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J. of Supercritical Fluids 56 (2011) 64–71

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

journa l homepage: www.e lsev ier .com/ locate /supf lu

upercritical fluid extraction of wormwood (Artemisia absinthium L.)

. Martína, A.M. Mainara,∗, A. González-Colomab, J. Burilloc, J.S. Urietaa

Group of Applied Thermodynamics and Surfaces (GATHERS), I3A (Aragón Institute of Engineering Research), Universidad de Zaragoza, C/ Pedro Cerbuna 12, Zaragoza 50009, SpainCSIC, Centro de Ciencias Medioambientales, Instituto de Ciencias Agrarias, C/Serrano 115-bis, Madrid 28006, SpainCentro de Investigación y Tecnología Agroalimentaria de Aragón (CITA), Avda. Montanana 930, Zaragoza 50059, Spain

r t i c l e i n f o

rticle history:eceived 14 September 2010eceived in revised form7 November 2010ccepted 29 November 2010

a b s t r a c t

The objective of the work was to optimize the extraction of wormwood oil by supercritical fluid extrac-tion (SFE) of growth-controlled plant material. Different extraction conditions, two growth techniquesand various crops were tested and the evolution of the extracted oil composition was screened chro-matographically. A comparison with conventional techniques such as hydrodistillation (HD) or organic

3

eywords:rtemisia absinthium L.upercritical fluid extractionovová model

solvent extraction (OSE) was also presented. Particularly, six CO2 densities ranging from 285.0 kg/m to819.5 kg/m3 were studied in the range of 9.0–18.0 MPa and 40–50 ◦C. A systematic study was carried outwith plant material from 2005, while SFE of 2006, 2008 and aeroponically grown crops was performedfor comparative purposes. The effect of ethanol as a modifier of the supercritical fluid extraction wasalso studied. The major compounds found in the SFE extracts as well as in the HD essential oils were

thenccess

ydrodistillationrganic solvent extraction

Z-epoxyocimene, chrysanthe Sovová model, was su

. Introduction

The need for clean processes and safe products is the essencef green chemistry [1] which is linked to environmental protectionnd the demand for quality and value added products. An advancedeparation technique like supercritical fluid extraction (SFE) notnly fulfils these conditions when extracting oils from natural prod-cts, but it also circumvents the problems related to traditionalethods. Hydrodistillation (HD) requires high operating temper-

tures (with its inherent energetic cost) that can damage naturalroducts present in the matrix. Organic solvent extraction (OSE)

s related to toxic residues such as VOC emissions and expensivend incomplete solvent–solute separation. By means of supercrit-cal CO2, an inert, nontoxic, cheap and recyclable solvent, thoseisadvantages can easily be overcome.

Wormwood (Artemisia absinthium L.) essential oils (obtained byD) and absolutes (obtained by OSE) have been widely used mainlyue to their antimicrobial [2], antiparasitic [3], antihelmintic [4]r hepatoprotective [5] properties. Documented medical use oformwood dates as far back as Ebers Papyrus, an Egyptian medi-

al document from 1552 B.C., the oldest existing medical document

6]. Despite its medical applications, wormwood is better known asraw material used in the manufacture of absinthe, a bitter spiritanned in several countries and re-destricted last century. Its toxi-ity and therefore its prohibition was based on its thujone content

∗ Corresponding author. Tel.: +34 976761195; fax: +34 976761202.E-mail address: ammainar@unizar.es (A.M. Mainar).

896-8446/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.supflu.2010.11.017

ol and chrysanthenyl acetate. A model based on mass transfer equations,fully applied to correlate the experimental data.

© 2010 Elsevier B.V. All rights reserved.

but there is still some controversy regarding this point [7]. Nev-ertheless, thujones are known for their toxicity [8] and methodshave already been developed to eliminate them from absinthe [9] oreven to cultivate thujone-free wormwood [10]. Another importantcharacteristic of the plant is its pesticidal nature. There is docu-mented use of the plant in organic agriculture as an aqueous slurryto combat ants, caterpillars and aphids [11]. Different wormwoodextracts have also proven to be efficient against pests like the Col-orado potato beetle (Leptinotarsa decemlineata) [12] or the granaryweevil (Sitophilus granarius) [13].

The chemical composition of wormwood oils determines thechemotaxonomy of the plant. Several chemotypes have previouslybeen described, their major components varying depending mainlyon the origin of the plant, and also on the growing conditions andthe development stage of the plant. Thus, controlled growth ofwormwood is vital in order to acquire homogeneous material whenconducting systematic experiments [14]. Chialva et al. [15] havedescribed four different chemotypes containing sabinyl acetate,epoxyocimenes, thujones or chrysanthenyl acetate, respectively,as the major compounds. Other authors have reported wormwoodessential oils whose major compounds were sabinene + myrcene[16], �-pinene + �-thujone [17], cis chrysanthenol [18], bornylacetate [19], caryophyllene oxide + p-cymene + 1,8-cineole [20] andZ-epoxyocimene + chrysanthenyl acetate or Z-epoxyocimene [21].

In addition to the interesting characteristics of the wormwoodoils already mentioned, both Z-epoxyocimene and chrysanthenylacetate have shown antifungal activity [22] and, in the case ofchrysanthenyl acetate, acts as antiprostaglandins due to their anal-gesic effect inhibiting the enzyme prostaglandin synthetase [23].

L. Martín et al. / J. of Supercritica

Nomenclature

a0 specific surface area per unit volume of extractionbed (1/m)

cu (=xu/(1 + xu)) solute content in the untreated solid(kg (solute)/kg (solid))

C1 fitting parameterC2 fitting parameterecal calculated extraction yield (kg (extract)/kg (insolu-

ble solid))eexp experimental extraction yield (kg (extract)/kg

(insoluble solid))E amount of extract (kg)kf fluid phase mass transfer coefficient (1/s)ks solid phase mass transfer coefficient (1/s)K (=y0/x1,0) partition coefficientKI Kovats IndexM solid charge in the extractor (kg)N number of separated fractions in the SFE curvesNm (=(1 − cu)M) charge of insoluble solid (kg)q relative amount of the passed solvent (kg (sol-

vent)/kg (insoluble solid))qc relative amount of the passed solvent when all the

solute in broken cells has been extracted (kg (sol-vent)/kg (insoluble solid))

Q solvent flow rate (kg/s)r grinding efficiency (fraction of broken cells)RSMD2 root squared mean deviationt extraction time (s)xu concentration in the untreated solid (kg (solute)/kg

(insoluble solid))y0 initial fluid phase concentration (kg (solute)/kg

(insoluble solid))Y global yield of the process (kg (solute) × 100/kg

(dried plant))Yr relative yield of the compound in each extract

(100 × mass of compound (kg)/mass of extract (kg))Ysp specific yield of each compound from 100 grams of

plant material (Yr × Y/100 g of plant material)

Greek letters� (=�fε/(�s(1 − ε))) solvent-to-matrix ratio in the bed

(kg of solvent/kg of insoluble solid)ε (=1 − �b/�s) bed porosity�b bed density (kg/m3)

cwtPdtyfg(darae

�f fluid density (kg/m3)�s solid density (kg/m3)

The aim of this study was to optimize and evaluate the extractionapability of the supercritical fluid over different growth controlledormwood. A. absinthium is an attractive plant because of its insec-

icidal properties and, to date, its SFE has not been studied in depth.articularly, six CO2 densities [24] were evaluated, working at threeifferent pressures and two temperatures ranging from 9.0 MPao 18.0 MPa and 40 ◦C and 50 ◦C, respectively. Three different cropears (2005, 2006 and 2008) were studied. Furthermore, two dif-erent growth methods (controlled field growth and aeroponicrowth) were also investigated. The range of densities studied low285.5 kg/m3 corresponding to 9.0 MPa, 50 ◦C) and relatively high

3 ◦

ensities (819.5 kg/m corresponding to 18.0 MPa and 40 C). Andditional extraction with ethanol as modifier of the SFE was car-ied out to study the degree of exhaustiveness of the matrix afterconventional SFE. The evolution of the oil composition for each

xperiment was screened by chromatographic analysis (GC–MS

l Fluids 56 (2011) 64–71 65

and GC) by quantifying the 3 major compounds found in the essen-tial oil and a fourth compound, nonacosane, a heavier compoundfound in OSE and SFE extracts. In addition, a comparison with theessential oils obtained by HD and extracts obtained by OSE withhexane and ethanol was performed.

Furthermore, it is very important to apply models that allow forthe correlation and extrapolation of experimental data for possibleindustrial application of the SFE process. The model used here wasthe one proposed by Sovová [25] which appears to be one of themost appropriate to describe the experimental data collected.

2. Materials and methods

2.1. Raw material characterization and preparation

Field wormwood was cultivated under controlled conditionsby CITA (Centro de Investigación y Tecnología Agroalimentaria deAragón) in Ejea de los Caballeros (Zaragoza, Spain), dry land withargillaceous-sandy soil in the north-east of the Iberian Peninsula.The amount of biomass, plant diameter and plant height wereregistered. The controlled-growth plant variety is currently beingpatented and is registered under No. 20090972 in the RegionalPlant Variety Office [26]. Also a patent application of the super-critical extraction procedure of this plant variety has been sent[27]. Plants were collected while in full bloom in 2005, 2006 and2008 and air dried. In order to compare growing techniques, worm-wood was also cultivated aeroponically where no soil is requiredand higher yields can be obtained [28]. The individuals for aero-ponic cultivation were obtained from a population donated by thenursery at the Sierra Nevada National Park (2003, Granada). Cut-tings of selected individuals were vegetatively multiplied, rootedin vermiculite, watered three days/week with a nutrient solution(Nutrichem 20:20:20 N, P, K – Miller Chemical & Fertlizer Corp.;3 g/l) and kept in a growth chamber (25 ◦C, 70% relative humidity,16:8 light:obscurity) until their transfer to the aeroponic cham-ber (10–15 cm plants). The aeroponic chamber (Apollo 3 system:33 plants, 240 L, 1750 mm × 1350 mm × 750 mm) was located inan environmentally controlled greenhouse (20–30 ◦C). The plantswere kept under constant pulverization with water at 26 ◦C supple-mented with 0.2 g/l Nutrichem and 0.03% H2O2 (33%, w/v, Panreac)and artificial light (16:8, light:obscurity). The plants were grownfor nine months, their aerial part and roots collected periodically[29]. Both CITA and CCMA supplied stalks with leaves and flow-ers with a water content below 10% (w/w). The water content wasdetermined to be 7–9% for the different crops, and was controlledappropriately by a desiccant stove (Selecta).

Leaves and flowers were ground in batches of 0.1 kg each forabout 5 s using an electric grinder. The plant material was cooledwith liquid nitrogen during milling to prevent the loss of volatilecompounds. Particle size selection (0.56 mm for all the experi-ments) was by vibrating sieve. This particle size was the result ofthe collected mass between sieve sizes with pore diameter of 0.71and 0.40 mm. Ground samples were stored at −15 ◦C.

2.2. SFE, solvent extraction and hydrodistillation apparatus

The SFE plant presented in previous works [30,31] was builtaccording to the layout reported by Reis-Vasco et al. [32]. Themain components are a compression pump, filter, 1 L extractionvessel and two separators (V: 0.18 L each), one for the recov-

ery of waxes [33] and the other for the recovery of volatile oil.Temperatures in the extraction vessel and separators were main-tained constant with the aid of jackets with circulating liquids fromthermostat-controlled baths. Other important elements include a0.5 L refrigerator cylinder, a heat exchanger, a back pressure regu-

6 critica

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dwiamotdCtw1le

epaittsmv

tHm

vrePr

2

cmcciluflwmwcosTic(sdmps

6 L. Martín et al. / J. of Super

ator and a flowmeter plus a CO2 totalizer. Several valves connectedhe different sections of the pilot plant. Pressure and temperatureere measured and/or controlled with manometers, a pressure

ransducer, thermopars, and temperature controllers.The experimental procedure adopted is similar to those

escribed elsewhere [28,29]. Approximately 90 g of ground worm-ood was introduced into the extraction vessel with several porous

nert materials (glass spheres, nickel sponges and glass frits), insymmetrical arrangement with the plant material placed in theiddle. The porous elements were used to achieve a uniform flux

f the supercritical fluid. Once the extraction vessel and separa-ors reach working temperature, the pump is turned on to theesired flow rate and the valves set at the desired pressure. TheO2 stream to the second separator is manipulated by openingwo needle valves at fixed time intervals in order to collect andeigh the recovered oil. All the SFE experiments ended either after

1 h of extraction or when the slope of the extraction curve wasower than 10% of the maximum slope in the initial steps of thexperiments.

In order to determine the exhaustiveness of the matrix, an SFExperiment was carried out in a similar fashion but in this case thelant material had previously undergone SFE extraction (18.0 MPat 40 ◦C), and 50 mL of ethanol (99.9% Scharlau) were added tot prior to this second extraction. The ethanol was added beforehe extraction, the extraction vessel de-pressurized, opened andhe cosolvent added. The experiment was also ended following theame criterion as in the case of the extraction without ethanol asodifier. Ethanol in the extracted fractions was removed with a

acuum evaporator.The essential oil was obtained by a Clevenger type hydrodis-

iller made at the Faculty of Sciences, Zaragoza, Spain.ydrodistillation was performed for 4 h using 40 g of plantaterial.OSE was performed in a Soxhlet apparatus with an extraction

essel of 0.125 L and a 0.1 L flask. About 10 g of ground plant mate-ial were put into a cellulose cartridge. Two different extractionxperiments (20 h each) were carried out, one with hexane (99%anreac) and one with ethanol (99.9% Scharlau). Both solvents wereemoved from the extract using a vacuum evaporator.

.3. Analysis of the extracts

The composition of the extracts was determined using a gashromatographer (GC) and a gas chromatographer coupled to aass spectrometer (GC–MS). Three replicates of each analysis were

arried out, obtaining an average deviation of less than 1% for eachomponent. A GC Trace GC 2000 Series Thermo Quest with a flameonization detector (FID) and a split injector equipped with a capil-ary column of fused silica (DB-5, 30 m × 0.25 mm × 0.25 �m) wassed. The carrier gas was helium (99.9990%, Abelló Linde) at aow rate of 1.3 mL/min. The injector and detector temperaturesere 310 ◦C. The temperature ramp was 65–300 ◦C at 6 ◦C/minaintaining the final temperature for 20 min. Samples of 0.1 �Lere injected with a split ratio of 1:50. The identification of the

ompounds was done by comparison with the retention timesf standards from the NIST 08 library database or by compari-on with the Kovats Retention Indexes [34] from the bibliography.he proportion of each compound in the oil was made throughts area percentage in the chromatogram. A GC Trace GC Ultrahromatographer equipped with a capillary column of fused silicaDB-5, 30 m × 0.25 mm × 0.25 �m) coupled to an electrospray mass

pectrometer ITQ 900 was also used to complete the compositionetermination. The temperature ramp was 65–300 ◦C at 6 ◦C/minaintaining the final temperature for 20 min. The injection tem-

erature was 270 ◦C. Helium was the carrier gas at 1.0 mL/min. Theample (0.1 �L) was injected with a split ratio of 1:50. The MS con-

l Fluids 56 (2011) 64–71

ditions were as follows: ion source temperature 230 ◦C, interfacetemperature 230 ◦C, mass range 50–450 u.

3. Results and discussion

3.1. Yield evaluation

The aim of this work was to evaluate the extraction capabil-ity of supercritical CO2 on wormwood. Three different pairs ofpressures and temperatures (Table 1) were chosen to optimizethe SFE of wormwood with densities ranging from 285.0 kg/m3 to819.5 kg/m3.

This optimization was carried out with plant material from2005. Once the extraction conditions were optimized, SFE experi-ments on plant material from 2006, 2008 and aeroponically grownwormwood were also carried out under the best extracting condi-tions to obtain high total yields to compare crops and cultivationmethods. An additional extraction with ethanol as SFE modifier wasalso performed to determine the completeness of the extraction.All the SFE experiments, except the one using ethanol as a modi-fier, were conducted with a particle size of 0.56 mm and CO2 flowrate of 1.08 kg/h which had proven to be the optimum conditionsin previous experiments. Three flow rates of CO2, namely 0.72, 1.08and 1.32 kg/h and three different particle sizes, namely 0.26, 0.56and 0.80 mm were used in several experiments with wormwoodand other plant material, concluding that the mentioned conditionswere the best ones [35–37]. When ethanol was used as an SFE mod-ifier, the CO2 flow rate was diminished to favour the contact timebetween modifier, CO2 and matrix. For comparison purposes, yieldsfrom conventional techniques such as HD or OSE are also shown inTable 1.

It is desirable to separate high molecular weight compoundsfrom lighter compounds when working with SFE to obtain extractssimilar to the hydrodistilled ones. For this purpose, two conden-sation separators were used in order to keep the heavier ones(nonacosane and analogous) in the first separator and the volatile(essential oil constituents) in the second [38]. The temperatureand pressure conditions of the separators were as follows: −5 ◦C,8.0 MPa in separator 1 (SP1) and 10 ◦C, 2.0 MPa in separator 2 (SP2).The extract of the first separator was collected only at the endof the complete experiment in order to evaluate the separationefficiency and assess volatile content. The compositions of the col-lected extracts for each experiment in SP1 are shown in Table 2.The total extracted mass between SP1 and SP2 is also shown.

Results showed that the amount of the condensed extracts inSP1 was always lower than 10% of the collected amount in SP2 inexperiments E1–E9. When ethanol as an SFE modifier was used, thisamount accounted for 28% of the extracted mass in SP2. The compo-sition of the extracts recovered in SP1, shown in Table 2, revealed alow content in volatiles while the proportion of heavy compounds isgreater. Furthermore, as it can be seen in Table 2 (composition of theextracts), as solvent density increased from 285.0 kg/m3 in experi-ment 4–819.5 kg/m3 in experiment 3 (seen in Table 1, experimentsand density), the proportion of volatiles diminished markedly, from46.11% in experiment 4–11.33% in experiment 3 or even 0% to theexperiment carried out with ethanol as entrainer.

The overall yield of recovered oil by SFE in SP2 or by traditionaltechniques was calculated as Y (%), Eq. (1):

Y = mass of extractmass of dried plant

× 100 (1)

The variation of the extraction yield with time for the differentexperiments is plotted in Fig. 1. The theoretical yields calculatedwith the Sovová model, explained in Section 3.3 are also shown.

From the data reported in Table 1 and Fig. 1, it is evident thatSFE yield increases as density rises. Yield increase is greater than

L. Martín et al. / J. of Supercritical Fluids 56 (2011) 64–71 67

Table 1Experimental conditions and yields of the different extractions of wormwood.

SFE

Experiment Plant material P (MPa) T (◦C) �sc (kg/m3) Flow rate (kg/h) Y (%)

E1 2005 9.0 40 485.5 1.08 0.75E2 2005 13.5 40 753.6 1.08 2.26E3 2005 18.0 40 819.5 1.08 2.65E4 2005 9.0 50 285.0 1.08 0.56E5 2005 13.5 50 655.5 1.08 2.16E6 2005 18.0 50 757.1 1.08 2.50E7 2006 18.0 40 819.5 1.08 1.61E8 2008 18.0 40 819.5 1.08 3.66E9 Aeroponical 18.0 40 819.5 1.08 1.91E10 2005 + EtOH 18.0 40 819.5 0.81 1.08

Conventional techniques

Experiment Plant material Y (%)

HD 2005 0.52OSE ethanol 2005 23.81OSE hexane 2005 8.78

Overall yield of recovered oil, Y (%) = mass of extractmass of dried plant × 100.

Table 2Major compounds found in the extracts collected in separator 1 in each SFE experiment.

Components IK % Composition (w/w)

E1a E2a E3a E4a E5a E6a E7a E8a E9a E10a

Z-Epoxyocimene 1130 24.97 13.33 5.96 24.62 6.00 3.39 5.03 9.43 11.59 –Chrysanthenol 1164 10.83 3.96 3.82 14.26 2.74 0.84 1.97 5.31 4.05 –Chrysanthenyl acetate 1263 5.11 5.09 1.55 7.23 1.52 0.42 4.12 0.58 0.85 –Nonacosane 2900 11.34 8.44 13.45 8.96 22.31 35.83 17.53 13.72 26.34 17.40

Collected mass in SP1 (g) 0.0615 0.1479 0.2406 0.0715 0.1132 0.2194 0.1780 0.1970 0.1750 0.27700.526

I

tbTaEa

Fi(5

Collected mass in SP2 (g) 0.6941 2.0290 2.5237

K: Kovats Retention Indexes.a Experiments.

hat corresponding to solubility increase with temperature and has

een certified in several extractions such as in Langa et al. [30,31].he comparison of the different wormwood crops was carried outt the highest density to obtain high yields similarly to experiment3 (18.0 MPa, 40 ◦C). 2006, 2008 and aeroponically grown cropsccounted for yields of 1.61%, 3.66% and 1.91%, respectively. Fur-

ig. 1. SFE curves. Overall yield, Y (mextract/mdried plant × 100), vs time, t (h). Normal-zed extraction yield, e (mextract/Nm) vs relative amount of the passed solvent, q. E1�): 9.0 MPa, 40 ◦C. E2 (�): 13.5 MPa, 40 ◦C. E3 (�): 18.0 MPa, 40 ◦C. E4 (♦): 9.0 MPa,0 ◦C. E5 (�): 13.5 MPa, 50 ◦C. E6 (�): 18.0 MPa, 50 ◦C. (—) Sovová model.

7 2.0290 2.3487 1.5051 3.4282 1.6846 0.9838

thermore, all the SFE experiments achieved higher yields than HDwhere a yield of 0.52% was obtained. Yield from hydrodistillationwas similar to those found in the literature [13,39]. However, inmost of the SFE experiments the asymptotic region of the extrac-tion was not reached due to the long extraction times (experimentswere ended after 11 h), which implied a difficulty compared withother materials.

The degree of matrix exhaustiveness for the 2005 crop wasinvestigated by conducting a second SFE experiment (E10) on theused plant material from experiment E3. The severe conditions(18.0 MPa, 40 ◦C, 50 mL ethanol) of the extraction yielded only1.08%. Adding this value to the yield from experiment E3, an overallyield of 3.73% is obtained for SFE. The analysis of the composition ofthe extracts from these experiments will be commented in Section3.2.

Global yields of OSE were also considerably higher as seen inTable 3. The reason for these yields is that the extracted compo-nents are noticeably different from those extracted with SFE orHD and, along with essential oil components, other heavier com-pounds such as waxes are coextracted. The major compounds ofHD and two SFE extracts (with the highest and the lowest densi-ties) are presented in Table 3 along with the composition for bothOSE extracts. The relative yields of the components in each extract(Yr) and the specific yield of each component from 100 g of plantmaterial (Ysp) are also presented in Table 3.

Table 3 shows that the relative yields of the major compoundsfor HD are similar to those of SFE and both are higher than thoseof the OSE. The specific yields of the SFE extracts are considerablyhigher than the HD ones and similar to those obtained by OSE. To theextent of their specific yields, those of SFE are considerably higher

68 L. Martín et al. / J. of Supercritical Fluids 56 (2011) 64–71

Table 3Major compounds found in essential oil and some SFE extracts (E3: 18.0 MPa, 40 ◦C (2005); E4: 9.0 MPa, 50 ◦C) (2005)), compared to their proportion in the OSE extracts.Relative (Yr), specific (Ysp) and global yields achieved for every technique (Y).

Compound IK HD SFE (E3) SFE (E4) OSE hexane OSE ethanol

Yr (%) Ysp (g) Yr (%) Ysp (g) Yr (%) Ysp (g) Yr (%) Ysp (g) Yr (%) Ysp (g)

Z-Epoxyocimene 1130 22.14 0.12 17.78 0.47 39.49 0.22 6.01 0.53 0.26 0.06Chrysanthenol 1164 15.67 0.08 8.82 0.23 9.33 0.05 2.90 0.25 2.94 0.70Chrysanthenyl acetate 1263 13.53 0.07 4.58 0.12 7.72 0.04 2.26 0.20 3.23 0.77Nonacosane 2900 – – 6.81 0.18 – – 5.65 0.50 13.02 3.09

R ecific

e

tmhdsme

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Y (%) 0.52 2.65

elative yield of the compound in each extract, Yr = mass of compoundmass of extract × 100; sp

xperimental yield, Y = mass of extractmass of dried plant × 100.

han the HD ones and similar to the obtained by OSE. OSE experi-ents produce higher global yields because they extract more and

eavier components from the plant matrix due to the higher resi-ence time of the solvent in it (20 h), due to the fact that the pureolvent recirculates approximately every 5 min through the plantatrix, due to their higher solvent powers and due to polarity when

thanol is added.

.2. Composition evaluation

The analysis of the different SFE fractions collected in SP2 inach experiment was used to observe the evolution of the com-osition of the supercritical extract during the extraction process.uantification of the three major compounds of the essential oil, Z-poxyocimene, chrysanthenol and chrysanthenyl acetate, and alsohat of nonacosane was performed. The major compounds found inur essential oil differ even from the ones found in wormwood fromimilar geographical origins (less than 200 km from our planta-ion) described by Arberas et al. [21,40]. Essential oil of wormwoodrom the Pyrenees presented as major compound 7-epoxyocimene21], while wormwood essential oil from Haro, La Rioja presentedhrysanthenyl acetate and Z epoxyocimene as major compounds40]. Those differences can be explained by the different chemo-ype of the wormwood plants. Nonacosane, a constituent of theo-called waxes, appears as the SFE conditions are stronger or inhe tail fractions in a single SFE experiment. Study of this evolutions important if the oil is intended to be enriched in certain specificompounds or if the completeness of the extraction is investigated.oextraction of heavier compounds such as long chain alkanes can-ot fully explain the increase in SFE yield since the extracted mass

n SP1 is not taken into account and since heavier compounds areeen just when SFE approaches its end and they account for as muchs 20% of the total yield under the most severe conditions.

Quantification of those four components in head and tail frac-ions of the SFE experiments is reported in Table 4 where theiruantification in the HD essential oil is also presented. The majoromponents of the essential oils, Z-epoxyocimene, chrysanthenolnd chrysanthenyl acetate, registered percentages of 22%, 16% and4%, respectively. In the SFE extracts, these three are also majorompounds, their percentages depending on the extraction con-itions, the year and the growth technique. For the 2005 crop,ead fractions of experiments E1, E2, E4 and E5 are enriched,especting to HD essential oils, in Z-epoxyocimene (tail frac-ions are also enriched in the case of experiments E1 and E4),hile the proportion of chrysanthenol and chrysanthenyl acetateiminishes slightly for every SFE extract of that crop. As the

xtraction processes came to a close, supercritical extracts werenriched in nonacosane and analogous heavier compounds which,n some fractions was the major compound as shown in Table 4.t is worth noting the absence of thujones, toxic compoundshat usually appear in wormwood but which were prevented

0.56 8.78 23.81

yield of each compound from 100 g of plant material, Ysp = Yr100 g of plant material × Y;

using a controlled growing technique cited in the introduction[10].

Comparison of SFE extracts from different year crops and grow-ing techniques shows that major compounds are the same as thosefrom 2005 but their proportion varies as shown in Table 4. In thecase of 2008 extracts, the major compound is chrysanthenyl acetateaccounting for 13.25% and in the case of aeroponically grown plantsthe proportion of Z-epoxyocimene reaches a value of 45.71%, dou-ble the proportion of the essential oil found in 2005.

As for the completeness of the extraction, after two consecutiveSFE experiments the composition of the extracts from experimentE10 shows the oil evolution. Compared to the extracts obtained byHD, OSE or those previously obtained by SFE, Z-epoxyocimene is nolonger the major component and its proportion in the tail fractionsis practically non-existent. It is therefore safe to say that the totalquantity of oil had been extracted. As will be explained in Section3.3, this experiment could be used to determine the total quantityof extractable oil by SFE (xu from Sovová model).

3.3. Modelling the supercritical extraction process

Following the Sovová model where the oil is treated as a singlecomponent [25], the experimental yield for each fraction, eexp, wascalculated according to Eq. (2) and the amount of solvent, q, wasobtained through Eq. (3).

eexp = E

Nm(2)

q = Qt

Nm(3)

where E was the amount of extract (kg), Nm the charge of insolublesolid (kg), Q the solvent flow rate (kg/s) and t the extraction time(s). The charge of insoluble solid, Nm, was calculated as describedin the following equations:

Nm = (1 − cu) · M (4)

Cu = xu/(1 + xu) (5)

where cu was the solute content in the untreated solid(kg(solute)/kg(solid)), M, the solid charge in the extractor (kg)and xu, the concentration in the untreated solid (kg(solute)/kg(insoluble solid)). The variation of eexp with q for the variousexperiments and the theoretical values are plotted in Fig. 1.

Knowledge of where the essential oil is located in the plantis quite a difficult issue. Wormwood, as it can be seen in Fig. 2,has glandular trichomes where essential oil is stored. Images ofwormwood after milling and before the extraction were obtained

by means of a scanning electron microscope (SEM) Hitachi S3400in the Instituto de Carboquímica de Zaragoza, Spain.

Sovová [25] has devised a model to show the essential oil dis-tribution in the complex plant structure in order to formulate theextraction process. This model proposed by Sovová provided us

L. Martín et al. / J. of Supercritica

Tab

le4

Maj

orco

mp

oun

ds

ofth

ees

sen

tial

oila

nd

ofth

eSF

Eex

trac

tsco

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inse

par

ator

2th

rou

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tth

eex

per

imen

ts.C

hea

din

dic

ates

the

init

ialp

oin

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the

extr

acti

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rves

wh

ile

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dic

ates

the

last

ones

.

Com

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nd

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E2E3

E4E5

E6E7

E8E9

E10

Ch

ead

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tail

Ch

ead

Cta

ilC

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dC

tail

Ch

ead

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tail

Ch

ead

Cta

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hea

dC

tail

Ch

ead

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hea

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tail

Z-Ep

oxyo

cim

ene

1130

22.1

439

.98

36.7

628

.65

14.6

720

.94

13.0

342

.88

31.5

932

.82

7.56

18.2

812

.67

13.0

54.

9228

.03

9.59

45.7

113

.85

6.18

0.85

Ch

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1164

15.6

711

.87

12.8

87.

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669.

587.

698.

8810

.37

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4.67

9.22

8.81

5.44

2.69

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26.

4915

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5.93

6.62

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Ch

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enyl

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1263

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114.

285.

253.

577.

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782.

936.

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2613

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1.80

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2.11

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Non

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251.

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l Fluids 56 (2011) 64–71 69

with satisfactory results. It is based on several mass balances in theextraction bed and provides that the extractable material is con-tained in a spherical structure with broken cells in an outer layerand intact cells in the nucleus, where r is the grinding efficiency(the volumetric fraction of broken cells). The solute is assumed tobe homogeneously distributed in the untreated solid. The easilyaccessible solute from the broken cells is transferred directly to thesolvent fluid phase while the solute from intact cells is first trans-ferred to the broken cells and then to the fluid phase. This leads toextraction curves (eexp vs q) with two parts, each corresponding toone of these mass transfer processes.

Sovová assumed that the balance between the solvent and thesolute within broken cells is established during extractor pressuri-sation, i.e., before the solvent begins to flow from the extractor.Moreover, concentration in the intact cells is assumed to remainunchanged and equal to the concentration in the untreated mate-rial, xu, up to the beginning of the extraction.

Sovová [25] took several types of flow patterns and plantmatrices into account and this leads to the description of severalsituations in her model. Particularly, when a solute–matrix inter-action exists, the solute never saturates the fluid phase; a smoothtransition appears between the first part of the extraction curveand its end.

According to the protocol proposed by Sovová and some previ-ous results obtained by Coelho et al. [41], the suitable equations forthe fitting of the experimental data of the SFE of wormwood were

those corresponding to the equilibrium model. In this equilibriummodel, ecal was related to the q parameter according to Eq. (6) for

Fig. 2. SEM images of wormwood tissue. (a) A glandular trichome (2000×); (b) groupof glandular trichomes (400×).

70 L. Martín et al. / J. of Supercritical Fluids 56 (2011) 64–71

Table 5Values of the main parameters obtained with the Sovová model and RMSD2 for each experiment.

Experiment �sc (kg/m3) xu y0 � r ksas × 106 (s−1) kfa0 (s−1) qc C1 C2 RSMD2

E1 485.5 0.031 0.00026 2.86 0.13 2.08 0.52 12 0.885 0.0026 0.048E2 753.6 0.031 0.00056 4.46 0.13 9.25 0.52 12 0.938 0.0118 0.0014E3 819.5 0.031 0.00079 4.85 0.13 10.8 0.52 12 0.970 0.017 0.011E4 285.0 0.031 0.00025 1.68 0.13 0.649 0.52 12 0.876 0.00097 0.038

710

t

e

e

wumsvspp

εpswi

tftm

R

bda

mfitiaoftcsxcabSoeditex

E5 655.0 0.031 0.00052 3.90 0.13E6 757.1 0.031 0.00065 4.48 0.13

he first part of the curve and to Eq. (7) for the second part.

cal = qKxu

1 + K(�/r)= qy0, for 0 ≤ q ≤ qc (6)

cal = Xu[1 − C1 exp(−C2q)], for q > qc (7)

here K is the partition coefficient, xu is the concentration in thentreated solid, r is the grinding efficiency, � is the solvent-to-atrix ratio in the bed, y0 is the so-called oil solubility in the

upercritical CO2 and it is the slope of the curve when eexp is plotteds q, qc is the relative amount of the passed solvent when all theolute in broken cells has been extracted, and C1 and C2 are fittingarameters. K, � , and r are obtained simultaneously with the fittingrocess.

Other properties needed to apply the model are bed porosity,, solid density, �s, and bed density (300.0 kg/m3), �b. The bedorosity (0.70) was calculated using the true and apparent den-ities according to Reis-Vasco et al. [32]. The apparent bed densityas calculated using the bed volume and the mass of plant material

n the bed.As several experiments with the same plant material are needed

o correlate the SFE process with the Sovová model, data correlationor the experiments was carried out only for the 2005 crop usinghe Solver Excel tool by minimizing a function RSMD2 (root squared

ean deviation) defined as

SMD2 =∑i=N

i=1 (eexp − ecal/eexp)2

N(8)

The estimated curves are plotted in Fig. 1. Good correlation cane observed between the model correlations and the experimentalata for all experiments. The values of the main fitting parametersre collected in Table 5 along with RSMD2 values.

The values of some of the parameters are worthy of further com-ent. Concentration in the untreated solid, xu, was obtained by

tting the experimental values and is in agreement with the extrac-ion curves shown in experiments E1–E6. The value obtained for xu

s 0.031, constant for all of the experiments and, as expected, isbove every individual yield for SFE experiments. When the yieldsf experiments E3 and E10 are added together and this value trans-ormed into xu, this combined empirical value is 20% higher thanhe values we obtained. This higher value can be explained by theoextraction of heavier compounds occurring when high CO2 den-ities and ethanol as entrainer of the SFE are used. However, ouru value was not obtained empirically but was evaluated numeri-ally by entering it as adjustable in the Solver function taking intoccount that it should be the same for all the SFE experimentsecause the same raw material and particle size was used [42].ome authors defend that xu represents the maximum amountf extract that can be recovered from a raw material at a givenxtraction pressure and temperature [43]. Some authors even use

ifferent xu values at fixed pressure and temperature but vary-

ng particle sizes [44]. Some authors have fixed xu as the quantityhat can be obtained by hydrodistillation [45], or by conventionalxtraction using an organic solvent [46]. Other authors incorporateu as an input parameter with no mention of its origin [47]. Thus,

.80 0.52 12 0.924 0.0097 0.0064

.1 0.52 12 0.9479 0.013 0.003

our xu value is quite reasonable because it is above that found inevery SFE experiment and it would not have been appropriate toobtain it through HD or OSE due to the different composition andaspect of the obtained extracts.

The initial fluid phase concentration of the oil, yo, assumed tobe its solubility in supercritical CO2, follows the expected patternfor every experiment, i.e. the higher the CO2 density the moresoluble the oil becomes. Increasing oil solubilities in CO2 appearfor experiments E4 < E1 < E5 < E2 < E6 < E3 arranged in order of theirCO2 densities. The solvent-to-matrix ratio in bed, � , which dependson the solid density, solvent density and porosity, also increaseswith solvent density following the same pattern. Grinding effi-ciency, r, remains constant due to the equal grinding process, andthe fluid phase mass transfer coefficient, kfa0, remains constantdue to the constant CO2 flow. The solid phase mass transfer ratio,ksas, behaves also in the expected manner, increasing with thedensity, because the higher the density of the CO2 in the experi-ment, the better the oil transfer from the intact cells to the brokenones.

4. Conclusions

Supercritical CO2 extraction of wormwood was carried out andthe influence of pressure, temperature, crop year, presence ofentrainer and growing technique on the yield and composition ofthe extracts was studied. The best conditions between the stud-ied ones to obtain higher yields were 18.0 MPa and 40 ◦C, beingthe 2008 crop the one that produced the highest yield. SFE usingethanol as a modifier was performed to investigate the degree ofexhaustiveness of the plant material after a typical SFE run. Con-ventional extraction techniques, such as hydrodistillation for 4 hor organic solvent extraction for 20 h with either ethanol or hex-ane were performed for comparative purposes. Yields from the SFEprocess were higher than for the HD. OSE yields were higher thanboth the others but their extracts differed greatly in compositionand appearance from those produced by HD. Major compounds inHD essential oils and SFE extracts were Z-epoxyocimene, chrysan-thenol and chrysanthenyl acetate; their evolution during the SFEexperiments was screened chromatographically.

The model proposed by Sovová was successfully applied to cor-relate the experimental data. The characteristic parameters of themodel, such as the so-called oil solubility in supercritical CO2, y0,the solid phase mass transfer coefficient, ksas and the concentrationin the untreated solid, xu, were obtained.

Acknowledgements

Authors are grateful for financial support from MICINN-FEDER

(CTQ2009-14629-C02-02), from Gobierno de Aragón (PI068-08and group E52) and from Gobierno de Aragón-La Caixa-Sumalsa(Proyecto Medio Ambiente Convocatoria La Caixa 2010). S. Carlinis acknowledged for language revision. L. Martín is grateful for anFPU PhD grant (AP2006-02054) from the MICINN.

rcritica

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L. Martín et al. / J. of Supe

eferences

[1] A.P.T. Anastas, J.C. Warner, Green Chemistry: Theory and Practice, Oxford Uni-versity Press, USA, 2000.

[2] F. Juteau, I. Jerkovic, V. Masotti, M. Milos, J. Mastelic, J. Bessière, et al., Composi-tion and antimicrobial activity of the essential oil of Artemisia absinthium fromCroatia and France, Planta Medica (2003) 158–161.

[3] G. Rücker, D. Manns, S. Wilbert, Homoditerpene peroxides from Artemisiaabsinthium, Phytochemistry 31 (1992) 340–342.

[4] K. Tariq, M. Chishti, F. Ahmad, A. Shawl, Anthelmintic activity of extracts ofArtemisia absinthium against ovine nematodes, Veterinary Parasitology 160(2009) 83–88.

[5] A.H. Gilani, K.H. Janbaz, Preventive curative effects of Artemisia absinthium onacetaminophen and CCl4-induced hepatotoxicity, General Pharmacology 26(1995) 309–315.

[6] Papyrus Ebers 7 (2b, 7-2b, 11), Universitätsbibliothek Leipzig,available from: http://www.medizinische-papyri.de/PapyrusEbers/1024/HTML/kolumne ii.htm.

[7] S. Padosch, D. Lachenmeier, L. Kroner, Absinthism: a fictitious 19th centurysyndrome with present impact, Substance Abuse Treatment, Prevention, andPolicy 1 (2006) 14.

[8] National Toxicology Program of The National Institute of Environmen-tal Health Sciences of the United States, Alpha-Thujone (546-80-5),available from: http://ntp.niehs.nih.gov/index.cfm?objectid=03DB8C36-E7A1-9889-3BDF8436F2A8C51F.

[9] E. Stahl, D. Gerard, Hochdruck-Extraktion von Naturstoffen mit überkritis-chen und verflüssigten Gasen, Zeitschrift Für Lebensmitteluntersuchung Und-Forschung A 176 (1983) 1–4.

10] J. Burillo, Cultivo Experimental de Ajenjo Artemisia absinthium L. como PotencialInsecticida de Origen natural, in: J. Burillo, A. González-Coloma (Eds.), Insecti-cidas y Repelentes de Origen Natural, CITA, Zaragoza, 2009, pp. 19–30.

11] J. Labrador, J.L. Porcuna, A. Bello, Manual de Agricultura y Ganadería Ecológica,Eumedia, Madrid, 2006, p. 209.

12] U. Uslu, Ó. Ertuerk, Antifeedant, growth and toxic effects of some plant extractson Leptinotarsa decemlineata (say.) (Coleoptera: Chrysomelidae), FreseniusEnvironmental Bulletin 16 (2007) 601–607.

13] S. Kordali, I. Aslan, O. Calmasur, A. Cakir, Toxicity of essential oils isolated fromthree Artemisia species and some of their major components to granary wee-vil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), Industrial Crops andProducts 23 (2006) 162–170.

14] I. Parejo, F. Viladomat, J. Bastida, A. Rosas-Romero, N. Flerlage, J. Burillo, et al.,Comparison between the radical scavenging activity and antioxidant activity ofsix distilled and nondistilled Mediterranean herbs and aromatic plants, Journalof Agricultural and Food Chemistry 50 (2002) 6882–6890.

15] F. Chialva, P.A.P. Liddle, G. Doglia, Chemotaxonomy of wormwood (Artemisiaabsinthium L.), Zeitschrift Für Lebensmitteluntersuchung Und -Forschung A 176(1983) 363–366.

16] A. Orav, A. Raal, E. Arak, M. Müürisepp, T. Kailas, Composition of the essentialoil of Artemisia absinthium L. of different geographical origin, Proceedings ofthe Estonian Academy of Sciences Chemistry 55 (2006) 155–165.

17] A. Rezaeinodehi, S. Khangholi, Chemical composition of the essential oil ofArtemisia absinthium growing wild in Iran, Pakistan Journal of Biological Sci-ences 11 (2008) 946–949.

18] A.P. Carnat, M. Madesclaire, O. Chavignon, J.L. Lamaison, cis-Chrysanthenol, amain component in essential oil of Artemisia absinthium L. growing in Auvergne(Massif Central) France, Journal of Essential Oil Research 4 (1992) 487–490.

19] J.A. Pino, A. Rosado, V. Fuentes, Chemical composition of the essential oil ofArtemisia absinthium L. from Cuba, Journal of Essential Oil Research 9 (1997)87–89.

20] A. Basta, O. Tzakou, M. Couladis, M. Pavlovic, Chemical compostion of Artemisiaabsinthium L. from Greece, Journal of Essential Oil Research 19 (2007) 316–318.

21] A. Arino, I. Arberas, G. Renobales, S. Arriaga, J.B. Dominguez, Essential oil ofArtemisia absinthium L. from the Spanish Pyrenees, Journal of Essential OilResearch 11 (1999) 182–184.

22] L.M.D. Badillo, R.E.M. Munoz, R.S. Garciglia, M.M.M. Pacheco, In vitroantioomycete activity of Artemisia ludoviciana extracts against Phytophthoraspp, Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas9 (2010) 136–142.

23] H. Hendriks, R. Bos, H.J. Woerdenbag, The essential oil of Tanacetum parthenium(L.) Schultz-Bip., Flavour and Fragrance Journal 11 (1996) 367–371.

[

[

l Fluids 56 (2011) 64–71 71

24] Thermophysical Properties of Fluid Systems, National Institute of Standardsand Technologies, available from: http://webbook.nist.gov/chemistry/fluid/.

25] H. Sovová, Mathematical model for supercritical fluid extraction of naturalproducts and extraction curve evaluation, The Journal of Supercritical Fluids33 (2005) 35–52.

26] A. Gonzalez-Coloma, J. Burillo (CSIC and CITA), European Community PlantVariety Office Patent Application 20090972 (2009).

27] A. Gonzalez-Coloma, J. Burillo, J.S. Urieta, J. Sanz, C.E. Díaz, B.M. Fraga, M. Reina,R. Cabrera, R. Martínez, A. M. Mainar, Spanish Patent Application 201031389(2010).

28] Y. Xu, M.T. Marron, E. Seddon, S.P. McLaughlin, D.T. Ray, L. Whitesell, A.A.Gunatilaka, 2,3-Dihydrowithaferin A-3[beta]-O-sulfate, a new potential pro-drug of withaferin A from aeroponically grown Withania somnifera, Bioorganic& Medicinal Chemistry 17 (2009) 2210–2214.

29] A. Gonzalez-Coloma, M. Bailen, C.E. Díaz, B.M. Fraga, R. Martínez-Díaz, G.Zuniga, R.A. Contreras, J. Burillo, Chemical and biological (antifeedant andantioxidant effects) profiles of two chemotypes of Artemisia absinthium L. culti-vated under different environmental conditions, Industrial Crops and Products,submitted.

30] E. Langa, G.D. Porta, A. Palavra, J. Urieta, A. Mainar, Supercritical fluid extrac-tion of Spanish sage essential oil: optimization of the process parameters andmodelling, The Journal of Supercritical Fluids 49 (2009) 174–181.

31] E. Langa, J. Cacho, A. Palavra, J. Burillo, A. Mainar, J. Urieta, The evolution ofhyssop oil composition in the supercritical extraction curve: modelling of theoil extraction process, The Journal of Supercritical Fluids 49 (2009) 37–44.

32] E.M.C. Reis-Vasco, J.A.P. Coelho, A.M.F. Palavra, Comparison of pennyroyal oilsobtained by supercritical CO2 extraction and hydrodistillation, Flavour andFragrance Journal 14 (1999) 156–160.

33] E. Reverchon, G. Donsi, L. Sesti Osseo, Modeling of supercritical fluid extrac-tion from herbaceous matrices, Industrial & Engineering Chemistry Research32 (1993) 2721–2726.

34] H. van Den Dool, P. Dec Kratz, A generalization of the retention index systemincluding linear temperature programmed gas–liquid partition chromatogra-phy, Journal of Chromatography 11 (1963) 463–471.

35] M.A. Martínez, Memoria para la obtención del Diploma de Estudios Avanzados,Universidad de Zaragoza, Spain, 2005.

36] A. Álvarez, Memoria para la obtención del Diploma de Estudios Avanzados,Universidad de Zaragoza, Spain, 2005.

37] E. Langa, Extracción con CO2 Supercrítico de Aceites Esenciales de PlantasAromáticas, Tesis Doctoral, Universidad de Zaragoza, Spain, 2007.

38] Q. Lang, C.M. Wai, Supercritical fluid extraction in herbal and natural productstudies—a practical review, Talanta 53 (2001) 771–782.

39] D. Lopes-Lutz, D.S. Alviano, C.S. Alviano, P.P. Kolodziejczyk, Screening of chemi-cal composition, antimicrobial and antioxidant activities of Artemisia essentialoils, Phytochemistry 69 (2008) 1732–1738.

40] A. Arino, I. Arberas, G. Renobales, J.B. Domínguez, Influence of extractionmethod and storage conditions on the volatile oil of wormwood (Artemisiaabsinthium L.), European Food Research and Technology 209 (1999) 126–129.

41] R.L. Mendes, J.P. Coelho, H.L. Fernandes, I.J. Marrucho, J.M.S. Cabral, J.M. Novais,A.F. Palavra, Application of supercritical CO2 extraction to microalgae andplants, Journal of Chemical Technology and Biotechnology 62 (1995) 53–59.

42] A. Berna, A. Tárrega, M. Blasco, S. Subirats, Supercritical CO2 extraction ofessential oil from orange peel; effect of the height of the bed, The Journal ofSupercritical Fluids 18 (2000) 227–237.

43] S. Quispe-Condori, D. Sánchez, M.A. Foglio, P.T.V. Rosa, C. Zetzl, G. Brunner,M.A.A. Meireles, Global yield isotherms and kinetic of artemisinin extractionfrom Artemisia annua L leaves using supercritical carbon dioxide, The Journalof Supercritical Fluids 36 (2005) 40–48.

44] N. Mezzomo, J. Martínez, S.R. Ferreira, Supercritical fluid extraction of peach(Prunus persica) almond oil: kinetics mathematical modeling and scale-up, TheJournal of Supercritical Fluids 51 (2009) 10–16.

45] E.M.C. Reis-Vasco, J.A.P. Coelho, A.M.F. Palavra, C. Marrone, E. Reverchon, Math-ematical modelling and simulation of pennyroyal essential oil supercriticalextraction, Chemical Engineering Science 55 (2000) 2917–2922.

46] J. Stastová, J. Jez, M. Bártlová, H. Sovová, Rate of the vegetable oil extraction withsupercritical CO2: III. Extraction from sea buckthorn, Chemical EngineeringScience 51 (1996) 4347–4352.

47] Y. Gao, B. Nagy, X. Liu, B. Simándi, Q. Wang, Supercritical CO2 extraction of luteinesters from marigold (Tagetes erecta L.) enhanced by ultrasound, The Journal ofSupercritical Fluids 49 (2009) 345–350.