Journal of Chromatography A, 1218 (2011) 84808489

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Application of ionic liquids based microwave-assisted simultaneous extraction

of carnosic acid, rosmarinic acid and essential oil from Rosmarinus ofcinalis
Tingting Liu 1 , Xiaoyu Sui 1 , Rongrui Zhang, Lei Yang , Yuangang Zu , Lin Zhang,
Ying Zhang, Zhonghua Zhang
Key Laboratory of Forest Plant Ecology, Northeast Forestry University, 150040 Harbin, China

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

Article history: An ionic liquid based microwave-assisted simultaneous extraction and distillation (ILMSED) method
Received 6 September 2011 has been developed for the effective extraction of carnosic acid (CA), rosmarinic acid (RA) and essential
Received in revised form oil (EO) from Rosmarinus ofcinalis. A series of 1-alkyl-3-methylimidazolium ionic liquids differing in
25 September 2011
composition of anion and cation were evaluated for extraction yield in this work. The results obtained
Accepted 26 September 2011
indicated that the anions and cations of ionic liquids had inuences on the extraction of CA and RA,
Available online 1 October 2011
1.0 M 1-octyl-3-methylimidazolium bromide ([C8mim]Br) solution was selected as solvent. In addition,
the ILMSED procedures for the three target ingredients were optimized and compared with other con-
Keywords:
Ionic liquids
ventional extraction techniques. ILMSED gave the best result due to the highest extraction yield within
Ionic liquid based microwave-assisted the shortest extraction time for CA and RA. The novel process developed offered advantages in term of
simultaneous extraction and distillation yield and selectivity of EO and shorter isolation time (20 min in comparison of 4 h of hydrodistillation),
(ILMSED) and provides a more valuable EO (with high amount of oxygenated compounds). The microstructures
Essential oil and chemical structures of rosemary samples before and after extraction were also investigated. More-
Carnosic acid over, the proposed method was validated by the stability, repeatability and recovery experiments. The
Rosmarinic acid results indicated that the developed ILMSED method provided a good alternative for the both extraction
Rosmarinus ofcinalis
of non-volatile compounds (CA and RA) and EO from rosemary as well as other herbs.
2011 Elsevier B.V. All rights reserved.

1. Introduction and chemopreventive activities [2]. Most of the reported results

associated functional properties of rosemary with the composi-
Rosemary (Rosmarinus ofcinalis L.) is a well-known aromatic tion of polyphenolic compounds, such as carnosic acid (CA) and
evergreen shrubby herb belonging to the family Lamiaceae, indige- rosmarinic acid (RA), which are fat-soluble phenolic diterpene and
nous to the Mediterranean region, rosemary is now widely water-soluble phenolic acid species, respectively [3]. Essential oil
distributed, and is cultivated in China, India Uttaranchal, Alge- (EO) is another main product from rosemary and is widely used as
rian Sahara, North America, Northern Europe and England. In a seasoning for foodstuffs such as meat dishes, salamiand sauces,
recent years, rosemary species, which in addition to being used and as part of perfume or cosmetic products because of its unique
as a food avoring, is also well-known medicinally for its pow- aroma and antibacterial activity [4].
erful antioxidant, antibacterial, antimutagenic properties and as a There are many literatures on the analysis and extraction
chemopreventive agent [1]. Furthermore, several extracts, essen- technique of the three representative ingredients. Traditionally,
tial oils and chemical constituents isolated from this species volatile EO and non-volatile CA, RA were extracted in separate
demonstrated a number of interesting biological activities includ- processes, respectively. The EO of rosemary has usually been iso-
ing antioxidant, antimicrobial, anti-inammatory, antitumorigenic lated by traditional hydrodistillation [5], steam distillation [6] or
organic solvent extraction. The conventional Soxhlet extraction
[7], heat reux extraction (HRE) [8] and maceration extraction [9]
Corresponding author at: Key Laboratory of Forest Plant Ecology, Ministry of
with volatile organic solvents, including methanol, ethanol, ace-
Education, Northeast Forestry University, Box 332, Hexing Road 26, Harbin, Hei-
tone, and some mixed solvents were utilized to extract CA and
longjiang Province, China. Tel.: +86 451 82191387; fax: +86 451 82102082. RA. These conventional extraction techniques are discommodious,
Corresponding author at: Key Laboratory of Forest Plant Ecology, Ministry of inefcient, time-consuming, energy-consuming, target ingredient
Education, Northeast Forestry University, Box 332, Hexing Road 26, Harbin, Hei- degraded and involved large volumes of toxic organic solvents
longjiang Province, China. Tel.: +86 451 82191517; fax: +86 451 82102082.
(in CA and RA extraction) and the recoveries are unsatisfactory
E-mail addresses: [email protected] (L. Yang), [email protected] (Y. Zu).
1
These two authors contributed equally to this work. [5,10].

0021-9673/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2011.09.073
 T. Liu et al. / J. Chromatogr. A 1218 (2011) 84808489 8481

Recently, microwave-assisted extraction (MAE) has been Pharmaceutical Co., Ltd., Hangzhou, China. All samples were dried,
accepted as a potential and powerful alternative to conventional milled, passed through a stainless steel sieve and stored in closed
extraction techniques in the extraction of target ingredients from desiccators at 4 C until use. The same batch of sample was used
plant materials. Moreover, there are many works have proved that here in the experiments.
MAE is a more effective technique compared to the conventional
method for the extraction of phenolic compounds from medici- 2.2. Apparatus
nal plant [8,11,12]. The extraction time was reduced, less solvent
was used and the amount of extracted phenolic compounds was A microwave oven (WP700, Glanz, Industries Inc Company,
increased. Several microwave-assisted distillation techniques have Guangdong, China) was used for ILMSED with 2.45 GHz microwave
also been used to extract EO from rosemary [5,10,13]. However, irradiation frequency [25,26]. The maximum output power of
there are no reports on the simultaneous extraction of non-volatile the oven was 700 W. The whole system was run at atmospheric
compounds and volatile oil in the literature to our best knowledge. pressure. The dimensions of the interior cavity of the oven are
Ionic liquids, which are composed of bulky organic cations and 215 mm 350 mm 330 mm. Round bottom ask is placed in the
inorganic or organic anions, are liquid near room temperature. oven and connected to a Clevenger-type apparatus [27]. In order
They have recently attracted much research interest for a variety of to examine the effect of microwave power, ve power levels, 100%
applications thanks to their excellent properties: negligible vapor (700 W), 77% (540 W), 55% (385 W), 33% (230 W), and 17% (120 W)
pressure, good thermal stability, wide liquid range, tunable viscos- were studied.
ity, and miscibility with water and organic solvents, as well as good The chromatographic system (Waters, USA) equipped with Mil-
solubility and extractability for various organic compounds, more- lennium 32 system software, 717 plus autosampler, 1525 pump,
over, ionic liquids can efciently absorb and transfer microwave 717 automatic column temperature control box and 2487 UV
energy [14,15]. Over the past few years, ionic liquids have been used detector was used for HPLC analysis. Chromatographic separa-
as attractive green alternatives to conventional volatile organic tion was performed on a Kromasil-C18 (4.6 mm 250 mm, 5 m)
solvents in various applications, including analytical applications, reversed-phase column (Akzol Nobel Inc., Sweden). A GCMS
organic synthesis, catalysis, and separation science [1619]. There instrument (6890N/5973, Agilent Technologies, Wilmington, DE,
are some efforts have been made in the eld of ionic liquids USA) equipped with a mass selective detector operating in the
aided sample treatment techniques, such as solidliquid extrac- electron impact mode (70 eV) was used to study the compositions
tion [20], solid-phase microextraction [21], liquidliquid extraction of the extracted EO. The GC part was equipped with an HP-5MS
[22], liquid-phase microextraction [23], and aqueous two-phase (Agilent Technologies) capillary column (30 m long, 0.25 mm inner
systems extraction [24]. diameter, and 0.25 m lm thickness). A FEI Quanta-200 scanning
In the present study, the potentiality of ionic liquids as alter- electron microscope (FEI Company, USA) was used to examine the
native solvents in the MAE of CA, RA, and EO from rosemary was morphological alteration of the sample. The FT-IR spectra of sam-
investigated. Eight ionic liquids with different cations and anions ples were obtained on an IRAfnity-1 FT-IR spectrophotometer
were investigated and various inuential parameters of the ionic (Shimadzu Co., Japan).
liquids based microwave-assisted simultaneous extraction and dis-
tillation (ILMSED) procedures were optimized systematically. The 2.3. HPLC analysis
results of ILMSED were evaluated by the determination of RA and
CA in the extracts as well as the quality and quantity of EO. The Before HPLC analysis, all standard solutions of rutin and extracts
ILMSED approach developed here was compared with conventional were ltrated through 0.45 m membranes (GuangFu Chemical
extraction approaches. The simultaneous extractions of CA, RA, and Reagents Co., Tianjin, China). HPLC analysis of CA and RA was
EO demonstrated that the proposed ILMSED approach exhibited done according to the procedure described by Wei Zheng [28]. The
great potential for the ease of quality assessment of rosemary as mobile phase consisted of acetonitrile (A) and 2% formic acid aque-
well as other aromatic medicinal plant. ous solution (B) using the following gradient elution program for
separation: 010 min, 30% (A); 1015 min, 3070% (A); 1525 min,
70% (A); 2530 min, 7030% (A). The wavelength used for CA and
2. Experimental RA with the diode detector was 285 nm and 330 nm, respectively.
The column temperature was maintained at 25 C, the ow rate was
2.1. Reagents and samples 1.0 mL/min and the injection volume was 10 L. Under such condi-
tions, the CA and RA were baseline separated. The chromatographic
Reference compounds of CA and RA were purchased from peaks of the analytes were conrmed by comparing its retention
SigmaAldrich Inc. (St. Louis, MO, USA). Acetonitrile and formic time with the reference standard.
acid of chromatographic grade were purchased from J & K Chemical The quantications of CA and RA were performed by the
Ltd. (Beijing, China). All Ionic liquids including 1-butyl-3-methyli- standard curve method. The calibration curves of CA and
midazolium chloride ([C4mim]Cl), 1-butyl-3-methylimidazolium RA showed good linearity over the ranges of 2.010.0 and
bromide ([C4mim]Br), 1-butyl-3-methylimidazolium tetrau- 0.22.0 mg/mL, respectively. The linear regression equations for
oroborate ([C4mim]BF4 ), 1-butyl-3-methylimidazolium nitrate CA and RA is YCA = 2657149X 202756 (r = 0.9997, n = 8) and
([C4mim]NO3 ), 1-ethyl-3-methylimidazolium bromide ([C2mim] YRA = 20470465X + 1625134 (r = 0.9998, n = 8). The chromatograms
Br), 1-hexyl-3-methylimidazolium bromide ([C6mim]Br), 1- of the two phenolic acids obtained from [C8mim]Br extraction
octyl-3-methylimidazolium bromide ([C8mim]Br), and 1-decyl- solution at different detection wavelength is shown in Fig. 1. No
3-methylimidazolium bromide ([C10mim]Br) were bought from inuence attributable to the ionic liquids used were observed on
Chengjie (Shanghai, China) and used without further purication. peak resolution, elution order and elution time.
All the other reagents obtained from Beijing Chemical Reagents
Co. (Beijing, China) were of analytical grade. Deionized water was 2.4. GCMS analysis
puried by a Milli-Q water purication system from Millipore
(Bedford, MA, USA). Analyses were carried out using helium as carrier gas at a ow
Fresh leaves of the cultivated rosemary were hand-harvested rate of 1.0 mL/min. Oven temperature program used was an initial
in September from the Botanical Garden of the Zhejiang Hisun hold at 40 C for 5 min and a rise to 160 C at 4 C/min followed
8482 T. Liu et al. / J. Chromatogr. A 1218 (2011) 84808489

Fig. 1. HPLC chromatogram of rosemary extract. (a) Detection wavelength at 285 nm and (b) detection wavelength at 330 nm. Inset: the molecular structures of CA and RA.

by additional rise to 240 C at 5 C/min, held at 240 C for 10 min. according to the European Pharmacopoeia [31] with 200 mL of
The injector was set at 280 C. The samples were diluted with ethyl water for 4 h (until no more EO was obtained). The system was
acetate (1:50, v/v) and a volume of 1.0 l was injected to the GC with brought to boiling point by heating at a xed power of 700 W; the
the injector in the split mode (split ratio, 1:50). The compounds of experiment was carried out at atmospheric pressure.
the EOs were identied by comparing their mass spectral fragmen-
tation patterns with those of similar compounds from a database 2.6.2. Microwave hydrodistillation (MHD)
(Wiley, Mass Finder 2.1 Library, NIST98) and MS literature data MHD has been performed using the microwave oven. 20 g of
[29,30] combined with comparison of GC retention indices (RI). For accurately weighed rosemary sample was heated using a xed
each compound on the chromatogram, the percentage of peak area power of 700 W for 60 min with 200 mL water.
relative to the total peak areas from all compounds was determined
and reported as relative amount of that compound.
2.6.3. Heat reux extraction (HRE)
20 g of accurately weighed rosemary sample were put into a
2.5. Ionic liquids based microwave-assisted simultaneous round-bottom ask by adding 200 mL of ethanolwater (85:15,
extraction and distillation (ILMSED) v/v), the ask was placed into a heating mantle and connected
with a reux condenser, and then allowed to reux for 2 h under a
ILMSED was performed in microwave oven. 20 g of accurately heating power of 700 W.
weighed rosemary sample was mixed with 200 mL of different The EO obtained from HD and MHD methods were collected,
ionic liquid solutions and then the suspension was irradiated by dried under anhydrous sodium sulphate and stored at 4 C until
microwave. Then the vessels were placed into the A modied analyzed. The extracts of HD, MHD and HRE were processed as
domestic microwave oven system followed by microwave extrac- described in Section 2.5.
tion one bye one. The optimum ionic liquid and its concentration,
solidliquid ratio, microwave irradiation power and time were sys-
2.7. Statistical analysis
tematically studied in this work. After ILMSED, 1 mL of extract
obtained was cooled to room temperature rapidly and ltered
The one-way ANOVA test was used to calculate the signicance
through a 0.45 m membrane (Guangfu Chemical Reagents Co.,
of the differences of extraction yields for the target analytes. The
Tianjin, China) for subsequent HPLC analysis. The EO was collected,
results of analysis were expressed as means of extraction yield SD.
dried under anhydrous sodium sulphate and stored at 4 C until
analyzed.
The extraction yields of target ingredients were determined as 3. Results and discussion
follows:
3.1. Optimization of ionic liquids solution
extraction yield (mg/g)
mean mass of CA, RA or EO in herb samples (mg) The structure of ionic liquids has signicant inuence on its
= physicochemical properties, which might greatly affect the extrac-
mean mass of herb samples
tion yield of target analytes. To nd out the optimal ionic liquids
The mean mass of CA, RA in herb samples was determined by HPLC and evaluate its performance on the ILMSED of CA, RA and EO from
analysis of three samples, respectively. The mean mass of EO was R. ofcinalis leaves, 1-alkyl-3-methylimidazolium type ionic liquids
the average mass of three independent tests. The mean mass of with different anion and the alkyl chain length of the cation were
the herb samples was the average mass of three samples before studied in the present work.
extracted. It is well known that the choice of anion affects water misci-
bility of ionic liquids [32]. Thus, the 1-butyl-3-methylimidazolium
2.6. Conventional reference extraction methods ionic liquids with four different anions (Cl , Br , NO3 , and BF4 )
were tested. The four ionic liquids are both hydrophilic and could
2.6.1. Hydrodistillation (HD) enhance miscible capacity of solute in water [18]. The results of
20 g of accurately weighed rosemary sample was weighed and Fig. 2a suggested that the anions of ionic liquids inuenced the
submitted to hydrodistillation with a clevenger-type apparatus extraction yield of target analytes. As shown in Fig. 2a, the ionic
 T. Liu et al. / J. Chromatogr. A 1218 (2011) 84808489 8483

concentration. But the increasing tendency of the extraction yield

of CA and RA was not similar. With increasing of [C8mim]Br con-
centration from 0.2 M to 1.0 M, the extraction yield of CA increased
rapidly and reached its maximum at concentration of 1.0 M. But as
for RA, the extraction yield increased when the [C8mim]Br con-
centration over the range of 0.20.6 M. Upon further increases,
however, a slight decrease in extraction yield was observed. The
difference of polarity between the two phenolcarboxylic acids may
bring out this result. The greater [C8mim]Br concentration led to
the greater solubility capacity of solvent for water insoluble com-
pound. As for EO, the concentration of [C8mim]Br had no signicant
effect. As a result, the extraction yields of CA, RA, and EO were rela-
tively stable when [C8mim]Br concentration was 1.0 M, therefore,
1.0 M [C8mim]Br was selected for the subsequent experiments.

3.2. Optimization of the ILMSED process

In the preliminary work, we found that selection of ionic liq-

uids has no remarkable inuence on the extraction yield of EO, but
the factors of ILMSED procedure have dominating effect on that of
EO. Therefore, the inuential factors of ILMSED procedure, includ-
ing solidliquid ratio, microwave irradiation power and time were
investigated in terms of extraction yields of CA, RA, and EO. The
inuence of each factor was studied.

3.2.1. Solidliquid ratio

The solidliquid ratio is a crucial factor and was also studied
to optimize extraction yield. Large solvent volumes could make
the procedure difcult and lead to unnecessary waste, while small
volumes may lead to incomplete extraction. In investigating the
inuence of solidliquid ratio on yields of CA, RA, and EO, a series of
experiments were performed with different solidliquid ratios (1:6,
1:8, 1:10, 1:12, 1:14, and 1:20 g/mL). It can be seen in Fig. 3a that
the extraction yields of CA and RA increased with increasing solvent
volume for solidliquid ratio of up to 1:12. Higher solvent volumes,
Fig. 2. Effect of ionic liquids anions (a), cations (b), and [C8mim]Br concentrations however, did not signicantly improve the extraction yield of CA
(c) on the extraction yields of target ingredients. 20 g dried sample was mixed with and RA. As for EO, a higher solidliquid ratio may give slightly lower
200 mL IL of different ionic liquids and then extracted for 10 min at 385 W irradiation extraction yield. This is probably due to dissolution of some oxy-
power. The extraction yields of EO were all divided by 10 in order to make the values
genated compounds of EO in ionic liquid aqueous solution. Hence, a
in the same coordinate.
solidliquid ratio of 1:12 was considered as the optimal solidliquid
ratio for the ILMSED process.
liquids based on NO3 and Br were the most efcient of the liquids
tested for CA, BF4 , and Br were the most efcient for RA, and in 3.2.2. Irradiation power
contrast, these four anions showed no signicant effect on the EO Optimization of the microwave irradiation power used dur-
extraction. This result indicates that extraction efciency of phe- ing ILMSED is very important to ensure efcient extraction, and
nolcarboxylic acids is anion-dependent, which is consistent with the effect of this variable was examined. The extraction yield of
previous study [33]. In order to gain better extraction yields of the CA and RA obtained at different powers (120, 230, 385, 540, and
target analytes simultaneously, we subsequently screened the ve 700 W) gradually increased as the microwave irradiation power
ionic liquids by increasing alkyl chain length. With the same anion was increased from 120 to 700 W. The ILMSED time was main-
of Br , a series of 1-alkyl-3-methylimidazolium cations including tained constant throughout this experiment at 10 min. As shown
[C2mim]+ , [C4mim]+ , [C6mim]+ , [C8mim]+ , and [C10mim]+ were in Fig. 3b, it was found that as the microwave irradiation power
evaluated. From Fig. 2b, the extraction yield of CA and RA was found was increased, the extraction yield of CA, RA, and EO increased con-
to increase signicantly when the alkyl chain length was increased sistently. We can attribute the result to the fact is that microwave
from ethyl to octyl. The ionic liquids tested, with alkyl chains rang- irradiation energy can enhance the penetration of the solvent into
ing from C2 to C10, are all water-soluble, although the lipophilicity the matrix and deliver efciently to materials through molecular
of these ionic liquids increases with increasing alkyl chain length interaction with the electromagnetic eld and offer a rapid trans-
[34]. This may be because the lipophilic organic compounds CA and fer of energy to the solvent and matrix, allowing the dissolution
RA are better dissolved by a lipophilic ionic liquid. [C8mim]Br ionic of components to be extracted. The maximum microwave irradi-
liquid may be able to aid expansion of cellulose, facilitating dis- ation power tested (700 W) was therefore the optimal power for
solution of CA and RA from plant cells. And as for EO, the type of maximum yield.
ionic liquids had no signicant effect. Having optimized both the
anion and cation of the ionic liquid, [C8mim]Br was selected for 3.2.3. Irradiation time
subsequent extraction parameter optimization studies. To select a proper irradiation time to guarantee completion of
The effects of different concentration of [C8mim]Br on extrac- simultaneous extraction of CA, RA, and EO. The extractions were
tion yield are shown in Fig. 2c. It can be observed the extraction carried out at 700 W when microwave irradiation was changed
yield of CA and RA were increased with the increase of [C8mim]Br within 20 min. As shown in Fig. 3c, when the irradiation time
8484 T. Liu et al. / J. Chromatogr. A 1218 (2011) 84808489

Fig. 4. Extraction kinetics of CA, RA (a) and EO (b) from rosemary under different
extraction methods. The extraction yields of RA were all multiplied by 10 in order
to make the two curves in the same coordinate.

Fig. 3. Effect of ILMSED conditions on the extraction yields of target ingredients

For the extraction of polyphenolic compounds from rosemary
with 1.0 M [C8mim]Br. (a) Solidliquid ratio, (b) microwave irradiation power, and
(c) extraction time. The extraction yields of RA were all multiplied by 10 in order to leaves using ILMSED, the extraction yields increased rapidly at rst
make the values in the same coordinate. and then reached an equilibrium concentration, and the two kinetic
curves obtained were similar. As shown in Fig. 4a, with an increase
of extraction time, the extraction yields of CA and RA increased
increased from 4 to 15 min, the extraction yields of CA and RA in the initial 15 min, and then did not signicantly change from
increased dramatically. When the variable was changed from 15 to 20 to 60 min. The results of the ILMSED kinetic mechanism indi-
20 min, slight improvements were observed. And extraction yield cated that about 15 min of extraction time was enough to obtain
of EO had a similar tendency. Therefore, 15 min was selected for high extraction yields of polyphenolic compounds from rosemary.
further experiments. In HRE, the yields of CA and RA reached its maximum in 90 min and
Generally, extraction temperature is a factor that is usually did not signicantly change from 90 to 120 min, but the yields were
studied to increase the effectiveness of extraction of analytes lower than those obtained by ILMSED. Microwave-assisted extrac-
employing microwave-assisted extraction. The EO can be extracted tion offers a rapid delivery of energy to a total volume of solvent
with vaporisation of water, which indicates the system tempera- and solid plant matrix with subsequent heating of the solvent and
ture must be reached to boiling temperature of solution. Hence, the solid matrix, efciently and homogeneously. Because ionic liquid
factor of temperature is not investigated in the consideration the s solution within the plant matrix absorbs microwave energy, cell
simultaneous extraction of EO. disruption is promoted by internal superheating, which facilitates
Based on the above experiments, the optimum ILMSED con- desorption of chemicals from the matrix, improving the recovery
ditions were found to be: 1.0 M [C8mim]Br as extraction solvent, of phenolic compounds as well as fastening the extraction process.
microwave irradiation power of 700 W, irradiation time of 15 min Fig. 4b shows the variation of the EO extraction yield according
and solidliquid ratio of 1:12 (g/mL). to the extraction time under HD, MHD, and ILMSED method. The
extraction yields of EO extracted from rosemary with the different
isolation methods are 23.5 0.78, 22.5 0.69, and 22.5 0.79 mg/g
3.3. Extraction kinetics for the HD, MHD, and ILMSED, respectively. As is shown in Fig. 4b,
an isolation time of 15 min with ILMSED provides yields similar to
In the extraction process, the extraction yield depends on both those obtained after 150 min by means of HD. In MAD, the yields
the extraction efciency and chemical change of the target ana- of EO reached its maximum in 30 min, but the extraction rate was
lytes. The chemical structure of the target analytes may change with a little slower than that obtained by ILMSED. Because the addi-
some extraction conditions after the target analytes being trans- tion of [C8mim]Br contributes to enhancing the penetrability of the
ferred into the extraction solvent, which can directly inuence the solvent to the sample matrixes, which was benecial to evapora-
extraction yield of the target analytes [35]. Hence, the extraction tion rate of EO. In the light of Fig. 4b, EO released from the sample
kinetics of CA, RA and EO under different extraction methods were generally took place at three different stages during the ILMSED
investigated in this study. process. A preliminary phase required about 4 min to heat phase
 T. Liu et al. / J. Chromatogr. A 1218 (2011) 84808489 8485

Table 1
Comparison of proposed ILMSED with the conventional methods.

Methoda Solvent Time Power (W) CA yield (mg/g) RA yield (mg/g) EO yield (mg/g)

HD Water 4h 700 0.49 0.02b 2.18 0.05 23.1 0.86

MHD Water 20 min 700 1.09 0.03 2.97 0.06 18.5 0.75
ILMHD 1 M [C8mim]Br 20 min 700 33.29 0.69 3.97 0.07 21.5 0.83
HRE Ethanolwater (85:15, v/v) 2h 700 28.30 0.71 3.86 0.05 c
ILHE 1 M [C8mim]Br 20 min 700 21.27 0.66 2.07 0.05
a
20 g dried sample was mixed with different solutions and then extracted with corresponding methods. The power and the solidliquid ratio were maintained 700 W and
1:12, respectively.
b
Data represent mean SD of three independent experiments.
c
Not obtained.

from room temperature to 100 C. The rst phase is represented 3.4.2. Effect of different extraction procedures on the chemical
by an increasing line which indicates the rst quantities extracted, composition of rosemary essential oils
located at the surface of plant particles representing approximately A total of 26 compounds accounting for about 92% of the total
86% of the yield obtained into 10 min. During this stage, EO rapidly oil (Table 2) were identied in rosemary EOs using the three
volatilized with water vapor and separated from plant matrixes, as techniques. ILMSED, MHD, and HD enabled the detection of most
a result of microwave energy enhancing the extraction efciency volatile active compounds in EO of rosemary such as -pinene,
and speed due to the localized heating, pressure builds up within camphor, 1,8-cineole and camphene. As is illustrated in Fig. 5,
the cells of the sample and nally leading to a fast transfer of the the representative ingredient proportions depend on the isolation
compounds from the cells. The second phase (realized into 25 min) techniques. Lightly higher amounts of oxygenated compounds are
is an increasing line which represents the intern diffusion of the present in the EOs of the rosemary plant isolated by ILMSED and
essential oil from the midst of the particles towards the external MHD in comparison with HD.
medium involved by the intern warming of the water located in The monoterpene hydrocarbons are the main chemical group in
the plant cells. In this stage, the EO amount extracted represents the HD, ILMSED, and MHD EOs. -pinene, a monoterpene hydro-
nearly 14% of the total yield. The third phase corresponds to a hori- carbon, was the main abundant component in the EO extracted
zontal line which marks the end of the extraction process. Bousbia
et al. [13] have used microwave extraction to get EO from rosemary
and found a yield prole similar to that described here. The con- Table 2
ventional extraction technique HD presents three similar aspects Chemical compositions of EOs obtained by ILMSED, MHD and HD.
but different phases to those obtained with ILMSED. The end of the No. Compounds Relative peak area (%)
extraction process is reached after 180 min.
ILMSED MHD HD

Monoterpene hydrocarbons 54.77 55.38 56.83

3.4. Comparison of different extraction procedures 1 -Pinene 37.14 37.98 38.32
2 Camphene 6.56 6.72 7.93
3.4.1. Effect of different extraction procedures on the yields of CA, 3 Sabinene 0.69 0.65 0.76
4 -Pinene 1.32 1.88 1.53
RA, and EO
5 3-Carene 0.61 0.4 0.95
In the present study, ILMSED, MHD, HD, ILHE, and HRE tech- 6 Myrcene 2.01 1.95 1.83
niques were compared for their extraction yields of CA, RA, and 7 -Phellandrene 0.58 0.43 0.78
EO from rosemary. The results are shown in Table 1, the proposed 8 -Terpinene 0.97 1.25 0.84
approach not only obviously increased the extraction yield, but also 9 Limonene 4.89 4.12 3.89

dramatically reduced the extraction time. Oxygenated monoterpenes 32.95 32.46 31.16
The results indicated that the yields of CA and RA by ILMSED 10 1,8-Cineole 10.84 10.22 11.46
11 -Ocimene 0.54 0.42 0.23
were slightly higher than that of organic solvent (85% ethanol) HRE
12 Linalool 0.83 0.93 0.51
method, and much higher than that of regular MHD and HD meth- 13 trans-Pinocarveol 1.27 1.23 1.45
ods. The yields of EO by the ILMSED, MHD, and HD methods were 14 Camphor 11.01 11.39 10.86
similar, while the extraction completeness by HD depended to a 15 Borneol 4.39 4.43 3.76
large extent on the extraction time (4 h), the completed extraction 16 Verbenone 1.83 1.36 1.69
17 4-Terpineol 0.85 0.96 0.51
was achieved only 20 min by ILMSED and MHD. In order to further
18 -Terpineol 0.77 0.94 0.26
demonstrate the impact of microwave in the extraction, the regular 19 cis-Jasmone 0.62 0.58 0.43
heating extraction (ILHE) process was used to compare at the same Sesquiterpene hydrocarbons 2.86 2.88 3.64
condition. The results of ILHE had unsatisfactory extraction yields. 20 -Caryophyllene 1.71 1.78 1.95
ILMSED has so unique extraction mechanism including superheat- 21 -Humulene 0.34 0.23 0.64
ing, mass heating and fast heating, which is very different from HD, 22 -Bisabolene 0.81 0.87 1.05
ILHE just having conventional heating. Moreover, the developed Oxygenated sesquiterpenes 1.36 1.12 1.04
method takes advantage of the good solubility and extractability of 23 Caryophyllene oxide 1.05 0.97 0.35
ionic liquids, which leads to the simultaneous extraction of CA and 24 -Bisabolol 0.31 0.15 0.69
RA and gets perfect extraction yield. Other oxygenated compounds 1.80 1.85 1.07

In conclusion, compared with regular HRE and MHD technique, 25 Bornyl acetate 1.49 1.53 0.74
the proposed approach used only small amount of ionic liquids 26 Thujone 0.31 0.32 0.33
could obtain higher extraction yield in shorter extraction time, Extraction time (min) 20 20 240
which indicated that the ionic liquids were excellent extractant and Total oxygenated compounds 34.75 34.31 32.23
ILMSED was a more rapid and effective method for simultaneous Total non-oxygenated compounds 57.63 58.26 60.47
Yield (%) 92.38 92.57 92.70
preparation of CA, RA, and EO from rosemary.
8486 T. Liu et al. / J. Chromatogr. A 1218 (2011) 84808489

Fig. 5. The GCMS information of essential oil from rosemary by the ILMSED, MHD and HD methods including four main compounds: -pinene, camphor, 1,8-cineole and
camphene. Total ion current chromatogram was obtained during GCMS analysis of ILMSED oil.

from rosemary with equivalent relative amounts for all extrac- 3.5. Structural changes after extraction
tion methods: 37.14%, 37.98%, and 38.32%, respectively for ILMSED,
MHD, and HD. Camphor was the main oxygenated component in In order to study the structural alteration during the different
the EO isolated from rosemary leaves but the relative amounts extraction techniques and to understand the extraction mech-
differed for the different isolation methods. It is the most abun- anism, the plant samples were examined by scanning electron
dant oxygenated component of the ILMSED oil and MHD oil microscopy. Different extraction techniques produced distinguish-
11.01% and 11.39%, respectively, whereas the HD oil contains able physical changes on rosemary. Fig. 6 shows the micrographs
10.86%. of samples of raw materials (RM), ILMSED, MHD, HRE, and HD,
A critical observation of the oil compositions revealed that respectively. After ILMSED and MHD (20 min only), cells and cell
higher amounts of oxygenated compound (34.31%) are present walls have been greatly affected, which led the target to bare in
in the EO isolated by MHD in comparison with the oil extracted extraction solution that would dissolve it. And it can be observed
by HD (32.23%). This is probably due to the dimunition of ther- damages of the oil-containing glands. Glandular trichomes were all
mal and hydrolytic effects compared with hydrodistillation which already ruptured and collapsed during microwave treatment, and
uses a large quantity of water and is time and energy consuming. the EOs have almost completely escaped from oil cells. When the
EOs contain organic compounds that strongly absorb microwave glands were subjected to more severe thermal stresses and local-
energy. Compounds with high and low dipolar moments could be ized high pressures, as in microwave heating, pressure build-up
extracted in various proportions by microwave extraction. Organic within the glands could have exceeded their capacity for expan-
compounds like oxygenated compounds that have a high dipo- sion and caused their rupture more rapidly. In HRE and HD,
lar moment will interact more vigorously with microwaves and plant materials have a partial destruction of secreting glands and
can be extracted more easily in contrast with aromatic com- a part of them are still lled. In the two methods, the solvent
pounds which have low dipolar moments (like monoterpene transfers into the sample and extracts the compounds by per-
hydrocarbons) [13]. Microwave irradiation highly accelerated the meation and solubilisation processed under higher temperatures.
extraction process, but without causing considerable changes in Hence, little destruction of the microstructure of sample occurs
the volatile oil composition, a phenomenon already described and a few of slight ruptures took place on the surface of the
by Okoh et al. [5]. Furthermore, the EO of rosemary leaves sample.
isolated either by ILMSED or MHD contains the same domi- As FTIR spectra can provide useful information for identifying
nant components. Similar distribution of the chemical groups the presence of certain function groups or chemical bonds in a
was described in EO of rosemary by the proposed method of molecule or an interaction system, it was applied here to investigate
ILMSED (monoterpenes 54.77%, oxygenated monoterpenes 32.95% the changes in chemical structures of rosemary before and after
and sesquiterpenes 2.86%), in comparison of those of MHD extraction by various methods, respectively. Generally, reectance
(monoterpenes 55.38%, oxygenated monoterpenes 32.46%, and peaks between 1800 and 700 cm1 were selected as representative
sesquiterpenes 2.88%). Combining the results of Fig. 4b, we can con- peaks for known carbohydrate compounds [36]. The results of FTIR
clude that the approach of ILMSED highly accelerated the isolation spectra (see Fig. 7) showed that the signal situations and inten-
process, without causing considerable changes in the volatile oil sity of absorption bands at 1622, 1452, 1361, 1249, and 1160 cm1
composition. for rosemary were not apparently changed after ILMSED, compar-
Thus, ILMSED method offers the possibility for a better pro- ing to those after MHD HRE, and HD methods, which indicated that
duction of EO from the aromatic plant, which could be a good the chemical structures of carbohydrate compounds, including cel-
alternative for the isolation of EO from rosemary leaves. lulose, hemicellulose, lignin, and insoluble starch, were unbroken
 T. Liu et al. / J. Chromatogr. A 1218 (2011) 84808489 8487

Fig. 6. The scanning electron micrographs (20.0 m, 15.0 kV) of rosemary leaves. (a) Untreated sample, (b) after ILMSED 20 min, (c) after MHD 20 min, (d) after HRE 2 h, and
(e) after HD 4 h.

after extracted by the three methods. The reason of the results was time of CA and RA. After one week, the average recovery of CA
probably related to that water segregated the strong interactions and RA was 98.5399.42% and 97.5797.60%, respectively, which
between ILs and carbohydrate compounds of matrixes [3739]. indicated that CA and RA were stable in [C8mim]Br ionic liquid
Therefore, the chemical bonding interactions between ILs and sam- solutions.
ple matrixes were not obvious, which coincides with the study of
Du et al. [34].

3.6. Method validation

To evaluate the proposed ILMSED approach, some parameters

such as stability, repeatability and recovery were determined under
the optimized conditions.

3.6.1. Stability
The stability of CA and RA in [C8mim]Br aqueous solution
was evaluated by determining standard solution of CA and RA
after ILMSED and one week later. The recovery of CA and RA
was taken to evaluate the stability of CA and RA at the obtained
operating extraction conditions. As is shown in Table 3, the
results indicated that average complete recovery at the oper-
ating extraction conditions varied from 100.41% to 101.14% for Fig. 7. The FTIR spectrograms of rosemary leaves. (A) Untreated sample, (B) after
CA and 98.40% to 98.48% for RA with no change in retention HD 4 h, (c) after ILMSED 20 min, (d) after MHD 20 min, and (e) after HRE 2 h.
8488 T. Liu et al. / J. Chromatogr. A 1218 (2011) 84808489

Table 3
Stability studies of standards CA and RA under optimum ILMSED conditions: 700 W microwave power, 15 min extraction time, 10 mL solution volume prepared with 1 M
[C8mim]Br.

Concentration Initial Recovered RSD (%), Average Recovered RSD (%), Average
level concentration concentration n=3 recovery (%) concentration n=3 recovery (%)
(mg/mL) after ILMSED after one week
(mg/mL) (mg/mL)

CA RA CA RA CA RA CA RA CA RA CA RA CA RA

1 5.25 1.25 5.31 1.23 0.80 1.14 101.14 98.40 5.19 1.22 0.81 1.71 99.42 97.60
2 9.58 3.30 9.62 3.25 0.99 1.07 100.41 98.48 9.44 3.22 1.04 1.73 98.53 97.57

Table 4
The recovery of CA and RA from rosemary (n = 3).

Sample Polyphenolic content The amount of added The amount of the sample Recovery (%)
of the sample polyphenolic standards determined with added
determined (mg) (mg) standards (mg)

CA RA CA RA CA RA CA RA

1 31.98 3.8 2.65 0.55 34.44 4.32 95.84 96.34

2 31.98 3.8 10.55 1.54 42.26 5.29 98.28 97.03
3 31.98 3.8 15.80 2.10 47.36 5.80 97.91 95.52

3.6.2. Recovery shows a great promising prospect in the extraction and the sepa-
To evaluate the accuracy of the proposed method, standard ration of natural products.
solution of CA and RA were added to rosemary samples, at three lev-
els respectively. Under optimized conditions, samples with added Acknowledgements
standard solution were extracted by ILMSED, respectively, and then
determined by HPLC to examine the recovery of the promoted The authors thank anonymous reviewers for their insight-
method. As shown in Table 4, satisfactory results were found, with ful comments and careful corrections. The authors also thank
recovery values between 93.5% and 98.28%, 95.52% and 97.73% (for
the National Key Technology R&D Program (2006BAD18B04)
CA and RA, respectively). and the China-Germany Inter-Governmental Cooperation Program
(CHN3/028) for nancial support.
3.6.3. Repeatability
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