Separation of aroma compounds from indus

() Industrial Crops and Products 58 (2014) 99–103 Contents lists available at ScienceDirect Industrial Crops and Products jo ur nal home p age www elsev ier com/ locate / indcrop Separation of aroma c[.]

j ou rn a l h o m epa g e :w w w e l s e v i e r c o m / l o c a t e / i n d c r o p

fractionation

Carla Da Porto∗, Deborha Decorti, Andrea Natolino

Department of Food Science, University of Udine, via Sondrio 2/A, 33100 Udine, Italy

Article history:

Received 24 December 2013

Received in revised form 18 March 2014

Accepted 31 March 2014

Available online 4 May 2014

Keywords:

Supercritical CO2 extraction

On-line fractionation,Cannabis sativaL.

GC–MS

HS-SPME/GC–MS

Theuseofsupercriticalcarbondioxide(Sc-CO2)extractionat10and14MPaand40◦Candon-line frac-tionationusingtwoseparators(Sep1:7MPa/25◦C;Sep2:5MPa/15◦C)torecoveryvolatilecompounds fromtheinflorescencesoffibertypeCannabis sativaL.wasinvestigatedbyHS-SPME/GC–MSanddirect GC–MSandcomparedwithhydrodistillation.ThebestresultswereobtainedbySc-CO2extraction car-riedoutat10MPaand40◦C.Undertheseoperatingconditions,cuticularwaxescoveringthesurfaceof flowerswerecollectedinthefirstseparatorandvolatilecompounds(100%)inthesecond.Thesuperior qualityofthislastextractwasprovedbytheperfectoverlappingofitsHS-SPME/GC–MSvolatileprofile

tothatofinflorescences.Therecoveryoffractionswithdifferentcompositionandbiologicalproperties, madetheinflorescencesoffibertypeCannabis sativaLsuitableforcosmeticand/orfoodindustry

©2014ElsevierB.V.Allrightsreserved

1 Introduction

IndustrialhempisanumberofvarietiesofCannabis sativaL

cul-tivatedforfiberand/orseedproduction.Onlyvarietiesofindustrial

hemppublishedbyEU(Regulation(EC)No1251/99andsubsequent

amendments)areapprovedforplantinginEurope.Thesevarieties

areeligiblefor cultivationonlyaftertheverification oftheir

␦-9-tetrahydrocannabinol(THC)content,theprincipalpsychoactive

constituentofthecannabisplant,whichmustbelessthan0.2%w/w

(RegulationECNo.1124/2008-12November2008).Inflorescences

offibertypeCannabis sativaL.cultivarsaregenerallyconsidered

wastepartsforfiberindustry,althoughtheinflorescences’volatiles

arepleasanttothehumansensorysystemandcouldbeusedas

fla-voringsforbeverages(foodindustry)oringredientsforbodycare

products(cosmeticindustry).Cannabisscent doesnot originate

fromtheterpenophenoliccannabinoids,producedbyglandular

tri-chomesthatoccuronmostaerialsurfacesoftheplant(Dayanandan

andKaufman,1976;Turneretal.,1978),butfromthemorevolatile

monoterpenesandsesquiterpenes(Turneretal.,1980)

Traditionally,therecoveryoffloral fragrancesfromplants is

byhydrodistillationorsteamdistillationtoproduceessentialoils

However,thesetechniquestakeatleastseveralhoursandrequire

∗ Corresponding author Tel.: +39 0432 558141; fax: +39 0432 558130.

E-mail address:carla.daporto@uniud.it (C Da Porto).

theapplicationofheating,whichcanproducethedegradationof thermolabilecompoundspresentinthestartingplantmaterial Among innovative process technologies, supercritical CO2 (Sc-CO2) extraction and fractionation can be applied as alter-native method to extract and isolate compounds from plant material (Reverchon and De Marco, 2006; Pourmortazavi and Hajimirsadeghi, 2007).Carbondioxideis economical,safe, non-toxic(itdoesnotleaveresiduesinextract)andreachessupercritical conditionseasily(32◦Cand7.38MPa).Furthermore,theuseofCO2

isacceptableinthefoodandpharmaceuticalindustries

Tothebestofourknowledge,therearenostudiesonthe sep-arationofvolatilecompoundsextractedbysupercriticalCO2from theinflorescencesofCannabis sativaL

TheaimofthisworkwastoapplysupercriticalCO2extraction andon-linefractionationprocesstoseparatehempvolatile com-pounds.TheSc-CO2 extractswerecompared totheessentialoil obtainedbyhydrodistillation

2 Materialsandmethods

FreshinflorescencesofCannabis sativaL.cv.Felina(THC<0.2%) were obtained from experimental trials carried out in Carnia (FriuliVenezia-Giuliaregion-Italy).OnAugust2013,fromatleast thirtyplantsofhemptheinflorescenceswereselectedrandomly http://dx.doi.org/10.1016/j.indcrop.2014.03.042

0926-6690/© 2014 Elsevier B.V All rights reserved.

Fig 1 SFE pilot plant flow sheet (B1) storage tank; (E1) Extraction vessel; (S1, S2) Separators; (H#) Heater exchangers; (C1) Condenser; (HV#) Hand valves; (MV1) membrane valve; (NVR#) No return valves; (P) Diaphragm pumps; (F1) Flowmeter; (M#) Manometers; (k) Safety devices; (FL1) Coriolis mass flowmeter; (D) Co-solvent storage tank and (X#) Mixer.

fromthecultivationarea,handpickedanddriedintheshade

(mois-turecontent9.60%w/w,±1.1)

Akitchen-typeknifemillwasemployedtocarryoutgrinding

oftheinflorescences.Theparticlesizedistributionwasdetermined

withavibratorysieveshaker.Particlesizeobtainedwasintherange

of200–600␮m

Analiquot(150g)ofdriedandgroundinflorescenceswas

sub-mittedtohydrodistillationwithaClevengertypeapparatusfor3h

Attheendofthedistillationprocesstheessentialoilwascollected,

driedoveranhydroussodiumsulphateandstoredat−18◦Cuntil

use.Theprocedurewasrepeatedthreetimes.Theyieldof

distilla-tionwasexpressedasthepercentageoftheessentialoilrecovered

fromtheplantmaterialused

SFEpilot-plant(SCF100model3PLC-GR-DLMP,SeparecoS.r.l,

Pinerolo,Italy)equippedwith1Lextractionvessel(E1),two0.3L

separatorsinseries(S1,S2),andatank(B1)whereCO2isstored

andrecycledwasused.Thesolventusedwascarbondioxide(Sapio

s.r.l,Udine,Italy).TheflowsheetofSFEpilotplantisgiveninFig.1

The extractor was filled with 0.15kg of inflorescences

dis-tributedinglassbeads(0.005m).Theextractionswereperformed

atpressureof10and14MPaandtemperatureof40◦C.On-line

fractionationoftheextractswasaccomplishedmaintainingS1at

7MPaand 25◦C andS2at 5MPaand 15◦C inboth

experimen-talassays.CO2 flowratewassetto3kg/hinboth experiments

(CO2/inflorescences=80kg/kg) Extractions were carried out by

duplicate.ThesamplesrecoveredinS1weresolid andpasty.S2

fractionswerecollectedintoacoldtrapcooledwithliquid

nitro-genandhadoilyappearance.ThefractionsobtainedinS1andS2

wererecuperatedandplacedinvials.Theywereweightedandkept

underN at−20◦Cinthedarkuntilanalysis

Headspacesolid-phasemicroextraction(SPME)isarapid, sol-ventlesssamplingprocedurewhich,combinedwithGC/MSanalysis

isausefulmethodfortheanalysisofvolatilecompounds(Zhang andPawlisyn,1993).InHeadSpaceSPME(HS-SPME)mode,a poly-mericfilmisexposedtothegasphasethatliesimmediatelyoverthe solidorliquidsample.Thisoperationstrategyhasanadvantageof beinganon-destructivetechniqueandallowstheevaluationofthe samplesatdifferentexperimentalconditions(Pawliszyn,1999) Volatile compounds of Cannabis sativa L inflorescences, essential oil and Sc-CO2 fractions were isolated by solid-phase microextraction (SPME) using a 1cm fiber coated with 50/30␮m divinylbenzene/carboxen/polydimethylsiloxane phase (DVB/CAR/PDMS)(Supelco,Milan,Italy)andanalyzedbyGC–MS The extraction temperature chosen was 30◦C in order togive

a better estimation of the volatile profile as perceived by the humannose.Theequilibriumofaromacompoundsbetweenthe SPMEcoatingfiberandheadspaceofeachsamplewasconsidered achievedafter50minofadsorption(DaPortoandDecorti,2012;

DaPortoetal.,2013)

GC–MSanalysisofthevolatilecompoundswasperformedusing

a Shimadzu gas chromatograph (model GC-17A) coupled to a Shimadzumassspectrometer(modelQP-5000) Thefusedsilica columnwasaDB-5fused-silicacolumn(Supelco,Bellafonte,PA) (30m×0.25mmi.d.,filmthickness0.25␮m).Workingconditions were:injector250◦C,transferlinetoMS250◦C,oventemperature: start45◦C,hold3min;programmedfrom45to190◦Cat3◦Cmin−1, hold5min,thenfurtherincreaseto250◦Cat20◦Cmin−1,holdfor

5min;carriergasheliumatflowrate2.0mlmin−1;ionization:EI

70eV;acquisitionparameters:scannedm/z:35–700.Splittingwas setinthesplitlessmodeforinflorescencesandthesplitratiowas 1/40(v/v)foressentialoilandSc-CO2fractions

Identificationof thevolatile compoundswas carried out by comparingtheKovats retentionindicesdeterminedbyinserting

a solutioncontaining thehomologous seriesof normal alkanes

Table 1

HS-SPME/GC–MS analysis of natural aroma compounds released by inflorescences

ofCannabis sativaL.

± RSD (%)

Bold values are referred to the main constituents.

a LRI = Linear retention indeces on DB5-column.

b GC peak area percentage Results expressed as mean of three replications.

(C7–C20)withthosereportedbyliterature(Bertolietal.,2010)and

withspectraoftheNISTandWILEYlibrariescoupledwiththe

soft-wareofGC–MSandAdams’library(Adams,2001).Theresultsare

expressedasGCpeakareaspercent

Thevolatilecompositionof essentialoiland ScCO2 fractions

weredeterminedbydirectGC–MSanalysis.GC–MSanalysiswas

performedusingaShimadzugaschromatograph(modelGC-17A)

coupledtoaShimadzumassspectrometer(modelQP-5000).The

fusedsilicacolumnwasa DB-5GCcolumn(Supelco,Bellafonte,

PA,USA)(30m×0.25mmi.d.,filmthickness0.25␮m).GC–MSdata

wereobtainedusingthefollowingconditions:carriergashelium

(He99.9995%);flowrate2.0mlmin−1;splitratio1/40(v/v)

Analiquotof50mg ofdistilledoilandSc-CO2fractionswere

dilutedwith25mln-hexaneand1.0␮lwasinjectedintotheGC–MS

system.Theoventemperatureprogramwas:45◦Cfor3min,from

45◦Cto250◦Cat3◦Cmin−1andholding250◦Cfor5min.The

injec-torandtransferlinetemperatureswere250◦C.Theelectronimpact

(70eV)spectrawererecordedat1s/scanwithafilamentemission

currentof10␮A

Identificationofthevolatilecompoundswascarriedoutas

pre-viouslyreportedforHS-SPMEanalysis.Theresultsareexpressed

asGCpeakareaspercent±RSD(%)

3 Resultsanddiscussion

Apreliminaryscreeningoftheheadspace(HD)bySPMEanalysis

ofinflorescenceswascarriedouttodefinetheoriginalvolatile

com-positionthatproducesthenaturalfragrance.Table1presentsthe

volatilecompoundsidentifiedaccordingtotheGC–MSanalysis.As

canbededucedfromtable,themain(moreabundant)compounds

identified in inflorescences were ␣-pinene (12.39%), ␤-pinene

(4.04%) myrcene (23.67%), terpinolene (10.17%), caryophyllene

(29.66%),␣-humulene(6.72%)andcaryophylleneoxide(4.70%),in

accordancewiththeliterature(Bertolietal.,2010)

0 10 20 30 40 50 60 70 80 90 100

Inflorescences HD ScCO2 10 MPa ScCO2 14 MPa

Oxyge nated sesquiter penes Sesquit erpene hy drocarbons

Oxygenated monoterpenes Monoterpene hydrocarbons

Fig 2 Comparison of HS-SPME/GC–MS analysis performed on inflorescences, essential oil (HD) and S2 fraction from Sc-CO2 extraction at pressure of 10 and

14 MPa and temperature of 40 ◦ C.

GC peaks were identified as hydrocarbon monoterpenes (52.73%)and oxygenatedmonoterpenes (2.22%),sesquiterpenes (36.96%),andoxygenatedsesquiterpenes(8.07%)

Thevolatilecompositionoftheessentialoil(HD)andthe dif-ferentfractions(S1andS2samples)obtainedbysupercriticalCO2 extractionwereanalyzedbydirectGC–MSanalysis(Table2) The main constituents of the essential oil were ␣-pinene (11.08%),␤-pinene(3.75%)myrcene(10.83%),terpinolene(5.83%), caryophyllene (41.14%),␣-humulene (9.85%) and caryophyllene oxide(5.27%) The essential oilcomposition showed significant quantitativedifferencesincomparisonwiththeessentialoilsfrom differentfiberhempinflorescencesreportedbyBertolietal.(2010), but these main constituents were confirmed In the essential oil,sesquiterpenes(52.63%),andrelatedoxygenatedcompounds (11.61%) were present in high percentage in comparison with hydrocarbon monoterpenes (34.31%) and oxygenated monoter-penes(1.44%)

Supercritical fluid extraction(SFE) with supercritical carbon dioxide(Sc-CO2)has beenwidely usedfor theextraction from naturalproducts.SFEisanenvironment-friendlytechnologythat representsanalternativetoconventionalextractionmethodsand offersseveraladvantagesoverclassicalsolventextractionmethods

CO2isthemostcommonlyusedsolventinSFEbecauseitischeap, inert,non-toxic,andallowsextractionatlowertemperatureand relativelylowpressure.(Brunner,1994)

SupercriticalCO2extractiononhempinflorescenceswere per-formedatpressureof10and14MPaandtemperatureof40◦C(CO2 densityhigherthanabout600kg/m3).On-linefractionationofthe extractswasachievedbydecreasingpressureandtemperaturein thetwoseparatorsS1andS2,withrespecttotheoperating condi-tionsusedduringsupercriticalextractions.InthefirstseparatorS1, pressurewasloweredto7MPaandtemperatureto25◦C,inthe sec-ondseparatorS2,pressurewasloweredto5MPaandtemperature

to15◦C.Undertheseconditionsofpressureandtemperature,CO2 densitywaslowerthan600kg/m3andthisallowedtoexcludeall butoneofthenonvolatilecompoundsfamiliesfromtheextract.The onlyexceptionwasrepresentedbyparaffinsconstitutingthe cutic-ularwaxes(Reverchonetal.,1995).Fig.2showsthattheextraction

Table 2

Direct GC–MS analysis of volatile compounds of essential oil (HD) and ScCO2 extracts (10, 14 MPa, 40 ◦ C)) ofCannabis sativainflorescences.

Bold values are referred to the main constituents.

a GC peak area percentage±RSD (%).

yield(massextracted/massloadedintheextractor×100)was

sig-nificantlyhigherinS1thaninS2forbothextractions.Itisapparent

thatcuticularwaxesprecipitatedinS1,duetotheirlowersolubility

insupercriticalCO2incomparisontoterpenesandtheirderivatives

(StahlandGerard,1985).Theextractionyieldobtainedinthe

sepa-ratorS1forinflorescencesprocessedat14MPa(1.39%w/w,±0.58)

wassignificantly higherthan inS1forinflorescences processed

at10MPa(1.03% w/w,±0.73) becauseof thehigherextraction

pressureemployed(Simandietal.,1999).Instead,lower

extrac-tionyieldswereachievedin theseparator S2for inflorescences

processed,respectivelyat10MPa(0.67%w/w,±0.18)and14MPa

(0.34%w/w,±0.11).However,boththeextractionyieldsobtained

inS2fractionsresultedhigherthanessentialoil(HD)yield(0.24%

w/w,±0.13).

TheSFEenergyconsumptionwasabout4.5kWhperkiloofplant

matter,taking intoaccount themechanical energy required by

thepumptoincreasethefluidpressure(1.2kWh)andtheheating

energytoincreasethefluidtemperatureandthecoolingenergyto

condensethefluidvapour(3.3kWh).Instead,thehydro-distillation

ofonekiloofplantmatterconsumedabout9.6kWh,duetothehigh

heatofvaporizationofwater.Itistobenotedthatextractionby

supercriticalCO2isparticularlyadvantageousintermsofenergy

consumptionbecauseofthesmallvolumeofsolventintroduced,

theseparationoftheextractbydecompression,plusthefactthat

itispossibletorecuperatethecaloriesproducedbythecoldgroup

(passagefromgasformtoliquidform)tofeedtheheatingsystem

(passagefromliquidformtosupercriticalstate)

Pereiraetal.(2010)reportedthattheCOM(manufacturingcost)

forSFEprocessisgenerallylowerthantheCOMofconventional

processesaswellastheCUT(utilitiescost)share(usuallybelow

1%).SFEiseconomicallyfeasibleafterappropriatelyoptimization

oftheprocess

AscanbeobservedinTable2,thedirect GC–MSanalysisof

thedifferentfractionscollected(S1andS2samples)indicatesthat

almostallvolatilecompoundswererecoveredinS2fraction.That

is,on-linefractionationwasasuitabletechniquetoachievethe

iso-lationofhempvolatilesinthesecondseparator.Itisinterestingto

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6

ScCO2 10MPa ScCO2 14MPa HD

S1

S1

S2

S2

Fig 3 Extraction yield (% w/w) obtained by Sc-CO2 extraction (10, 14 MPa and 40 ◦ C)

in the separators S1 and S2, and by hydrodistillation (HD).

notethevolatilecompositionofthedifferentS2fractionsinterms

ofthepercentageofterpenes,withrespecttothevolatile compo-sitionofessentialoil.Forinflorescencesprocessedat10MPaand

40◦C,thehighermolecularweightcompounds,namely hydrocar-bonsesquiterpenes(caryophyllene,␤-farnesene,␣-humulene)and oxygenatedsesquiterpens (caryophylleneoxide,␤-eudesmol, ␤-bisabololand␣-bisabolol)werefoundinlowerpercentage(45.56%) than at14MPaand 313.15K (63.89%).Thiscouldbeattributed

tothefactthatatconstanttemperature,theincreaseofpressure enhancestheCO2densityand,consequentlyitssolvationpower andthesolubilityofthesecompoundsinSc-CO2.TheS2fraction obtainedforinflorescencesprocessedat14MPaand40◦Chada chemicalprofilesimilartothatobtainedbyhydrodistillation(HD)

AcomparisonoftheresultsobtainedbyHS-SPME/GC–MS anal-ysisperformedoninflorescences,essentialoil(HD)andS2fractions collectedisshowninFig.3.Ascanbeobserved,thereisaperfect

inflorescencesprocessedbySc-CO2extractionat10MPaand40◦C

and inflorescencesin terms of thepercentage ofterpenes.This

provesthesuperiorqualityofthisextractincomparisonwiththe

otherone

4 Conclusions

SupercriticalCO2extractioncarriedoutat10MPaand40◦C

on-linefractionationoftheextractofCannabis sativainflorescences

allowedtherecoveryoffractionswithdifferentcompositionand

biologicalproperties,suitableforcosmeticand/orfoodindustry

Thelowprocessingtemperatureresultedinnon-damagedvolatile

compounds,givingtothearomaticextractsuperiorquality

ThesupercriticalCO2extractionprocessofhempinflorescences

resultedparticularlyadvantageousintermsofenergyconsumption

incomparisonwithhydrodistillation

References

Adams, R.P., 2001 Quadrupole Mass Spectra of Compounds Listed in Order of

Their Retention Time on DB-5 Identification of Essential Oils Components by

Gas Chromatography/Quadrupole Mass Spectroscopy Allured Publishing, Carol

Stream, IL, USA.

Bertoli, A., Tozzi, S., Pistelli, L., Angelini, L.G., 2010 Fibre hemp inflorescences: from

crop-residues to essential oil production Ind Crops Prod 32, 329–337.

Brunner, G., 1994 Gas Extraction: An Introduction to Fundamentals of

Supercriti-cal Fluids and The Application to Separation Processes Darmstadt: Steinkopff,

Springer, New York.

Da Porto, C., Decorti, D., 2012 Analysis of the volatile compounds of aerial parts and essential oil fromThymus serpyllumL, cultivated in North East Italy by HS-SPME/GC–MS and evaluation of its flavouring effect on ricotta cheese J Ess Oil Bear Plants 15, 561–571.

Da Porto, C., Decorti, D., Natolino, A., 2013 Ultrasound-assisted extraction of volatile compounds from industrialCannabis sativaL inflorescences Int J Appl Res Nat Prod 7, 1–14.

Dayanandan, P., Kaufman, B., 1976 Trichomes ofCannabis sativaL.(Cannabaceae).

Am J Bot 63, 578–591.

Pawliszyn, J., 1999 Applications of Solid Phase Microextraction Royal Society of Chemistry, Cambridge.

Pereira, C.G., Prado, J.M., Meireles, M.A.A., 2010 Economic evaluation of natural product extraction processes In: Rostagno, M.A., Prado, J.M (Eds.), Natural Prod-uct Extraction Principles and Applications RSC Publishing, United Kingdom, pp 442–469.

Pourmortazavi, S.M., Hajimirsadeghi, S.S., 2007 Supercritical fluid extrac-tion in plantessential and volatile oil analysis J Chromatogr A 1163, 2–24.

Reverchon, E., Della Porta, G., Senatore, F., 1995 Supercritical CO 2 extraction and fractionation of lavender essential oil and waxes J Agric Food Chem 43, 1654–1658.

Reverchon, E., De Marco, I., 2006 Supercritical fluid extraction and fractionation of natural matter J Supercrit Fluids 38, 146–166.

Simandi, B., Oszagyan, M., Lemberkovics, E., Kéry, A., Kaszacs, J., Thyrion, F., Matyas, T., 1999 Supercritical carbon dioxide extraction and fractionation of oregano oleoresin Food Res Int 31, 723–728.

Stahl, E., Gerard, D., 1985 Solubility behavior and fractionation of essential oils in dense carbon dioxide Perf Flav 10, 29–33.

Turner, C.E., Hemphill, P., Mahlberg, G., 1978 Quantitative determination of cannabi-noids in individual glandular trichomes ofCannabis sativaL.(Cannabaceae).Am.

J Bot 65, 1103–1106.

Turner, C.E., Elsohly, M.A., Boeren, E.G., 1980 Constituents ofCannabis sativaL XVII.

A review of the natural constituents J Nat Prod 43, 169–234.

Zhang, Z., Pawlisyn, J., 1993 Headspace solid-phase microextraction Anal Chem.

65, 1843–1852.