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Industrial Crops and Products 34 (2011) 1615– 1621

Contents lists available at ScienceDirect

Industrial Crops and Products

journa l h o me page: www.elsev ier .com/ locate / indcrop

omparative chemistry and insect antifeedant action of traditional (Clevengernd Soxhlet) and supercritical extracts (CO2) of two cultivated wormwoodArtemisia absinthium L.) populations

uis Martína, Luis F. Juliob, Jesus Burilloc, Jesus Sanzd, Ana M. Mainara, Azucena González-Colomab,∗

GATHERS, Aragon Institute for Engineering Research (I3A), Faculty of Sciences, Universidad de Zaragoza, SpainInstituto de Ciencias Agrarias, CSIC, Serrano 115-dpdo, 28006 Madrid, SpainCentro de Investigación y Tecnología Agroalimentaria, CITA, Avda. Montanana 930, 50059 Zaragoza, SpainInstituto de Química Orgánica General, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain

r t i c l e i n f o

rticle history:eceived 11 March 2011eceived in revised form 17 May 2011ccepted 3 June 2011vailable online 24 June 2011

a b s t r a c t

A comparison between traditional extraction techniques (hydrodistillation and organic solvent extrac-tion) and supercritical fluid extraction was made for two different populations and crops of Artemisiaabsinthium L., cultivated in the field and aeroponically. The composition of the extracts, volatile and nonvolatile oils, was analyzed by GC–MS and HPLC–DAD, respectively. The antifeedant and phytotoxic activ-ity of the extracts was tested on insect pests (Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi)

eywords:upercritical fluid extractionrtemisia absinthiumntifeedantydrodistillation and organic solvent

and plants (Lactuca sativa and Lolium perenne). The supercritical extracts exhibited stronger antifeedanteffects than the traditional ones (up to 8 times more active) with moderate selective phytotoxic effectson L. perenne root growth (<50% inhibition).

© 2011 Elsevier B.V. All rights reserved.

xtraction

. Introduction

The genus Artemisia consists of about 500 species distributedhroughout the world, Artemisia absinthium L. exhibiting aide range of biological activities including traditional medicine

Lachenmeier, 2010). The essential oils of A. absinthium haveeen reported as having antifungal (Juteau et al., 2003; Valdest al., 2008), free radical scavenger (Canadanovic-Brunet et al.,005), hepatoprotective (Gilani and Janbaz, 1995), antihelminticTariq et al., 2009), antiprotozoal and anti trichinellosis effectsCaner et al., 2008). Thujone-rich oils have been shown to havecaricidal (Chiasson et al., 2001) and insecticidal (Kordali et al.,006; Kaul et al., 1978) effects; myrtenol-rich oils repelled fleas,ies, mosquitoes (Erichsen-Brown, 1979) and ticks (Jaenson et al.,005). Additionally, organic extracts of this plant species exhibitedntifeedant and toxic effects on Leptinotarsa decemnlineata (Erturknd Uslu, 2007) and antifeedant effects on Rhopalosiphum padiHalbert et al., 2009). Moreover, water extracts were phytotoxic

o Lolium perenne and Bromus inermis (Corbu and Cachita-Cosma,009).

∗ Corresponding author.E-mail address: azu@ccma.csic.es (A. González-Coloma).

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.06.006

Among the major components reported in its essential oil are� and �-thujone (Carnat et al., 1992; Chialva et al., 1983), (Z)-epoxyocimene (Chialva et al., 1983), chrysanthenyl acetate (Chialvaet al., 1983), sabinyl acetate (Karp and Croteau, 1982) or a mix-ture of the latter ones (Carnat et al., 1992; Chialva et al., 1983),depending on the origin of the plant. The characteristic bitternessof wormwood is due to the presence of sesquiterpene lactones suchas absinthin, the main bitter constituent, anabsin, ketopelenolideb, and anabsinthin. Lignans, polyphenols and flavonoids are alsopresent in A. absinthium extracts (Aberham et al., 2010; Ivanescuet al., 2010).

A. absinthium is abundant in the mountains of Spain as a rud-eral species. There are seven chemotypes described in the IberianPeninsula according to the chemical composition of its essentialoil, some of which are thujone-free (Arino et al., 1999). Spanishpopulations of wormwood from Teruel (Aragón) and Sierra Nevada(Granada) have been experimentally cultivated (in the field andunder controlled conditions, Burillo, 2009; Gonzalez-Coloma et al.,submitted for publication). The chemistry and biological effects(insect antifeedant action and antioxidant effect) of the organic sol-vent (OSE) extracts of cultivated wormwood have been studied as

a function of the population and crop type over time (Gonzalez-Coloma et al., submitted for publication). The sesquiterpene lactonehydroxypelenolide was the major component followed by theflavones artemetin, and casticin. Plants grown under variable

1 s and Products 34 (2011) 1615– 1621

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Table 1Experimental conditions of the supercritical extraction experiments.

Experiment Origin Crop P (MPa) T (◦C) �sc (kg/m3) Flowrate(kg/h)

SCA Teruel 2005 9.0 40 485.5 1.08SCB 2005 13.5 40 753.6 1.08SCC 2005 18.0 40 819.5 1.08SCD 2005 + EtOH 18.0 40 819.5 0.81SCE 2006 18.0 40 819.5 1.08

616 L. Martín et al. / Industrial Crop

nvironmental conditions showed lower sesquiterpene:flavonoidatios than plants grown under controlled conditions. Casticin con-entrations correlated with the antifeedant and antioxidant effectsf wormwood extracts. (Gonzalez-Coloma et al., submitted forublication).

Supercritical fluid extraction (SFE) is an advanced separationethod that complies with the principles of Green Chemistry and

as advantages when extracting bioactive compounds from a nat-ral matrix (Reverchon and De Marco, 2006). Particularly, these of CO2 as a supercritical (SC) solvent, allows for mild extrac-ion conditions due to the low critical temperature (31 ◦C) andressure (7.1 MPa) of the solvent, thus circumventing the ther-al degradation of the extracted compounds (Pourmortazavi andajimirsadeghi, 2007).

Supercritical extraction of wormwood with carbon dioxideas been previously applied for the quantitative removal of thu-

one (Stahl and Gerard, 1983). Furthermore, the A. absinthiumopulations used in this study of have been submitted to SFEsing different extraction pressures and temperatures. The anal-sis of the hexane-soluble fraction of the SFE and hydrodistilledxtracts showed the absence of thujone and the presence of (Z)-poxyocimene, chrysanthenol and chrysanthenyl acetate as theajor compounds (Martín et al., 2011).As part of our ongoing search for improved botanical biopes-

icides the chemical composition of the dichloromethane andethanol soluble fraction of wormwood HD, OSE and SFE extracts

as been analyzed in this study using GC–MS and HPLC–DADlong with their antifeedant effects against a panel of insect pestsSpodoptera littoralis, Myzus persicae and R. padi) and their phyto-oxicity against L. sativa and L. perenne.

. Materials and methods

.1. Plant material and cultivation

Plant material for field cultivation was selected from wild pop-lations growing in Teruel, 2001 (Spain). The individuals for fieldultivation were obtained from seeds. The experimental fields wereocated in Barrio de San Blas (Teruel) and Ejea de los CaballerosZaragoza). A detailed description of these fields and the cultivationarameters has already been published (Burillo, 2009). Floweringlant samples from 30 randomly selected plants were collected andried in the shade for 8 days.

The individuals aeroponically produced were obtained from aopulation donated by the nursery at the Sierra Nevada Nationalark (2003, Granada). Cuttings of selected individuals were rootedith IAA in vermiculite, watered 3 days/week with a nutrient

olution (Nutrichem 20:20:20 N, P, K – Miller Chemical & Fertl-zer Corp.; 3 g/l) and kept in a growth chamber (25 ◦C, 70% rh,6:8, L:O) until their transfer to the aeroponic chamber (10–15 cmlants). The aeroponic chamber (Apollo 3 system: 33 plants, 240

, 1750 mm × 1350 mm × 750 mm) was located in an environmen-ally controlled greenhouse (20–30 ◦C). The plants were kept underonstant pulverization with water at 26 ◦C supplemented with.2 g/l Nutrichem and 0.03% H2O2 (33% w/v Panreac) and artificial

ight (16:8, L:D). The plants were grown for 9 months, their aerialart and roots collected periodically (from 20 to 30 cm plants), dried35 ◦C) and grounded for extract preparation (leaves and roots).

.2. SFE, HD and OSE

The SFE plant was built according to Reis-Vasco et al. (1999)nd Langa et al. (2009). The main components are a compressionump, filter, 1 L extraction vessel and two separators (0.18 L each),ne for the recovery of so-called waxes (heavy compounds) and the

SCF SierraNevada

Aeroponic 18.0 40 819.5 1.08

other for the recovery of volatile oil (Reverchon et al., 1993). Tem-peratures in the extraction vessel and separators were maintainedconstant. Approximately 90 g of grounded wormwood was intro-duced in the extraction vessel with several porous inert materials(glass spheres, nickel sponges and glass frits) to achieve a uniformflux of the supercritical fluid. All the SFE experiments were termi-nated after 11 h of extraction or when the slope of the extractioncurve was lower than 10% of the maximum slope in the initial stepsof the experiment. The extract collected in separator 1 was gatheredat the end of the experiment, while several fractions were collectedfrom separator 2 during the experiment.

In order to determine the thoroughness of the matrix, an extraSFE experiment was performed with previously extracted plantmaterial (18.0 MPa at 40 ◦C) with the addition of 50 ml of EtOHas a co-solvent as described by (Martín et al., 2011). Representa-tive samples of different SFE experiments were selected to analyzeboth composition and activity. The experimental conditions of theselected experiments are shown in Table 1.

HD was performed on 100 g plant samples in a Clevenger-typeapparatus according to the method recommended by the EuropeanPharmacopoeia (http://www.edqm.eu/en/Homepage-628.html).

OSE was performed in a Soxhlet apparatus with ethanol andconcentrated in vacuo.

2.3. GC–MS analysis

The essential oils were analyzed by GC–MS using an Agilent6890N gas chromatograph (Agilent Technologies, Palo Alto, CA,USA) coupled to an Agilent 5973N mass detector (electron ioniza-tion, 70 eV) (Agilent Technologies, Palo Alto, CA, USA) and equippedwith a 25 mm × 0.20 mm i.d. capillary column (0.2 �m film thick-ness) HP-1 (methyl silicone bonded) (Hewlett-Packard). Workingconditions were as follows: split ratio (30:1), injector temperature,260 ◦C; temperature of the transfer line connected to the mass spec-trometer, 280 ◦C; column temperature 70 ◦C for 5 min, then heatedto 270 ◦C at 4 ◦C min−1. EI mass spectra and retention data wereused to assess the identity of compounds by comparing them withthose of standards or found in the Wiley Mass Spectral Database(2001). Quantitative data were obtained from the TIC peak areaswithout the use of response factors.

2.4. HPLC–DAD analysis

Quantification of the three major components of A. absinthium,the sesquiterpene lactone hydroxypelenolide (I) and the flavonesartemetin (II) and casticin (III) (Fig. 1), was carried out in anShimadzu-LC 20AD HPLC equipped with a diode-array detector(SPD-M20A) and a Waters Novapack-C18 column (15 cm × 3.9 cmand 4 �m particle size). The compounds were eluted with an

Acn:1% H3PO4 25:100% gradient for 20 min and 100% Acn for 15 minat 1 mL/min flow rate and a column temperature of 25 ◦C. Thecompounds were detected at 340 nm (artemetin and casticin) and204 nm (hydroxypelenolide). Three calibration curves were built

L. Martín et al. / Industrial Crops and

OH3CO

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ig. 1. Chemical structure of compounds I–III. I, Hydroxypelenolide; II, artemetin;II, casticin.

ith known concentrations (1.0–0.001 mg/mL) of compounds I–IIIor their quantification. The calculated limits of detection (LOD) anduantification (LOQ) were 24.7, 1.8 and 2.8 ng/ml (LOD) and 301.8,4.5 and 37.5 ng/ml (LOQ) for I–III, respectively.

.5. Insect bioassays

S. littoralis, M. persicae and R. padi colonies were reared on arti-cial diet, bell pepper (Capsicum annuum) and barley (Hordeumulgare) plants, respectively, and maintained at 22 ± 1 ◦C, >70% rel-

tive humidity with a photoperiod of 16:8 h (L:D) in a growthhamber. The bioassays were conducted with newly emerged S.ittoralis L6 larvae or ten M. persicae/R. padi adults as describedBurgueno-Tapia et al., 2008).

able 2ompounds identified by GC–MS in the extracts.

M+ Base peak Main fragments IK Compou

116 43 59-58-101 815 4-Hydro136 71 93-41-43-55-69 1083 Linaloola

152 79 81-77-41-39-53 1111 (Z)-Epox152 81 91-109-41-79-39 1144 Chrysant170 43 55-59-71-84-82 1220 Dimethy170 43 55-71-59-84-81 1232 Dimethy170 43 119-43-71-91-109 1263 Dimethy170 43 55-74-59-84-81 1266 C10H18O194 59 43-81-67-82-79 1242 trans-Ch

204 93 133-91-79-69-41 1409 trans-Car204 93 41-79-91-105-107 1473 �-Seline220 41 79-93-43-69-91 1559 Caryoph

? 69 41-57-93-68-121 1579 Unidenti222 43 41-161-67-81-204 1630 Sesquite

? 96 41-55-43-81-107 1638 Unidenti248 215 43-159-230-119-91-248 1997 Absilacto250 232 43-217-162-150-77 1927 Hydroxy

268 43 58-71-41-57-59 1828 6,10,14-T284 119 132-145-41-69-105 1934 Diterpen284 119 132-145-105-41-123 1986 Diterpen286 119 121-93-105-41-91 1989 Diterpen284 119 132-145-105-91-41 1993 Diterpen

a Wiley database.b Bailen (2008).c Arino et al. (1999).

Products 34 (2011) 1615– 1621 1617

2.6. Phytotoxic activity

The experiments were conducted with Lactuca sativa cv Teresa(Fito, Espana), and L. perenne seeds. 2.5 cm diameter filter paperwith 20 �L of the test compound (10 �g/�L for extracts and 5 �g/�Lfor pure compounds) were placed on 12-well plates (Falcon).500 �L H2O/well and 10/5 seeds (L. sativa/L. perenne pre-soakedin distilled water for 12 h) were added and the covered platesplaced in a plant growth chamber (25 ◦C, 70% RH, 16:8 L:D). Ger-mination was monitored for 6 days and the rootlet/leaf lengthmeasured at the end of the experiment (25 plantlets randomlyselected for each experiment and digitalized with the applicationImageJ 1.43, http://rsb.info.nih.gov./ij/). A non parametric analysisof variance (ANOVA) was performed on radical length data. Juglone(JU) (5 �g/�L) was included as a positive control.

3. Results and discussion

3.1. Composition evaluation

Table 2 shows the compounds identified from thedichloromethane soluble fraction of different extracts and Table 3shows their composition. The composition of the HD extracts wasincluded for comparative purposes.

The HD extracts from A. absinthium Teruel (HD05-06) werecharacterized by the presence of the monoterpene dimethyl-octadienol (7), previously isolated from the same population(Bailen, 2008; supporting information), chrysanthenol (4, HD05-06), an absilactone-type sesquiterpene (16, HD05) (Bailen, 2008,supporting information), two unknown diterpenes (22 and 20,HD05, Arino et al., 1999), a hydroxypelenolide isomer (17, HD05-06) also isolated from A. absinthium (González Coloma et al.,submitted, supporting information), caryophyllene oxide (12,HD05-06), t-chrysanthenil acetate (9, HD05-06), and a sesquiter-pene alcohol (14, HD05-06, Arino et al., 1999). All these compounds

were found in the HD extracts of the same population culti-vated in previous years (2002–2004) (Bailen, 2008). The HDAextract from the aeroponic plants (SN population) was character-ized by (Z)-epoxyocimene (3) and chrysanthenol (4).The evolution

nd Number Group

xy-4-methyl-2-pentanonea 1 A Lower terpenes2

yocimenea 3henola 4l-octadienol C10H18O2 isomerb 5l-octadienol C10H18O2 isomerb 6l-octadienol C10H18O2 isomerb 72 isomer 8rysanthenyl acetatea 9

yophyllenea 10 B Sesquiterpenesnea 11yllene oxidea 12fied 13rpene alcohol C10H18Oc 14fied 15ne type sesquiterpeneb 16pelenolide isomerb 17

rimethyl-2-pentadecanonea 18 CKetones and diterpenese C20H28Oc 19e C20H28Oc 20e C20H30Oc 21e C20H28Oc 22

1618L.

Martín

et al.

/ Industrial

Crops and

Products 34 (2011) 1615– 1621

Table 3Composition of the different extracts analyzed by GC–MS.

Abundance (% area)

Compound Teruel Sierra Nevada

05HD SCA SCB SCC SCD HD06 SCE HDA SCF

H T H T W H1 H2 H T W H T W H T W

1 – – 1.62 – – – – – – – – – – – – – – –2 – – 1.40 0.37 – – – – – – 1.60 – – – 0.44 – – –3 1.07 1.44 15.10 5.92 4.07 4.75 – – – – 1.96 – 2.18 – 75.10 0.96 8.38 –4 9.31 9.44 13.94 4.88 11.90 7.58 2.79 3.38 3.66 8.92 – 19.34 6.89 1.33 5.81 10.56 5.29 5.71 –5 – 1.95 – 0.85 – 1.21 – – – – – – – – 2.53 0.81 1.02 –6 – 2.60 – 1.06 – 1.54 – – – – – – – – – 1.03 1.38 –7 30.03 3.55 2.78 1.75 2.82 2.90 1.64 2.33 – – 2.71 54.57 – 1.10 – 0.95 2.08 2.36 –8 – 14.97 16.94 9.77 16.74 12.88 7.66 10.49 7.85 13.36 – – 13.55 3.95 12.13 – 11.37 13.09 4.179 2.37 10.99 12.50 5.02 5.72 5.42 1.67 – – – 4.38 – 3.92 – – 1.97 3.09 –

10 – 3.22 2.73 1.48 – 1.63 – – – – – – – – 1.76 1.12 2.03 –11 – 7.37 6.35 3.21 3.06 3.58 1.92 2.22 – – – – – – – 0.70 1.69 2.06 –12 2.60 7.38 4.82 4.97 4.74 5.70 4.63 5.70 – – – 5.41 6.65 2.84 – 0.67 4.80 5.25 –13 – 2.36 0.58 1.52 – 1.61 – – – – – – – – 0.34 – – –14 2.67 4.70 4.46 4.62 4.41 4.60 4.70 6.23 4.87 5.57 2.67 4.57 6.61 3.21 – 0.87 5.10 4.85 3.6215 – 1.60 – 1.58 – 1.49 1.72 – – – – – 1.20 – – 1.54 1.33 –16 17.72 – 5.82 11.04 11.11 12.29 24.40 24.40 32.07 28.94 39.72 – 21.90 38.77 29.57 – 8.89 15.74 34.0717 6.21 1.71 – 3.60 4.51 3.62 7.28 7.23 10.42 9.10 11.56 5.02 8.14 7.44 11.54 – 2.68 2.20 9.7418 2.22 1.21 – 1.69 – 1.48 2.58 2.85 3.36 – 2.70 3.29 1.71 – – 1.34 0.98 2.8419 – – – 1.14 – 1.06 1.59 1.62 – – – – – – 13.83 – – – –20 7.53 4.24 2.86 6.39 – 5.85 9.50 10.25 12.14 11.13 8.32 – 11.22 5.43 18.59 – – – 10.4621 – – – 1.70 7.26 1.58 2.49 2.84 – – – – – 3.28 – – 5.94 3.99 2.6322 10.62 5.17 1.62 8.70 – 8.47 13.60 14.51 17.15 15.75 13.01 – 15.43 8.11 – – 4.70 6.61 15.02

L. Martín et al. / Industrial Crops and Products 34 (2011) 1615– 1621 1619

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f the composition of the SC extracts during the extraction processhowed that different combinations of pressure and temperatureffected the extract composition. For the 2005 crop, the increase inressure (from SCA to SCC) resulted in decreased extraction levelsor monoterpenes (up to compound 9) and an increased extractionf the sesquiterpene hydroxypelenolide isomer (17) and the heav-er compounds (18–22). The addition of EtOH (SCD) resulted in a

ore efficient extraction of heavier compounds (17–22). The headractions, H (collected from separator 2 at the initial stages of thextraction) and the tail fractions, T (collected from separator 2 at thenal stages from the extraction) were richer in volatile compoundshan the so-called waxes, W fractions (collected in separator 1 alonghe whole extraction).

The SCA extracts were composed mostly of group A compoundslower terpenes,) with (Z)-epoxyocimene (3), chrysanthenol (4),

and trans-chrysanthenyl acetate (9) being the major ones,heir group B compounds (sesquiterpenes) being dominated by-selinene (11) and caryophyllene oxide1 (12) followed by theesquiterpene alcohol (14) and the sesquiterpene 16 (in fraction) while their group C (ketones and diterpenes) had low amountsf diterpenes 20 and 22 in fraction H. The SCB extracts had a similaromposition in group A compounds with 8 and 4 being the majornes; their group B compounds consisted of 16 (the major one),2 and 14, 11, hydroxypelenolide 17 and small amounts of trans-aryophyllene (10) and their group C showed higher amounts of0 and 22 with the presence of 21 in fraction T. The SCC extractsere quite similar in composition since H1 and H2 are consecutive

xtraction fractions (T was not available), and they had a simpleromposition in group A compounds with 8 being the major one;heir group B composition had 16 as the major component followedy 17, 14 and 12, while their group C was similar to that of SCB-With higher amounts of 20 and 22. The SCD extracts had a simpler

roup A (8 and 4) and B (16, 17 and 14) compositions than SCC withigher concentrations of 16, 17, 20 and 22 than the previous super-ritical extracts. The SCE extracts had a similar composition to SCDn group A compounds with the addition of 9; in group B with theddition of 12 and in group C with the addition of 19. SCF extractsad a similar composition in group A and B compounds to SCB and

different composition in group C compounds with the presencef 21 in the W fraction.

.2. Quantification of compounds I–III

Fig. 2 shows the quantification of compounds I–III in the dif-

erent extracts. Overall, artemetin II was the most abundant inhe OS extracts. As previously shown, plants grown under fieldonditions had higher concentrations of I–III than plants grownnder controlled conditions (OSEA). hydroxypelenolide I was the

ds I–III in different extracts.

most abundant in SCB-H, therefore the SCB experiment had thebest conditions to selectively extract sesquiterpene I. Overall, theconcentrations of these compounds increased during the extrac-tion process. However, none of the SC experiments was effective inselectively extracting flavones artemetin (II) and casticin (III).

These flavones are antioxidant components of A. absinthium(Gonzalez-Coloma et al., submitted for publication) and their syn-thesis may be induced by abiotic stress. Compound II has beendescribed as an antioxidant that neutralizes peroxide radicals(Dugas Jr. et al., 2000), while a recent study reported weak radicalscavenging activity for III while II was inactive (Azizuddin et al.,2010). However, we found similar weak antioxidant effects forthese flavonoids and stronger ones for the sesquiterpene lactoneI (Gonzalez-Coloma et al., submitted for publication).

3.3. Biological activities

3.3.1. Antifeedant effectsThe supercritical extracts are considerably more active against

S. littoralis than the traditional ones (Table 4). Previous resultshave shown that HD and OSE extracts from A. absinthium (Teruelpopulation) showed moderate-low antifeedant effects on theseinsect species (Bailen, 2008; Gonzalez-Coloma et al., submitted forpublication; supporting information).

Almost all of the supercritical extracts exhibited an antifeedantactivity of higher than 80%. A similar pattern was found for theeffects of these extracts on M. persicae and R. padi, aphids being lesssensitive than S. littoralis. The SCA and SCC extracts were the mostactive against both aphids, while SCF from the aeroponic plants hada selective effect on R. padi. Overall, the H and T fractions were moreactive than the W one for experiments SCC–SCF with the exceptionof SCE (the most active), while experiments SCA and SCB did not fol-low this pattern. We cannot correlate the antifeedant effects of theSC extracts with the presence of any given compound. Therefore, asynergistic effect of these mixtures is suggested.

3.3.2. Phytotoxic bioassaysTable 5 shows the phytotoxic effects of the HD, OSE and super-

critical extracts on L. perenne and L. sativa. Overall, the effects weremoderate and selective against the monocotyledonous weed L.perenne.

L. perenne root growth was selectively affected by the SCextracts, while leaf growth was not significantly altered. Someextracts, however, namely SCB-T, OSEA and SCF-H, affected both

root and leaf growth.

L. sativa germination at 24 h was affected by most treatments.From 48 h to the end of the experiment, the germination was notsignificantly affected by any extract. The traditional extracts did not

1620 L. Martín et al. / Industrial Crops and Products 34 (2011) 1615– 1621

Table 4Antifeedant effects of the different A. absinthium extracts on the target insect species.

Extract S. littoralis (100 �g/cm2) M. persicae (100 �g/cm2) R. padi (100 �g/cm2)

FI%b SI%c %Cd %Tew SI%c %Cd %Te

HD05a 19.8 ± 16.7 30.8 ± 6.8 53 ± 5 47 ± 5 34.7 ± 7.9 40 ± 5 60 ± 5OSE05a 21.3 ± 9.8 16.5 ± 6.3 54 ± 5 46 ± 5 30.7 ± 7.6 59 ± 4 41 ± 4SCA-H 82.8 ± 4.7* 79.5 ± 6.1* 84 ± 4 16 ± 4 81.7 ± 4.0* 86 ± 3 14 ± 3SCA-T 93.7 ± 4.0* 63.4 ± 7.2* 75 ± 4 25 ± 4 90.6 ± 2.0* 92 ± 2 8 ± 2SCA-W 92.0 ± 7.2* 48.1 ± 11.6 67 ± 7 33 ± 7 29.8 ± 6.0 58 ± 3 42 ± 3SCB-H 93.5 ± 3.7* 46.0 ± 6.7 66 ± 4 34 ± 4 55.4 ± 6.0 70 ± 3 30 ± 3SCB-T 85.6 ± 10.0* 59.6 ± 7.7* 75 ± 4 25 ± 4 40.2 ± 7.2 61 ± 4 39 ± 4SCB-W 86.4 ± 8.4* – – – 28.1 ± 7.6 55 ± 5 45 ± 5SCC-H1 88.6 ± 4.9* 96.7 ± 3.3* 97 ± 3 3 ± 3 83.1 ± 6.4* 87 ± 5 13 ± 5SCC-H2 64.7 ± 9.3 94.3 ± 2.2 95 ± 2 5 ± 2 58.9 ± 7.1 73 ± 4 27 ± 4SCD-H 90.9 ± 8.4* 83.1 ± 4.3* 87 ± 3 13 ± 3 68.8 ± 5.8* 78 ± 3 22 ± 3SCD-T 85.7 ± 8.7* 78.5 ± 5.2* 84 ± 3 16 ± 3 68.0 ± 4.9* 77 ± 2 23 ± 2SCD-W 84.2 ± 9.3* 72.4 ± 3.7* 80 ± 2 20 ± 2 34.8 ± 6.8 61 ± 3 39 ± 3

HD06a 21 ± 4.7 34.5 ± 6.2 60 ± 3 40 ± 3 40.2 ± 7.3 63 ± 4 37 ± 4OSE06a 6.4 ± 6.4 35.9 ± 9.2 52 ± 7 48 ± 7 42.6 ± 9.0 56 ± 8 44 ± 8SCE-H 94.9 ± 3.1* 64.5 ± 5.8* 76 ± 3 24 ± 3 91.6 ± 4.5* 94 ± 3 6 ± 3SCE-T 96.2 ± 2.8* 60.6 ± 6.4* 74 ± 3 26 ± 3 69.9 ± 4.4* 78 ± 2 22 ± 2SCE-W 97.7 ± 1.1* 31.2 ± 8.1 55 ± 5 45 ± 5 – – –

HDA 69.9 ± 6.3* 32.0 ± 9.1 55 ± 6 44 ± 6 54.6 ± 6.0 71 ± 3 29 ± 3OSEAa 52.4 ± 3.3 38.1 ± 7.8 40 ± 5 60 ± 5 72.2 ± 7.5* 80 ± 5 20 ± 5SCF-H 96.4 ± 1.7* 76.4 ± 5.6 84 ± 3 16 ± 3 99.4 ± 0.6* 99 ± 1 1 ± 1SCF-T 69.7 ± 9.1* 72.3 ± 5.9* 81 ± 3 19 ± 3 87.2 ± 3.1* 90 ± 2 10 ± 2SCF-W 49.8 ± 14.4 59.2 ± 8.8* 73 ± 5 27 ± 5 82.9 ± 3.9* 87 ± 3 13 ± 3

SC: supercritical extract, conditions shown in Table 1. H refers to head extract, T to tail extract, W to waxes. HD: hydrodistillation. OSE: organic solvent extract. A: aeroponicgrowth. 2005, 2006, 2008: different crops.

a From Bailen (2008).b FI%: feeding inhibition index: [1 − (treatment consumption/control consumption)] × 100.c SI%: settling inhibition index: [1 − (treatment/control)] × 100.d % C: percentage of individuals remaining on the control surface.e % T: percentage of individuals remaining on the treated surface.* P < 0.05, Wilcoxon Paired Rank Test.

Table 5Phytotoxic effects of the A. absinthium extracts on L. perenne and L. sativa.

Lolium perenne Lactuca sativa

Extract (100 �g/cm2) Growth (%C) Germination (%C) Growth (%C)

Radicular Leaf 24 h 48 h Radicular

HD05a 73 ± 0.19* 98 ± 0.13 51 ± 0.58 98 ± 0.40 111 ± 0.11OSE05a 68 ± 0.20* 91 ± 0.18 100 ± 0 100 ± 0 108 ± 0.09SCA-H – – 30.0 ± 4.1* 87.5 ± 2.5 70 ± 0.07*

SCA-T – – 15.0 ± 5.0* 97.5 ± 2.5 106 ± 0.1SCA-W 88 ± 0.23 99 ± 0.16 30.0 ± 4.1* 97.5 ± 2.5 123 ± 0.13SCB-H 60 ± 0.22* 80 ± 0.22 20.0 ± 4.1* 90.0 ± 4.1 89 ± 0.10SCB-T 44 ± 0.18* 61 ± 0.20* 15.0 ± 5.0* 85 ± 6.45 78 ± 0.08*

SCB-W 78 ± 0.13 97 ± 0.12 12.5 ± 4.8* 92.5 ± 4.8 97 ± 0.15SCC-1 67 ± 0.16* 81 ± 0.13 – – –SCC-2 85.2 ± 0.21 95.4 ± 0.16 – – –SCD-H 80 ± 0.16 78 ± 0.17 52.5 ± 4.8* 100 ± 0.0 97 ± 0.13SCD-T 88 ± 0.22 99 ± 0.20 46.3 ± 13.8* 100 ± 0.0 109 ± 0.15SCD-W – – 60.0 ± 4.1* 100 ± 0.0 116 ± 0.14

HD06a 67 ± 0.16* 106 ± 0.16 43 ± 1.47 100 ± 0.0 97.6 ± 0.07OSE06a 76 ± 0.21* 87 ± 0.18 102 ± 2.1 100 ± 0 87 ± 1.9SCE-H 84 ± 0.18 85 ± 015 85.0 ± 5.0 100 ± 0.0 76 ± 0.09*

SCE-T 72 ± 0.22* 72 ± 024* 87.5 ± 2.5 97.5 ± 2.5 101 ± 0.14SCE-W – – 85.0 ± 6.5 100 ± 0.0 115 ± 0.10

HDA 84 ± 0.24 113 ± 0.19 – – –OSEAa 41 ± 0.13* 35 ± 0.56* 98 ± 1.2 100 ± 0 83 ± 3.6SCF-H 64 ± 0.21* 72 ± 0.17* 45.0 ± 2.9* 100 ± 0.0 72 ± 0.11*

SCF-T 75 ± 0.24* 84 ± 0.18 45.0 ± 6.5* 100 ± 0.0 120 ± 0.17SCF-W 83 ± 0.23 88 ± 0.16 60 ± 4.1* 100 ± 0.0 115 ± 0.18

%C: percentage of control. SC: supercritical extract, see Table 1. H head extract, T tail extract, W waxes. HD: hydrodistillation. OSE: organic solvent extract. A: aeroponicgrowth. 2005,2006, 2008: different crops.

a Bailen (2008).* P < 0.05, Mann Whitney test.

s and

ae

4

ttsFtaeLesommpce

A

1(SgMSt

A

t

R

A

A

A

B

B

B

C

C

L. Martín et al. / Industrial Crop

ffect L. sativa root growth, while some of the SC had a moderateffect (SCA-H, SCB-T, SCE-H and SCF-H).

. Conclusions

Supercritical fluid extraction, compared to traditional extractionechniques, improved the yield of (Z)-epoxyocimene (3), chrysan-henol1(4), dimethyl-octadienol (8), trans-chrysanthenyl acetate 9,esquiterpene 16, diterpenes 20 and 22 and hydroxypelenolide I.urthermore, supercritical extracts were more active than tradi-ional ones against S. littoralis and both aphid species, reachingntifeedant values of over 90%. Their phytotoxic effects were mod-rate (<50%) and selective towards the monocotyledonous weed. perenne. We cannot correlate the antifeedant effects of the SCxtracts with the presence of any given compound. Therefore, aynergistic effect of these mixtures is suggested. The developmentf extraction processes with supercritical CO2 allows for the enrich-ent of desired bioactive compounds in A. absinthium extracts byeans of an environmentally friendly technique regardless of the

lant origin since both populations (Teruel and Sierra Nevada),ultivated under different conditions, produced more active SCxtracts than the traditional ones.

cknowledgements

This work has been supported by MICINN-FEDER (CTQ2009-4629-C02-01 and CTQ2009-14629-C02-02), Gobierno de AragónPI068-08 and group E52) and Gobierno de Aragón-La Caixa-umalsa (Proyecto Medio Ambiente Convocatoria La Caixa 2010)rants. L. Martín and L.F. Julio gratefully acknowledge their FPU-ICINN (AP2006-02054) and JAE-CSIC predoctoral fellowships. L.

antos and I. Calvillo are acknowledged for their technical assis-ance and S. Carlin for language revision.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.indcrop.2011.06.006.

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