A

sdaa©

K

1

rupcvtMtbr

tTtls

0d

Available online at www.sciencedirect.com

J. of Supercritical Fluids 45 (2008) 37–42

Supercritical fluid extraction of bioactive compounds from sunflowerleaves with carbon dioxide and water on a pilot plant scale

L. Casas a,∗, C. Mantell a, M. Rodrı́guez a, A. Torres b,F.A. Macı́as b, E.J. Martı́nez de la Ossa a

a Department of Chemical Engineering, Food Technology and Environmental Technologies, Faculty of Science,University of Cadiz, Box 40, 11510 Puerto Real, Cadiz, Spain

b Department of Organic Chemistry, Faculty of Science, University of Cadiz,Box 40, 11510 Puerto Real, Cadiz, Spain

Received 14 June 2007; received in revised form 16 November 2007; accepted 7 December 2007

bstract

An analysis of the supercritical fluid extraction process on Helianthus annuus L (sunflower) using carbon dioxide and water on a pilot plantcale has been carried out. The possibility of using the resulting extract as an herbicide was analyzed by bioassays. The influence of time for

ifferent solvent flow rates was investigated. The extraction pressure and temperature were optimized in previous studies and a pressure of 380 barnd a temperature of 50 ◦C were selected. The bioactivities and extraction yields obtained under the different conditions were compared. The bestctivity profiles were obtained for 40 g/min CO2 + 1 g/min H2O and 80 g/min CO2 + 2 g/min H2O.

2008 Elsevier B.V. All rights reserved.

pound

cp

hcdaCais

ossu

eywords: Sunflower; Supercritical carbon dioxide; Extraction; Bioactive com

. Introduction

The traditional methods for the extraction of plant mate-ials include steam distillation and organic solvent extractionsing percolation, maceration or Soxhlet techniques. Theserocedures, however, have distinct drawbacks such as time-onsuming and labour-intensive operations, handling of largeolumes of hazardous solvents and extended concentration stepshat can result in the loss or degradation of target analytes.

oreover, there is increasing interest in alternative extractionechnologies that consume smaller quantities of organic solventecause of the rising solvent acquisition and disposal costs andegulatory restrictions [1].

Supercritical fluids have been shown to exhibit several advan-ages in the extraction of natural products from plant matrices.he combined liquid-like solvating capabilities and gas-like

ransport properties of supercritical fluids make them particu-arity suitable for the extraction of diffusion-controlled matricesuch as plant tissues. Moreover, the solvent strength of a super-

∗ Corresponding author. Tel.: +34 956 016579; fax: +34 956 016411.E-mail address: lourdes.casas@uca.es (L. Casas).

b(tcoT

896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.supflu.2007.12.002

s

ritical fluid can be easily tuned by simply changing the appliedressure and/or temperature.

Carbon dioxide, the most commonly used supercritical fluid,as the advantages of being non-flammable, fairly non-toxic,ost-effective and easily removed from the extract followingecompression. Finally, due to its relatively low critical temper-ture (31.1 ◦C), thermal sample decomposition is reduced. PureO2, however, is not an appropriate extraction fluid for polarnalytes and retentive matrices. In order to enhance the solvat-ng power of CO2, the addition of a small amount of a modifierolvent is required [2].

Supercritical fluid extraction (SFE) can be applied to systemsn a range of different scales, for instance from an analyticalcale (less than a gram to a few grams of sample) to pilot plantcale (several hundred grams or kilograms of sample) and evenp to large industrial scale (tons of raw material).

In previous studies we began to address the extraction ofioactive substances from sunflower leaves at the analytical scale10 ml extraction vessel). Studies were carried out on the pre-

reatment of samples [3] and the effect of the presence of aosolvent (water, methanol and dimethyl sulphoxide (DSMO))n both the yield and the bioactivity of the resulting extracts [4].he use of methanol, water and DMSO as modifiers increased

3 critical Fluids 45 (2008) 37–42

tootndbiw

t(cvflst3wt

2

2

21p(

ta

••

g

2

Tw

moeiAi

thsao

a

TR

R

CWACPSD

8 L. Casas et al. / J. of Super

he extraction efficiency of the process but the best yields werebtained using water as a modifier. Furthermore, the resultsbtained show that the properties under investigation (pressure,emperature and pre-treatment of the samples) do not have a sig-ificant influence on the bioactivity profiles of the products. Thery sample extracted at 500 bar with CO2 + 5% H2O gave theest yields and a satisfactory activity profile. It is therefore ofnterest to continue to develop the process using carbon dioxideith water as a cosolvent.The work described here involved the extraction of bioac-

ive compounds from sunflower leaves (Helianthus annuus L)both congealed samples and dried samples) with supercriticalarbon dioxide and water on a pilot plant scale (2 l extractionessel). The aim of the study was to assess the effect that theow rate, percentage of water and extraction time had on theupercritical extraction process. The extraction pressure andemperature were optimized in previous studies and values of80 bar (pressure) and 50 ◦C (temperature) [3] were selected,ith water chosen as the cosolvent [4]. The extraction yield and

he bioactivity of the extracts were analyzed.

. Materials and methods

.1. Samples and chemicals

Leaves of H. annuus L (variety Aitana) were collected in July005 during the third plant development stage [5] (plants were.2 m tall with flowers, 1 month before harvest) and plants wererovided by Rancho de Merced, Agricultural Research StationCIFA), Junta of Andalucı́a, Jerez, Spain.

The sample was stored under two sets of conditions in ordero evaluate the behaviour of each in terms of extraction yieldnd bioactivity of the extracts:

sample congealed at −25 ◦C,sample dried at room temperature (25 ◦C ± 1 ◦C) until a con-stant weight was reached.

The specifications of the other chemical reagents used areiven in Table 1.

.2. Extraction at high pressure

The extractions were carried out in a pilot plant from Tharechnology (Pittsburgh, PA, USA, model SF2000) providedith an extraction vessel (capacity of 2 l) and two pumps with a

oait

able 1eagents

eagent Purity

arbon dioxide 99.995%ater Milli Qcetone PAitric acid monohydrate PAotassium phosphate di-basic 3-hydrate PAucrose PAimethyl sulfoxide PA

Fig. 1. Schematic diagram of the equipment used for the SFE.

aximum flow rate of 150 g/min of carbon dioxide and 50 g/minf cosolvent. A thermostated jacket allowed control of thextraction temperature. The cyclonic separator allowed period-cal discharge of the extracted material during the SFE process.

schematic diagram of the SFE apparatus used in this researchs shown in Fig. 1.

The operating methodology involved loading the extrac-ion cartridge with approximately 190 g of the sample, whichad previously been homogenized in order to maintain a con-tant apparent density in all experiments. The valves MV-1nd MV-2 were closed and the valves MX-1 and MV-4 werepened.

When the heat exchanger (HE1) was at a temperature ofpproximately 1 ◦C the CO2 cylinder was opened. All of the

perating conditions apart from the flow of CO2 were establishednd when the system had stabilized the extractor was pressur-zed with CO2. When the pressure in the extractor was near tohe desired extraction pressure valve MV-2 was opened to start

Source Use

Carburos metalics Extraction at high pressureCosolvent

Panreac Collection of extractsPanreac Preparation of bufferPanreac Preparation of bufferPanreac Preparation of bufferPanreac Dissolution of the extracts

critic

tB

tcwa

i

pp648

2

ies

1goacaada

tadbbtw

wtof

d

2

paf

te

di

3

3

rtistio

ldflopC

pre-treatment. According to our experimental data (Fig. 2), thebest extraction yields were obtained from the dried samples.The moisture from the congealed samples seems to be a factorthat diminishes the extraction yield, with the water acting as a

L. Casas et al. / J. of Super

he flow of solvent. When a balanced state had been attained,PR1 was opened automatically and the process began.

The extracts were collected in S1 and acetone was addedo dissolve the extract. Valve MV-3 was subsequently used toollect the extracts in glass bottles and these were stored at 4 ◦Cith the exclusion of light. The acetone was later evaporated

nd the resulting extract weighed.The experiments on each sample were carried out in duplicate

n order to evaluate the variability of the measurements.The extraction pressure and temperature were optimized in

revious studies and values of 380 bar (pressure) and 50 ◦C (tem-erature) were used. The extraction times selected were: 30,0, 90, 120, 180 and 300 min at 25 g/min CO2 + 1 g/min H2O,0 g/min CO2 + 1 g/min H2O, 40 g/min CO2 + 2 g/min H2O,0 g/min CO2 + 2 g/min H2O and 80 g/min CO2 + 4 g/min H2O.

.3. Coleoptile bioassay

Bioassays constitute an important tool to evaluate the inhibit-ng or stimulating activity in terms of growth of the substancesxtracted according to the conditions described in the previousection.

Wheat seeds (Triticum aestivum L cv. Duro) were sown in5 cm diameter Petri dishes moistened with water and wererown in the dark at 22 ± 1 ◦C for 3 days. The roots and cary-pses were removed from the shoots. The latter were placed inguillotine and the apical 2 mm sections were cut off and dis-

arded. The next 4 mm lengths of the coleoptiles were removednd used for bioassays. All manipulations were performed undergreen safelight. Compounds were dissolved in DMSO and

iluted to the final bioassay concentration. Parallel controls werelso run [5].

A sample (16 mg) of each extract obtained under the condi-ions described in Section 2.2 was weighed out. The extracts to bessayed for biological activity were added to test tubes and wereissolved in 16 ml of an aqueous solution of phosphate/citrateuffer (pH 5.6) containing 2% sucrose. The extracts were insolu-le in water and DMSO (5 �l per ml of plug) was therefore addedo ensure total dissolution. Solutions of 500, 250 and 125 ppmere prepared in a similar way for each extract.Five coleoptiles were placed in each test tube and the samples

ere rotated at 6 rpm in a roller tube apparatus for 24 h at 22 ◦C inhe dark. The coleoptile lengths were measured by digitalizationf their images. Data are presented as percentage differencesrom the control.

Each assay was performed four times and on two differentays.

.4. Cluster analysis

Statistical treatments were performed using the SPSS 10.0rogram (Statistical Package for Social Sciences). Associationnalysis of the data based on the bioactivity profile was per-

ormed for each of the different sets of extraction conditions.

To further clarify the relationships between the clusters andhose individuals forming the clusters, a dendrogram was gen-rated by hierarchical cluster analysis; the squared Euclidean

al Fluids 45 (2008) 37–42 39

istance between normalised data was used to measure the sim-larity between samples.

. Results and discussion

.1. Extraction yields

Interesting extraction characteristics are often obtained onunning the extraction experiment at different flow rates. In ordero determine the controlling stage of the extraction process, its necessary to study the effect of the solvent flow rate. If theolvent flow rate is relatively low, the controlling stage will behe solubility of the solute in the solvent. If the solvent flow rates relatively high, then the controlling stage will be the diffusionf the solvent into the matrix.

The extraction yields expressed as mg of extract/100 g of dryeaves are shown in Fig. 2 for different extraction times and underifferent conditions in terms of pre-treatment of sample, solventow rates and solvent–CO2 mixtures. Each experimental pointn the extraction curve represents the averages of the two inde-endent experiments with a reproducibility of approximately 5%V (coefficient of variation).

It can be seen that the recovery is very dependent on the

Fig. 2. Extraction yields for different extraction times.

4 critical Fluids 45 (2008) 37–42

srpS

ttCHooy

edwt

atea

wssrasi8efl

idrtOtp

3

ssbvaa

femct

Ft

iem

gdwa7a

es1−(

concluded that variations in extraction times do not cause appre-ciable changes in the bioactivities of the samples at low flow rate.Furthermore, the congealed samples give rise to better activityprofiles than the dried samples. The most appropriate extraction

0 L. Casas et al. / J. of Super

olvent that competes with supercritical CO2. If excess wateremains in the extraction vessel, highly water-soluble solutesrefer to partition into the aqueous phase and, consequently, theFE recovery will be low.

It can also be seen that the recovery is very dependent onhe flow rates. The experimental results indicate a higher extrac-ion yield for the process with a solvent flow rate of 80 g/minO2 + 4 g/min H2O than that obtained at 25 g/min CO2 + 1 g/min2O. Generally, an increase in the flow rate leads to higher yieldsf extracts. This effect is due to the presence of larger amountf solvent in the operation, a factor that improves the extractionield.

The addition of modifiers to supercritical CO2 not onlynhanced the solubility of the analyte in the fluid but alsoecreased the interaction of the analyte and the matrix. In thisay, different extraction results can be obtained by manipulating

he ratios of liquid modifier.The experimental results obtained indicate that the most

ppropriate extraction time is approximately 120 min for all ofhe selected flow rates. Above this range the increase in thextraction yield is not significant and therefore there is littledvantage in prolonging the extraction.

The increase in the extraction yields for different flow ratesas more marked for the dried samples than for the congealed

amples and the best extraction yields were achieved using driedamples. For the congealed samples an increase in the flowates to 80 g/min CO2 and 2 or 4 g/min H2O did not lead ton improvement in the extraction process, despite the fact thatolvent consumption was doubled. On the basis of these results,t is not advisable to carry out the extraction at a flow rate of0 g/min CO2. However, it is worth noting that the increase inxtraction yield obtained with dried samples on increasing theow rate is much more marked than for congealed samples.

In both cases, on using flow rates of 40 and 80 g/min anncrease in the percentage of water present in the solvent systemid not give rise to significant increases in the yields. For thiseason it was appropriate to carry out general biological assayso select the best flow rates and extraction times in the process.ther aspects, such as economic considerations, must also be

aken into account when choosing appropriate parameters for arocess of this type.

.2. Bioassay

It was necessary to perform a general bioassay in order toelect the conditions (extraction times, solvent flow rates andolvent–CO2 mixtures) that provide the extracts with the bestioactivities. The aim of this study was not to determine specificalues, but to attempt to obtain activity profiles on the basis thatn extract will be more bioactive when its activity levels persists the sample is diluted.

The results of the bioactivity assays are shown in Figs. 3 and 4or congealed and dried samples, respectively. The data are

xpressed as percentage differences from the control, whicheans that a value of zero represents an identical value to the

ontrol. On the other hand, a positive value represents stimula-ion of the parameter in question and a negative value represents

Ft

ig. 3. Bioactivities of extracts obtained from congealed samples (extractionime/CO2 flow + H2O flow).

nhibition of the growth of the wheat coleoptiles under the givenxperimental conditions. The experimental error was approxi-ately 8.4%.It can be seen from Fig. 3 that the extracts obtained from con-

ealed samples at 25 g/min CO2 + 1 g/min H2O do not show anyifference in terms of the activity levels of the extracts obtainedith different extraction times; at 1000 ppm the extracts showed

n activity of around −63% and values near to −20% for the5-ppm dilution. According to the cluster analysis (Fig. 5) thesessays are united in a sub-group.

It can be seen from Fig. 4 that the activity profiles of thextracts obtained at 25 g/min CO2 + 1 g/min H2O from driedamples are also very similar for different extraction times; at000 ppm the extracts showed −63% activity and values near to10% for the 75 ppm dilution. According to the cluster analysis

Fig. 5) these assays also form a well-defined sub-group.On the basis of the results described above, it can be

ig. 4. Bioactivities of extracts obtained from dried samples (extractionime/CO2 flow + H2O flow).

L. Casas et al. / J. of Supercritic

Fs

tp

awaeHiHaitc

ippstsettd−1s

teoesebra

CcoCt

pcd

btNptoo––

4

t

•

•

•

ta

•

•

R

[1] Q. Lang, C.M. Wai, Supercritical fluid extraction in herbal and natural

ig. 5. Cluster analysis (where S is for dried sample and C is for congealedample).

ime can be defined according to the yields and not the activityrofiles.

In the case of the congealed samples extracted for 120 min,n increase in flow to 40 g/min CO2 with a constant flow rate ofater (1 g/min) improves the activity of the extract because the

ctivity level does not decrease drastically with dilution. How-ver, an increase in extraction times at 40 g/min CO2 + 1 g/min2O proved detrimental to the activity of the extract. The activ-

ty profiles of the extracts obtained at 40 g/min CO2 + 2 g/min2O from congealed samples are also proved detrimental to the

ctivity of the extract with an increase in extract time. Compar-son of the activity profiles of the extracts obtained with thesewo flow rates shows that an increase in the percentage of wateraused a decrease in the activity profile (Fig. 3).

It can be seen from Fig. 5 that for dried samples an increasen the percentage of water also leads to a decrease in the activityrofile of the extracts. At this point we must consider that studiesreviously [4] the flow of solvent was down and in the currenttudy did above. Because working conditions of water are denserhan that of carbon dioxide and that part of the bioactive sub-tances are soluble in water, they are left accumulated in thexhaust, thus decreasing the bioactivity profiles of the extractso increase percentage of water. At 40 g/min CO2 + 1 g/min H2Ohe extracts show a small decrease in activity on increasing theilution; 1000 ppm shows −100% activity and 125 ppm shows45% activity. Nevertheless, at 40 g/min CO2 + 2 g/min H2O,

000 ppm shows inhibition levels of −78% and this decreasedignificantly to −38% at a dilution of 500 ppm.

It can be seen from the cluster analysis in Fig. 5 thathere is a clear difference between the activity levels of thextracts obtained with different pre-treatment regimes and flowsf 40 g/min CO2 + 1 g/min H2O and CO2 + 2 g/min H2O. Thextracts obtained at 40 g/min CO2 + 1 g/min H2O from dryamples and those obtained from congealed samples with anxtraction time of 120 min and the same flow show the best

ioactivity profiles. These assays form a conglomerate that cor-esponds to the best activity profiles found in this study, i.e. onlysmall decrease in the activity on increasing the dilution.

[

al Fluids 45 (2008) 37–42 41

In the extraction of congealed samples conditions of 80 g/minO2 + 4 g/min H2O are extremely unfavourable to the pro-ess, as evidenced by the marked decrease in the activity leveln dilution. An increase in the extraction times at 80 g/minO2 + 2 g/min H2O and 80 g/min CO2 + 4 g/min H2O gave rise

o a marked decrease in the activity level on dilution.The cluster analysis shown in Fig. 5 indicates that dried sim-

les extracted at 80 g/min CO2 + 2 g/min H2O also formed aonglomerate with the best activity profiles, i.e. only a smallecrease in the activity on increasing the dilution.

When the process was performed with congealed samples,oth the extraction time and the percentage of water were foundo have a significant influence on the bioactivity of the extracts.evertheless, when the process was performed with dried sam-les the number of significant variables was reduced and onlyhe percentage of water is a significant factor for the bioactivityf extracts. Therefore, low water contents and extraction timesf 120 min are the best conditions to obtain the extracts becausedespite the fact that they do not give rise to the maximum yieldthese extracts do show the best bioactivity profiles.

. Conclusions

The study described above allows the following conclusionso be drawn concerning the yield of extract:

The extraction yields from congealed samples are low com-pared to those obtained from previously dried samples undera given set of conditions of flow rate, extraction time andpercentage of water.It is not advisable to work with a flow rate above 25 g/minCO2 in the case of congealed samples because the increasedsolvent consumption is not compensated by increases in theyields.From dried samples it is advisable to work at 80 g/min CO2on the basis that the extraction yields are higher. Neverthe-less, significant differences in the extraction yields were notdetected for different flow rates of water.

As far as the biological activity is concerned, the extractionimes and percentage of water present in the solvent system haven influence on the bioactivity of the extracts:

The best activity profiles are obtained with an extraction timeof 120 min.An increase in the percentage of cosolvent, at constant flowrates (40 g/min CO2 and 80 g/min CO2), proved detrimentalto the extraction process in terms of the bioactivity of theextracts.

eferences

product studies—a practical review, Talanta 53 (2001) 771–782.2] J. Liu, B. Han, G. Li, Z. Liu, J. He, G. Yang, Solubility of the non-ionic

surfactant tetraethylene glycol n-laurel ether in supercritical CO2 with n-pentanol, Fluid Phase Equilib. 15 (2001) 247–254.

4 critic

[

[

of bioactive compounds from Helianthus annuus L, J. Supercrit. Fluids 41

2 L. Casas et al. / J. of Super

3] L. Casas, C. Mantell, M. Rodrı́guez, M.D. Gordillo, A. Torres, F.A. Macı́as,

E. Martı́nez de la Ossa, Effect of the pre-treatment of the samples on thenatural substances extraction from Helianthus annuus L. using supercriticalcarbon dioxide, Talanta 67 (2005) 175–181.

4] L. Casas, C. Mantell, M. Rodrı́guez, A. Torres, F.A. Macı́as, E. Martı́nez de laOssa, Effect of the addition of cosolvent on the supercritical fluid extraction

[

al Fluids 45 (2008) 37–42

(2007) 43–49.5] F. Macı́as, A. Torres, T. Galindo, R. Varela, J. Alvarez, J.M.G. Molinillo,

Bioactive terpenoids from sunflower leaves cv Peredovick, Phytochemistry61 (2002) 687–693.