Analytica Chimica Acta 707 (2011) 6975

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Analytica Chimica Acta

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Ionic liquids based microwave-assisted extraction of lichen compounds with

quantitative spectrophotodensitometry analysis
Sarah Bonny a , Ludovic Paquin b , Daniel Carri b , Jol Boustie a , Sophie Tomasi a,
a
Equipe PNSCM Produits naturels Synthses Chimie Mdicinale, UMR CNRS 6226 Sciences chimiques de Rennes, UFR Sciences Pharmaceutiques et Biologiques, Univ. Rennes 1,
Universit Europenne de Bretagne, 2 Avenue du Pr. Lon Bernard, F-35043 Rennes, France
b
Equipe ICMV Ingnierie Chimique et Molcules pour le Vivant, UMR CNRS 6226, Univ. Rennes 1, Universit Europenne de Bretagne, 263 Avenue du General Leclerc, F-35042
Rennes, France

a r t i c l e i n f o a b s t r a c t

Article history: Ionic liquids based extraction method has been applied to the effective extraction of norstictic
Received 10 June 2011 acid, a common depsidone isolated from Pertusaria pseudocorallina, a crustose lichen. Five 1-alkyl-3-
Received in revised form 26 August 2011 methylimidazolium ionic liquids (ILs) differing in composition of alkyl chain and anion were investigated
Accepted 12 September 2011
for extraction efciency. The extraction amount of norstictic acid was determined after recovery on HPTLC
Available online 16 September 2011
with a spectrophotodensitometer. The proposed approaches (IL-MAE and IL-heat extraction (IL-HE)) have
been evaluated in comparison with usual solvents such as tetrahydrofuran in heat-reux extraction and
Keywords:
microwave-assisted extraction (MAE). The results indicated that both the characteristics of the alkyl chain
Pertusaria pseudocorallina
Lichens, Polyphenolic compounds
and anion inuenced the extraction of polyphenolic compounds. The sulfate-based ILs [C1 mim][MSO4 ]
Ionic liquids and [C2 mim][ESO4 ] presented the best extraction efciency of norstictic acid. The reduction of the extrac-
Microwave-assisted extraction (MAE) tion times between HE and MAE (2 h5 min) and a non-negligible ratio of norstictic acid in total extract
Heat-extraction (HE) (28%) supports the suitability of the proposed method. This approach was successfully applied to obtain
additional compounds from other crustose lichens (Pertusaria amara and Ochrolechia parella).
2011 Elsevier B.V. All rights reserved.

1. Introduction can efciently absorb microwave energy, it is rather an inter-

Room-temperature ionic liquids (ILs) are salts, with melting
temperature below the boiling point of water, resulting from com-
binations of bulky organic cations and inorganic or large organic
anions [1]. These neoteric solvents are a class of novel green chem-
icals which are designed to replace traditional volatile organic
solvents in industrial processes. The potential of ILs in both aca-
demic and industrial elds is due to their negligible vapor pressure,
good thermal sensibility, low or virtually no volatility, good misci-
bility with water and organic solvents, as well as extractability for
various organic compounds [24].
ILs are regarded as potentially environmentally benign sol-
vents although recent publications indicated that the toxicity
of imidazolium based ILs increases with the alkyl chain length.
Such compounds have been shown to be either more toxic to
freshwater algae [5] or less toxic to watercress [6] than the
conventional solvents. Additionally, microwave-assisted extrac-
tion (MAE) has received increasing attention as an alternative to
classical solidliquid extraction methods since traditional extrac-
tion techniques require long extraction times. Considered that ILs
Corresponding author. Tel.: +33 0 2 23 23 48 17; fax: +33 0 2 23 23 47 04.
E-mail address: sophie.tomasi@univ-rennes1.fr (S. Tomasi).
esting challenge to use ILs as solvent for the MAE of various
biomolecules from solid samples. 1-Alkyl-3-methylimidazolium
based ionic liquids aqueous solutions were investigated as sol-
vents for the extraction of polyphenolic compounds from Psidium
guajava leaves and Smilax china tubers [7], of lignans from
Schisandra chinensis [8] and of alkaloids from medicinal plants
[9], indicating that ILs had potential applications in the MAE
of bioactive substances from various sources. Recently, protic
ionic liquids (N,N-dimethyl-N-(2-hydroxyethoxyethyl)ammonium
propionate (DMHEEAP) and N,N-dimethyl(cyanoethyl)ammonium
propionate (DMCEAP)) were used as solvents in MAE to extract
lactones from Ligusticum chuanxiong [10]. While the extraction
mechanism of MAE using IL was similar to traditional organic sol-
vents, the efciency of MAE was found much higher than standard
extraction.
Lichens are fascinating organisms, known as a stable, self-
supporting symbiont involving algae or cyanobacteria and fungi
[11]. They produce characteristic secondary metabolites which are
unique and allow them to survive under various harsh environ-
mental conditions (e.g., at high altitude, direct sunlight). These
compounds have various biological activities such as: antivi-
ral, antitumor, anti-inammatory, antioxidant properties [12,13].
Among them, depsides, depsidones and depsones are the major
representative groups. However they are often extracted in low

0003-2670/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2011.09.009
70 S. Bonny et al. / Analytica Chimica Acta 707 (2011) 6975

O
CH 3 O CH3 O
O H3 C O
H3C OH O OH
O
O OH
O O HO OH
OH O
O CH 3 O
HO HO
R OH
R = CHO : norstictic acid variolaric acid lecanoric acid
R = CH2OH : connorstictic acid

C 5H11
O O
O R
OH
O
O
O OH
O
O OCH3
O COOH
HO
R
HO C 5H11
-alectoronic acid R = C 5 H 11 :picrolichenic acid
R = C 5 H 11 or C 3H 7 : picrolichenic acid derivatives

Fig. 1. Chemical structures: norstictic acid; connorstictic acid; variolaric acid; lecanoric acid; -alectoronic acid; picrolichenic acid and derivatives.

quantities due to a reduced availability of the material and their
limited solubility in organic solvents. Pertusaria pseudocorallina is a
crustose lichen collected on seashore rocks and described to contain
as major compounds, two depsidones corresponding to norstic-
tic acid and its methyl alcoholic derivative connorstictic acid [14]
(Fig. 1). Norstictic acid has already demonstrated signicant antiox-
idant activities [15]. These polyphenolic compounds are usually
insoluble in water and are extracted with volatile organic solvents.
Previously we demonstrated their stability under MAE at temper-
atures even up to 100 C for 15 min without degradation using
tetrahydrofuran or acetone [16].
In this work, ILs were investigated as alternative solvents in
MAE for the extraction of norstictic acid with regard to the bal-
ance of their extraction yields, extraction time and selectivity.
The ionic liquids based microwave-assisted extraction (IL-MAE)
performed using ve 1-alkyl-3-methylimidazolium ionic liquids
were compared to classical extraction approaches. The efciency
of the methods was evaluated by quantication after recovery of
norstictic acid in the extracts from P. pseudocorallina using HPTLC
coupled to a Camag spectrophotodensitometer. This approach
was then extended to other crustose lichens Pertusaria amara [17]
and Ochrolechia parella [18] for the extraction of picrolichenic acid
along with derivatives and variolaric, lecanoric and -alectoronic
acids, respectively. The structures of these isolated metabolites are
reported in Fig. 1.
2. Experimental
2.1. Plant materials and reagents
P. pseudocorallina (Sw.) Arn. (Pertusariaceae) was collected on
seashore rocks near Avranches, France, in September 2008. Voucher
specimens have been deposited at the herbarium of Pharmacog-
nosy and Mycology, Rennes, France (REN-ABB) with the reference
number JB/07/e9. After cleaning and identication, lichens were
air-dried, ground to ne powder just before extraction and stored
at room temperature in a closed glass.
Acetone, tetrahydrofuran, isopropanol were purchased from
Carlo Erba (Milano, Italy); toluene and diethyl ether and formic
acid from Prolabo . 1-Methylimidazole, diethylsulfate, dimethyl-
sulfate, chlorobutane, bis((triuoromethyl)sulfonyl)imide lithium
(Fluka) were used as received.
Norstictic acid standard of 98% purity used in quantitative
analysis was isolated from P. pseudocorallina after purication
procedure using ash chromatography with cyclohexane/diethyl
ether/tetrahydrofuran/formic acid (50/40/50/2) as a mobile phase
and a 15 mL min1 ow rate. Norstictic acid was conrmed by com-
parison of its 1 H NMR and 13 C NMR spectra with previous data [17].
HREIMS in a negative ion mode conrmed the molecular formula
of norstictic acid of C18 H12 O9 with a typical molecular ion cluster
at m/z 371.04086 (calcd 371.0408).
Picrolichenic acids (PA and PA derivatives) and variolaric (VA),
lecanoric (LA), -alectoronic acids were obtained with a 98% purity
after extraction with THF from P. amara [17] and with acetone
from O. parella [18], respectively and successive precipitation and
column chromatography on silica gel.
2.2. Apparatus
A CEM Explorer 24, a focused monomode microwaves appa-
ratus, was used for the experiments. The apparatus was run under
pressure in closed system with programmable temperature, heat-
ing power, and irradiation time.
A Camag automatic sampler III (ATS3) was used for sam-
ple application in TLC. The samples (2 mL) and standard solutions
(5 mL) were spotted in the form of bands of 4 mm width, positioned
at 15 mm from the bottom of TLC plates (Merck silica gel 60F254,
20 cm 10 cm), with a Camag microliter syringe. The mobile
phase was toluene/ethyl acetate/formic acid (70/25/5). Camag TLC
Scanner II was used for densitometric evaluation of thin-layer chro-
matograms and quantication was carried out under UV light at
270 nm (deuterium lamp) in absorbance/reectance mode.
The HPLC system equipped with a Kontron 325 pump and a
diode array detector (DAD) was used for some measurements. The
injector is tted with a 20 L external loop. A C18 Spherisorb col-
umn (250 4.6 mm, 5 m) was used as analytical column.
1 H and 13 C NMR spectra were performed using a Bruker Avance
III 300 NMR spectrometer.
2.3. Synthesis of ILs
The structures of ILs used here are reported in Table 1.
1,3-Dimethylimidazolium methylsulfate [C1 mim][MSO4 ] and 1-
ethyl-3-methylimidazolium ethylsulfate [C2 mim][ESO4 ] were syn-
thesized according to the literature procedure [19]. Another pro-
cedure was used for the bis{(trioromethyl)sulfonyl}imide based-
ionic liquids ([C2 mimOH][NTf2 ], [C3 mim][NTf2 ], [C4 mim][NTf2 ]
[20,21]. Briey, the synthesis of [C2 mimOH][Cl], [C3 mim][Cl] and
[C4 mim][Cl] were realized under microwave irradiations focused
at 120 C (150 C for [C4 mim][Cl]) during 30 min. The NTf2 based
ILs were prepared by reaction of the three corresponding chlorides
and LiNTf2 at a molar ratio 1:1.
 S. Bonny et al. / Analytica Chimica Acta 707 (2011) 6975
Table 1
Chemical structures and properties of ionic liquids.
Ionic liquids Cations Anions
[C1 mim][MSO4 ] N N CH3 SO4
[C2 mim][ESO4 ] N N C2 H5 SO4
[C2 mimOH][NTf2 ] N N NTf2
OH
[C3 mim][NTf2 ] N N NTf2
[C4 mim][NTf2 ] N N NTf2
These ILs were dried at 70 C under vacuum during several hours
and their purity was then checked by 1 H and 13 C NMR spectra at
room temperature in D2 O or CDCl3 . NMR spectra are identical to
those previously described [22] and indicated their good purity
(>95%). The water content was determined using Karl Fischer titra-
tion (Table 1).
2.4. MAE procedure
The IL-MAE procedure was performed using CEM Explorer 24.
P. pseudocorallina, P. amara or O. parella (0.25 g) were mixed with
5 mL of each ionic liquid, and then the suspensions were irradi-
ated under microwave heating for 5 min at 100 C. Regular MAE
was performed using 5 mL of THF as solvent for 5 min at 100 C. All
extraction experiments were repeated three times. After the irradi-
ation, the obtained extracts were ltered through cotton to remove
lichen residue and were cooled down to room temperature.
2.5. Conventional extraction method
2.5.1. Organic solvents
Heat-reux extraction (HE) was chosen as the reference process
for extraction of norstictic acid. 0.5 g sample was mixed with 10 mL
of THF in a round-bottom ask and the mixture was boiled at 70 C
for 2 h under reux. The extraction matrix was re-extracted two
times using solvent to perform the total extraction of norstictic acid.
The suspensions obtained after extraction were ltered through a
paper and the ltrate evaporated to dryness. The nal extracts were
diluted with identical solvent to 1 mg mL1 concentration.
2.5.2. ILs
0.25 g samples (P. pseudocorallina) were extracted with 5 mL of
different IL solutions, heated in an oil bath for 2 h at 100 C. The
resulted extracts were ltered through cotton, and then the ltrate
cooled down at room temperature.
2.6. HPLC analysis
The extracts diluted in THF at 5 mg ml1 were ltered before
injection. The mobile phase consisted of solvent A (0.6% acetic acid
aqueous solution) and solvent B (MeOH) (60/40) with a ow rate
xed at 0.8 mL min1 . The UV detection wavelengths were 270 nm
and each injection volume was 20 L. The column temperature
was ambient. Norstictic and connorstictic acids identication was
71
MW Form (25 C) Water miscibility Water content %
160 Solid Totally miscible 1.20
188 Liquid Totally miscible 1.62
454 Liquid Non miscible 1.06
466 Liquid Non miscible 1.09
480 Liquid Non miscible 1.37
carried out by comparing their retention time with corresponding
peaks in the standard solution.
2.7. Recovery of compounds and quantication
2.7.1. Recovery
The proposed process involves liquidliquid extraction with
water and diethyl ether followed by separationprecipitation with
a mixture of acetone and water (1:1, v/v) (Fig. 2). After the extrac-
tion process, the raw extracts were ltered with cotton and the
ltrate was kept at room temperature to allow the precipitation
of compounds. The precipitate obtained (precipitate 1) was sepa-
rated from the ltrate then water was added as co-extractant (2:3,
v/v) and diethyl ether as extractant (0.5 mL 2 mL). The whole was
stirred during 15 min then put to centrifugation at 4000 tr min1
during 15 min. The superior organic phase which contains some
metabolites (terpenoids as apolar metabolites, depsidones. . .) was
evaporated. A mixture of acetone/water (1:1, v/v) was added to
the aqueous phase to precipitate targeted metabolites (precipitate
2). Both precipitates are mixed, washed with acetone then dried
under reduced pressure (Fig. 2). The nal extracts and the dried
organic extract were prepared in tetrahydrofuran, corresponding
to 0.5 mg mL1 crude extract and were analyzed by HPTLC coupled
to a spectrophotodensitometer and by HPLC.
2.7.2. Quantication using Camag
spectrophotodensitometer-method validation
Standard solutions for each compound (0.050.3 mg mL1 in
THF) were used for the preparation of a 5-point calibration curve
corresponding to an amount of 0.21.2 g. The 0.5 mg mL1 stock
solutions in THF are quantied according to the procedure already
described [16].
The method validation of the chromatographic determination
with TLC scanner II was studied. A series of standard solutions with
the concentration range 0.0110 mg mL1 was prepared to deter-
mine the linearity of this method. The linear range was from 0.02
to 0.5 mg mL1 . The correlation coefcient of the calibration curve
was 0.9989. The limit of detection and the limit of quantication
were 0.01 and 0.02 mg mL1 , respectively. The repeatability of the
chromatographic determination with TLC scanner II, expressed in
RSDr, was also considered in order to determine the experimental
errors and evaluated as 3.2%.
72 S. Bonny et al. / Analytica Chimica Acta 707 (2011) 6975

Fig. 2. Process of IL-based extraction from P. pseudocorallina, recovery of depsidones (NA) and possible regeneration of ILs.

The extraction efciency of the compounds in samples was to evaluate the inuence of the size of the alkyl chain along with
dened by the determination of the extraction yields as follows: the presence of a hydroxyl group for the extraction efciency.

mass of the compound in extract

yield (%) = 100 3.2. Effect of IL structures
mass of the material
The ratio in the total yield of the compounds was dened as Previous studies have demonstrated the inuence of the struc-
follows: ture of ILs on their physicochemical properties [25,26], which affect
ratio in the total yield (%) the extraction efciency of target analytes. The effects by change in
mass of the compound in extract anions and aliphatic chain length on the extraction yields were thus
= 100 investigated in this work. In the series of ILs studied here, the anion
sum of mass of the compound of the extract
identity greatly inuences the water miscibility [27,28]. Thus, the
NTf2 anion was tested with three imidazolium cations as well as
The total mass of norstictic acid in sample was determined by
sulfate anions (Table 1).
analysis of the total extraction.
The sulfate based-ILs presented the best norstictic acid extrac-
tion yield with similar yields for [C1 mim][MSO4 ] using MAE (3.2%)
3. Results and discussion
and [C2 mim][ESO4 ] under HE (3.8%) (Fig. 3). The extraction process
used inuenced the recovery of norstictic acid from crude extract
3.1. Choice of ILs

Preliminary tests were performed using 18 ILs correspond-

ing to combinations of 1-alkyl-3-methylimidazolium alkylsulfate,
1-alkyloxy-3-methylimidazolium, ammonium and phosphonium
based ionic liquids with chloride, hexauorophosphate and
bis(triuoromethylsulfonyl)imide as anions. The solidliquid ratio
(1:20) was kept the same for all experiments and determined
from previous studies [16]. A 2-fold increase in the solid amount
decreased the extraction efciency due to uneasy ltration.
Regarding their TLC proles, [C1 mim][MSO4 ], [C2 mim][ESO4 ],
[C4 mim][PF6 ], Aliquat 336, [PBu4 ][Cl], [PBu4 ][PF6 ], [C3 mim][NTf2 ],
[C3 mimOH][NTf2 ] and [C6 mimOH][NTf2 ] appeared to be the best
suitable ILs for the extraction of the tested lichens. Indeed,
some of them allowed selective extraction of norstictic acid
([C2 mim][ESO4 ] and [C3 mimOH][NTf2 ]) or along with other
compounds ([C1 mim][MSO4 ]). Considering the possibility of ILs
regeneration, their easy synthesis and aromatic feature able to
interact with -systems [23,24] of the required lichen compounds,
imidazolium cations were selected to extract compounds from P.
pseudocorallina in order to evaluate their performance in HE and
MAE approaches.
Thus, the most interesting ILs according to the given objec-
tives remain [C1 mim][MSO4 ], [C2 mim][ESO4 ] and [C3 mim][NTf2 ]
because besides extracting the targeted substances, most of them
Fig. 3. Effect of ILs on the extraction efciency of norstictic acid from P. pseudoco-
allowed to get back them and to envisage their quantication. rallina. Sample: 0.25 g, extractant volume: 5 mL, extraction time: 5 min (MAE)2 h
Moreover, we decided to use [C4 mim][NTf2 ] and [C2 mimOH][NTf2 ] (HE). No result was obtained with [C4 mim][NTf2].
 S. Bonny et al. / Analytica Chimica Acta 707 (2011) 6975
of [C2 mim][ESO4 ] as indicated by the efciency decrease between
HE (with 3.8% yield) and MAE (0.5% yield) (Fig. 3). The low extrac-
tion efciency under microwave irradiations could be explained by
the high ability of this IL to absorb and transfer microwave energy
leading to the degradation of lichen sample. It is for this reason that
the temperature for IL-MAE was limited to 100 C and was applied
to IL-HE. As shown in Fig. 3, the extraction yield was also decreased
from RSO4 to NTf2 due to the decrease in the water miscibility
(Table 1).
In the case of NTf2 -ILs, the increase in alkyl chain from propyl
to butyl dramatically decreased the extraction efciency (Fig. 3).
Thus, [C3 mim][NTf2 ] led to extraction of norstictic acid (1.6%)
whereas the extraction yields of [C4 mim][NTf2 ] were not measur-
able. These results suggested that alkyl chain length has inuence
on the extraction and the recovery of norstictic acid and could be
related to the hydrophobicity which increased with the length of
the chain (Table 1). The substitution of a terminal methyl group
by a hydroxyl group did not however improve the extraction yield
(Fig. 3). Similar extraction yields for each extraction method used
were thus obtained between [C2 mimOH][NTf2 ] and [C3 mim][NTf2 ].
A 0.5% extraction yield was performed under HE for these two ILs
while this efciency was 3-fold higher using MAE (around 1.5%)
(Fig. 3), suggesting their ability to heat up to a good extent under
microwaves effect.
3.3. Comparison of the proposed IL-MAE procedure with classical
methods
In order to compare the extraction efciency using ILs with
organic solvents in MAE and HE processes, tetrahydrofuranwas
Fig. 4. HPLC chromatograms of extracts using tetrahydrofuran under MAE (A), raw extract of [C1 mim][MSO4 ] under MAE (B), precipitates of [C1 mim][MSO4 ] under MAE
(C), and [C2 mim][ESO4 ] under HE (D), respectively. Peak: norstictic acid-NA; column: Spherisorb C18 column (150 mm 4.60 mm, 5 m); mobile phase A (methanol) and B
(wateracetic acid 0.6%): 60/40; ow rate: 0.8 mL min1 ; UV wavelength: 239 nm.
73
used to extract norstictic acid from P. pseudocorallina. We have
already demonstrated that THF led to the best extraction of this
sample specially under MAE [16] and this solvent avoids the degra-
dation of the lichen samples sensitive to hydrolysis by aqueous or
polar solvent such as MeOH [29]. The major constituents of heat-
reux and MAE extracts have been characterized using HPLC. The
chromatograms shown in Fig. 4 indicated that THF and some ILs e.g.
[C1 mim][MSO4 ] had a different extraction selectivity. It is apparent
that MAE using THF led to an efcient extraction yield of norstic-
tic acid (11.3%) but along with other substances in comparison
to a more selective IL extraction (Fig. 4). The extraction yield of
norstictic acid reached 3.2% with [C1 mim][MSO4 ] using the IL-MAE
approach. Compared to HE using THF, the proposed process was 3-
fold less efcient (3.29.0%) (Table 2) but was compensated by a
drastic reduction of the total extraction time (from 2 h to 5 min).
ILs were also used in HE during 2 h and [C2 mim][ESO4 ] appeared to
be the most effective with a 3.8% yield. As shown in Fig. 4, the purity
of norstictic acid recovered from these two IL extracts is similar but
in favor of [C1 mim][MSO4 ] in regard to the extraction time.
Except for [C2 mim][ESO4 ], IL-MAE process was 1.5- to 3-fold
more efcient than IL-HE (Fig. 3). These results suggested that most
of ILs were more efcient under microwaves, due to their abil-
ity to transfer microwave energy in a good manner. Nevertheless
MAE using THF was around 4-fold more efcient (11.3% yield and
97.8% ratio of norstictic acid in total extract) than IL-MAE (Table 2).
This difference could be explained by a partial recovery of norstic-
tic acid from ILs. Therefore, the recovery process of norstictic acid
from ILs has to be optimized along with further extraction condi-
tions. Despite this partial recovery, the best advantages of ILs in
comparison to THF are their selective extraction, possible regener-
ation and no ammability. These ILs are also more safe than THF
74 S. Bonny et al. / Analytica Chimica Acta 707 (2011) 6975

Table 2
Comparative study of extraction efciency of norstictic acid using different extraction methods.

Proposed method Conventional methods

IL-MAE [C1 mim][MSO4 ] Heat-reux extraction (THF) Regular MAE (THF)

Yield (mean S.D %) Ratio in total yielda Yield (mean S.D Ratio in total yielda Yield (mean S.D Ratio in total yielda
(mean S.D %) %) (mean S.D %) %) (mean S.D %)

3.2 0.5 28.0 9.0 78.5 11.3 98.7

0.3 0.3 6.2 1 8.4

a
This ratio is the ratio of norstictic acid in total extract and was determined using the mean of total mass of norstictic acid in P. pseudocorallina which was 114.4 5.6 mg g1 .

Fig. 5. TLC proles with UV detection of lichen metabolites in the [C1 mim][MSO4 ] extracts. (A) variolaric acid (VA), -alectoronic acid (AA) and lecanoric acid (LA) from
Ochrolechia parella (B) picrolichenic acid (PA) and derivatives from Pertusaria amara. Mobile phase: tolueneethyl acetateformic acid 70/25/5. UV detection at 270 nm.

because they do not form highly-explosive peroxides on storage in [C1 mim][MSO4 ] extracted the major compounds of P. amara
air. (picrolichenic acid and derivatives), which belong to the group of
depsones, as well as it extracted variolaric acid, -alectoronic acid
(depsidones) and lecanoric acid (depside) from O. parella (Fig. 5). It
3.4. Recovery of norstictic acid and regeneration of ionic liquids was found that [C1 mim][MSO4 ] was an IL able to extract a great
variety of lichen compounds such as depsides, depsidones and
The general extraction process of norstictic acid and the poten- despones without degradation. Compared to HRE using THF, the
tial regeneration of ionic liquid solvents are depicted in Fig. 2. IL based process thus appears to be a soft extractive method, what
Previous studies proved that some organic molecules could be is particularly advantageous for the study of the depsides which
extracted from ionic liquids with organic solvent [30]. We thus used are common in lichens but also among the most easily hydrolyzed
water as co-extractant for solubilizing partially the extract and var- molecules.
ious organic solvents as extractants, the co-extractant leading the
passage of products towards the extractant. This procedure was
studied for [C1 mim][MSO4 ], [C2 mim][ESO4 ] and [C3 mimOH][NTf2 ]
4. Conclusion
using diethyl ether, acetone and ethyl acetate to nd the best
extractant. This one must not be miscible with water and rather
In this work, ILs were proved to be alternative solvents for the
polar to extract most of the compounds. We chose the diethyl
extraction of major lichen compounds even if the recovery from ILs
ether instead of the ethyl acetate extractant because of the low-
is not complete. They also allowed extraction to be carried on with a
est amount of IL remaining in dry extracts. The following addition
small quantity of raw material (0.25 g dried lichen). The cations and
of acetone/water mixture allowed us to elaborate a process to
anions due to their difference in their physiochemical properties
recover the compounds of interest by precipitation and to regener-
such as ability to interact with -systems of the analytes and water
ate ILs. The extraction yields were then evaluated from precipitates
miscibility had inuences on the extraction efciency. The best
using HPTLC coupled to a spectrophotodensitometer. Concerning
results were thus obtained using sulfate anions ([C1 mim][MSO4 ]
the more hydrophobic ILs such as [C4 mim][NTf2 ] the recovery of
and [C21 mim][ESO4 ]) associated to the imidazolium cation bear-
NA was difcult as this IL is not soluble in water. We demon-
ing a short chain related to a low hydrophobicity. Compared with
strated here that the miscibility of ILs with water had a positive
conventional HE extraction procedures, the proposed approach
inuence on their regeneration and the recovery of analytes. The
provides sufcient extraction yields in a much shorter extraction
possibility to regenerate more hydrophilic ILs by easy evapora-
time. Further experiments are in progress to improve the total
tion of water/acetone fraction could be in favor of this process in
recovery of compounds from IL extracts and to determine the inu-
comparison to classical organic solvents.
ence of the sulfate anions on the extraction efciency.

3.5. Extension of the method to other crustose lichens

In the light of previous results, we investigated the IL-MAE
extraction process with other crustose lichens (P. amara and O. par-
ella) containing different secondary metabolites [17]. Assays were
performed under microwave irradiations with the most efcient IL
which was [C1 mim][MSO4 ] (Fig. 5).
Acknowledgements
We thank the team of Prof. J.P. Bazureau (Rennes, France) for the
use of the microwave apparatus and for their helpful comments. We
are also deeply grateful to Isabelle Rouaud and Aurelie Sauvager for
their helpful assistance.
 S. Bonny et al. / Analytica Chimica Acta 707 (2011) 6975 75

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