableshowsthecombinationsof1mlsolvent(e.g.water,ether,and
methanol)and1mlsolutesolution(e.g.oliveoil,glycerol,andvegetableshortening)and
showifthesolutewassolubleorinsolubletowardsitscorrespondingsolvent.Itwas
determinedthatoliveoil,glycerol,andvegetableshorteningwereinsolubleinwaterhowever,
thesesamesolventweresolubleinether.Also,vegetableshorteningwassolublein
methanol.
Solvent
Solute
Solubility
Water
Oliveoil
Insoluble
Water
Glycerol
Insoluble
Water
Vegetableshortening
Insoluble
Ether
OliveOil
Soluble
Ether
Glycerol
Soluble
Ether
Vegetableshortening
Soluble
Methanol
Vegetableshortening
Insoluble
Table3.Thefollowingtableshowsthedifferentcombinationbetweenthewateranddetergent
mixwitholiveoilorbilesaltsolution.Also,theseparationpropertyofsuchmixwasrecorded.
Itwasdeterminedthatoliveoilandbilewereseparableinbothwateranddetergent.
Solvent
Solute
SeparationResult
2.5mlWater
0.2mlOliveoil
ClearSeparation
2.5mlWater
2.5mlBile
ClearSeparation
2.5mlDetergent
0.2mlOliveoil
Unclearseparation
2.5mlDetergent
2.5mlBile
ClearSeparation
Alipidextractionwasperformedinwhichtheinitialweightoftheflaskwas74.4g.
Approximately5gofpowdernutmegwasplaceintotheflaskforisolationandpurification
purpose.Apaleyellowcolorextractedsamplewasobtained.Theweightoftheflaskandthe
crudeproductwas75.6gand,therefore,theweightofthecrudeproductwas1.20ggivinga
24.0%recoveryyield.Also,basedontheRf(Table1)values,theresultindicatedthatnutmeg
possessthelipidstriolein,tripalmitin,phosphatidylcholine,andmyristicacid.
Furthermore,thesolubilityofdifferentlipidsindifferentsolventwasdetermined(Table
2).Itwasidentifiedthatoliveoil,glycerol,vegetableshorteningwereinsolubleinwater.
However,itwasdeterminedthatbotholiveoilandglycerol,butthevegetableshorteningwas
insolubleinetherandmethanol.
Thenexttestwasthetheemulsificationtest(Table3).Theemulsificationtestis
determineifthelipidwouldbeabletobebrokendownintosmaller,uniformlydistributed
particles.Aseparationwasobservedbetweenamixtureofwaterandoliveoilwaterandbile
detergentandbileanddetergentandoliveoil.
Theiodineabsorptiontestisusedtodetectthepresenceofunsaturatedfattyacidin
whichthedecolorizationofthebrowniodinesolutionindicatesthepresenceofunsaturation.It
wasobservedthatthebotholiveoilandthesolidfatdecolorizetheiodinereagentindicating
thatbotholiveoilandthesolidfatareunsaturatedfats.
Thegreasespottestforwasperformed.Inthistest,itwasobservedthatthepaper
becometranslucentindicatingthepresenceoffat.
Thelasttestusedwasthesaponificationtest.Thistestisusetoidentifythepresence
offatbytheformationofsoapyfoam.Itwasobservedthatthemixturewascloudy.
Discussion:
Therearetwoclassofoils:essentialandfixedoils.Essentialoilsarehighlyvolatile
aromaticcompoundfromplant.Ontheotherhand,fixedoilsarenonvolatileoils.Examplesof
essentialoilsincludesbutnotlimitedtosabinene,4terpineol,myristicin,andmyristicacid
(Muchtaridi,Subarnas,Apriyantono,&Mustarichie,2010).
Itwasreportthatnutmegscontainedpalmiticacidandhighlevelofmyristicacid
(Spricigo,Pinto,Bolzan,&Novais,1999Subbaram&Youngs,1967).Infact,Niyas,Variyar,
Gholap,andSharma(2003)determinedthatmyristicacidaccountedfor90%offreefattyacid
(FFA)innutmeg,whilepalmiticacidaccountedfor6%.Thisisconsistentwiththe
experimentalresult.Also,theresultconfirmedthepresenceofpalmiticacidandshowedthat
thenutmegwasrichinmyristicacidasindicatedbythedarkestbandontheTLCplate.
Furthermore,about1.20gofextractedoilwasobtainedgiving24.0%recoveryyield.
Theamountofoilyieldisinconsistentwithotherstudies.Jukicet.al.yielded4.92g/100
gramofpowderedseedgivinga4.92%recoveryyield.Also,Spricigoetal.reportedayieldof
oilas6.9gram/100gram(6.9%recoveryyield)whileRahmanetal.showedayieldof2.28
g/100g(2.28%recoveryyield)(ascitedinAlJumaily&AlAmiry,2012).Thedifferenceinthe
amountofoilextractedcanbeexplainbytheorigininwhichtheseedsoriginatefrom.
However,theamountofoilextractedwaslargerthanexpected.Itcanbepostulatedthatthe
oilextractioncontainsimpurityleadingtoalargerrecoveryyield.
Next,variousoflipidtestingtechniqueswereperformedtodeterminethepropertyof
thedifferentlipidmolecules.Thesetechniquesincludedthesolubilityoflipids,greasespot
testforfats,saponificationoffats,iodineabsorptiontest,andtheemulsificationoflipids.
First,thesolubilityofvariouslipidsindifferentsolventwasdetermined.Itwas
examinedthatbotholiveoilandglycerolareinsolubleinwater.Thisisduetothefactthat
botholiveoilandglycerolarenonpolarcompoundsandunabletomixwithpolarwater
molecule.Furthermore,itwasobservedthatoliveoilissolubleinether.Thisisduetothefact
thatbotholiveoilandetherarenonpolarandabletomixwithoneanother.
Secondly,itwasobservedingreasespottestthatthepaperbecometranslucent
indicatingthepresenceoffat.
Third,saponificationistheprocessbywhichafattyacidandalkaliformsoap.Inthe
saponificationtest,itwasobservedthatthemixturewascloudyindicatedthattherewasfatty
acidpresenceinthesolutionhowever,notalargequantityoffattyacidwaspresencesince
therewasnotagreatyieldofbubbleformation.
Fourth,theiodineabsorptiontestwasconductedtodeterminetheunsaturationof
variousoflipids.Itwasdeterminedthattheoliveoilandvegetableshorteningwere
unsaturatedfats.Infact,botholiveoilandvegetableshorteningcontainsboth
monounsaturatedandpolyunsaturatedfattyacid(Mailer,2006Ritvanen,Putkonen,&
Peltonen,2012).
Fifth,aseparationawasobservedintheemulsificationtestforacombinationofwater
andoliveoilwaterandbiledetergentandoliveoilanddetergentandbile.Theseparation
occurredduetodifferencesindensitiesofthetwocompounds.McClementsandDecker
(2000)notedthatemulsionstendtoseparatethelipidlayer(lowerdensity)atthetopofwater
(highdensity)tominimizecontactbetweenoilandwater.
Improvementcanbemadeforfutureexperiment.Particularly,additionaltrialsmustbe
conductedtodetermineiftheexperimentisvalid.Also,theutilizationofadvancedtechnology
cangiveamoreaccurateresultonthecompositionofthelipids.
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