Recent Extraction Techniques for Natural

Products: Microwave-assisted Extraction and

Pressurised Solvent Extraction
Beatrice Kaufmann and Philippe Christen*
University of Geneva, School of Pharmacy, Laboratory of Pharmaceutical Analytical Chemistry, 20 bd dYvoy, CH-1211 Geneva 4,
Switzerland
In the last 10 years there has been an increased interest in using techniques involving microwave-assisted
extraction and pressurised solvent extraction in analytical laboratories. This review gives a brief overview
of both methods, and reports on their application to the extraction of natural products. The inuence of
parameters such as the nature of the solvent and volume, temperature, time and particle size of the matrix is
discussed. Through numerous examples, it is demonstrated that both techniques allow reduced solvent
consumption and shorter extraction times, while the extraction yields of the analytes are equivalent to or
even higher than those obtained with conventional methods. Copyright # 2002 John Wiley & Sons, Ltd.
Keywords: Microwave-assisted extraction; solidliquid extraction; supercritical uid extraction; pressurised solvent
extraction; accelerated solvent extraction; enhanced solvent extraction; natural products.
INTRODUCTION
A broad spectrum of solidliquid extraction (SLE)
techniques is widely used for the early purication of
natural products from plant material and micro-organ-
isms. Classically, SLE can be divided into traditional and
recent methods. Traditional methods include Soxhlet
extraction, maceration, percolation, turbo-extraction
(high speed mixing) and sonication. These techniques
have been used for many decades; however, they are very
often time-consuming and require relatively large
quantities of polluting solvents. Supercritical uid
extraction (SFE), microwave-assisted extraction (MAE)
and pressurised solvent extraction (PSE) are fast and
efcient unconventional extraction methods developed
for extracting analytes from solid matrixes. Numerous
review articles and textbooks have been published on
SFE (Lee and Markides, 1990; Westwood, 1993; King
and Bott, 1993) and a number are more particularly
dedicated to SFE of natural products (Castioni et al.,
1995; Smith, 1995, 1996; Jarvis and Morgan, 1997). The
present review deals with the application of MAE and
PSE to the extraction of secondary metabolites from plant
material.
MICROWAVE-ASSISTED EXTRACTION
Microwaves have been used since World War II
following the development of radar technology, and later
the rst commercial application of microwaves con-
cerned domestic ovens. The use of microwave energy as
a heating source in analytical laboratories started in the
late 1970s and was applied to acid digestions (Abu Samra
et al., 1975). The development of microwave assisted
extractions was rst reported by Ganzler and co-workers
(Ganzler et al., 1986a; Ganzler and Salgo, 1987).
Principle of the method and heating mechanism
Microwaves are electromagnetic radiations with a
frequency from 0.3 to 300 GHz (Camel, 2001). In order
to avoid interferences with radio communications,
domestic and industrial microwaves generally operate
at 2.45 GHz (Fig. 1). Owing to their electromagnetic
nature, microwaves possess electric and magnetic elds
which are perpendicular to each other. The electric eld
causes heating via two simultaneous mechanisms,
namely, dipolar rotation and ionic conduction (Thuery,
1992; Demesmay and Olle, 1993; Sinquin et al., 1993).
Dipolar rotation is due to the alignment on the electric
eld of the molecules possessing a dipole moment (either
permanent or induced by the electric eld) in both the
solvent and the solid sample. This oscillation produces
collisions with surrounding molecules and thus the
liberation of thermal energy into the medium. With a
frequency of 2.45 GHz, this phenomenon occurs
4.9 10
9
times per second (Ganzler et al., 1990; Sinquin
et al., 1993; Barnabas et al., 1995; Onuska and Terry,
1995) and the resulting heating is very fast. Indeed, the
larger the dielectric constant of the solvent (see Table 1),
the more optimal the heating (Jassie et al., 1997).
Consequently, unlike classical conductive heating
methods, microwaves heat the whole sample simulta-
neously (Fig. 2). In the case of extraction, the advantage
of microwave heating is the disruption of weak hydrogen
bounds promoted by the dipole rotation of the molecules.
A higher viscosity of the medium lowers this mechanism
by affecting molecular rotation (Sinquin et al., 1993;
Camel and Bermond, 1999). Furthermore, the migration
of dissolved ions increases solvent penetration into the
PHYTOCHEMICAL ANALYSIS
Phytochem. Anal. 13, 105113 (2002)
DOI: 10.1002/pca.631
Copyright # 2002 John Wiley & Sons, Ltd.
* Correspondence to: P. Christen, University of Geneva, School of Pharmacy,
Laboratory of Pharmaceutical Analytical Chemistry, 20 bd dYvoy, CH-1211
Geneva 4, Switzerland.
Email: philippe.christen@pharm.unige.ch
Received 26 October 2001
Revised 17 December 2001
Accepted 20 December 2001
matrix and thus facilitates the solvation of the analyte
(Ganzler et al., 1990). Ionic currents are also induced in
the solution by the electric eld. As the medium resists
these currents, frictions occur and heat is liberated by a
Joule effect. This phenomenon depends on the size and
charge of the ions present in the solution.
The effect of microwave energy is strongly dependent
on the nature of both the solvent and the solid matrix.
Solvents generally used cover a wide range of polarities,
from heptane to water. Most of the time, the chosen
solvent possesses a high dielectric constant and strongly
absorbs microwave energy, however, the extracting
selectivity and the ability of the medium to interact with
microwaves can be modulated by using mixtures of
solvents (Renoe, 1994). In some cases, the matrix itself
interacts with microwaves while the surrounding solvent
possesses a low dielectric constant and thus remains cold
(Jassie et al., 1997). This latter situation presents some
obvious advantages in the case of thermosensitive
compounds and has been successfully used for the
extraction of essential oils (Pare, 1990, 1991; Chen and
Spiro, 1994; Romele and Polesello, 1997). Indeed,
microwaves interact selectively with the polar molecules
present in glands, trichomes or vascular tissues. Loca-
lised heating leads to the expansion and rupture of cell
walls and is followed by the liberation of essential oils
into the solvent (Pare et al., 1994; Jassie et al., 1997;
Majors, 1998). This situation can also be obtained when a
dry sample has been re-hydrated before extraction (Pare,
1990; Pare et al., 1994; Majors, 1998). In fact, moisture
content is essential in MAE because water locally
superheats and promotes analytes to be liberated into
the surrounding medium (Onuska and Terry, 1993;
Budzinski et al., 1996; Jassie et al., 1997). In addition,
control of the water content of the matrix allows more
reproducible results.
Instrumentation
Two types of instruments are commercially available and
they use different approaches. The most common
procedure involves extraction in a closed vessel under
controlled pressure and temperature, whilst an alternative
approach uses an open extracting vessel under atmos-
pheric pressure. It must be strongly stressed that using a
domestic microwave oven for laboratory purposes should
not be considered. Application of microwave energy to
highly ammable organic solvents may cause serious
hazards. Furthermore, reproducibility may be poor with a
domestic device because of the lack of homogeneity of
the microwave eld. It is therefore seriously recom-
mended that only equipment approved for MAE be used.
Closed vessel systems. Such systems are generally
advised for digestions or acid mineralisations or for
extractions under drastic conditions, since the solvents
may be heated to ca. 100C above their atmospheric
boiling point (Barnabas et al., 1995; Jassie et al., 1997).
Both extraction speed and efciency are enhanced in this
procedure. Hazards occasioned by heating highly am-
mable solvents are overcome through the use of recent
security techniques such as high capacity exhaust fans,
solvent vapour detectors, or pressure-burst safety mem-
branes placed on each vessel (Demesmay and Olle, 1993;
Jassie et al., 1997). Figure 3 shows a schematic diagram
of a closed vessel system from CEM Corporation
(Matthews, NC, USA). In order to overcome the non-
homogeneity of the eld, the cells are placed on a rotating
carousel as in a domestic oven. The solvents can be
varied, and the pressure in the vessels essentially depends
on the volume and boiling point of the solvents used. The
temperature can be set at a xed value by adjusting the
microwave power. Typically, the cells are made of
Teon. In closed vessel systems, the maximal power
delivered is about 6001000 W (Pare, 1990, 1991;
Young, 1995), but the chosen power has to be set
correctly to avoid excessive temperatures leading to
possible solute degradation and overpressure problems.
The vessel must be cooled to room temperature before
opening: this is particularly important in the presence of
volatile solutes which can partition into the head-space,
but this step can considerably increase the overall
Figure 1. The electromagnetic spectrum.
Table 1. Dielectric constants and dipole moment values of
some commonly used solvents
Solvent
Dielectric constant
(20C)
Dipole moment
(25C) (Debye)
Hexane 1.89 <0.1
Toluene 2.4 0.36
Dichloromethane 8.9 1.14
Acetone 20.7 2.69
Ethanol 24.3 1.69
Methanol 32.6 2.87
Water 78.5 1.87
Figure 2. Scheme of the heating principle by conduction in
the classical method of extraction and by microwave
irradiation in microwave-assisted extraction.
106 R. KAUFMANN AND P. CHRISTEN
Copyright # 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 105113 (2002)
extraction time. Furthermore, an additional ltration or
centrifugation step is necessary in order to remove the
solid residue.
Open cells. These cells are quartz vessels topped by a
vapour condenser (Fig. 4). The system works at atmos-
pheric pressure, and the maximum temperature is
determined by the boiling point of the solvent used
(Renoe, 1994; Letellier et al., 1999). The solvent is
heated and reuxed through the sample, and in this case
the microwaves are focused on the sample placed into the
vessel allowing homogeneous and very efcient heating
(Demesmay and Olle, 1993). The sample to be extracted
can be placed into a Soxhlet-type cellulose cartridge in
order to avoid ltration steps, or may be directly dipped
into the solvent. Compared to closed vessel extractions,
open cells offer increased safety in sample handling and,
furthermore, they allow larger samples to be extracted. A
typical system is the Soxwave 3.6 commercialised by
Prolabo (Fontenay-sous-Bois, France) which operates at
percentage power increments from 10 to 100%, corre-
sponding to a maximum power of 250 W.
Applications to the extraction of natural products
Although many of the initial publications dealt with
natural product extraction, the majority of the applica-
tions concern the extraction of pollutants from environ-
mental matrixes. The use of MAE for environmental
analysis has recently been exhaustively reviewed (Dean
et al., 1999; Camel, 2000), and, therefore, only applica-
tions to natural products are reported in this review and
these are summarised in Table 2.
Closed vessels
Pyrimidine glycosides. The rst papers to report the use
of MAE for natural products were published by Ganzler
and co-workers (Ganzler et al., 1986a, b; Ganzler and
Salgo`, 1987) and concerned the extraction of vicine and
convicine from faba beans: these toxic pyrimidine
glycosides preclude the use of faba beans as a source of
nutritional proteins. Ground beans were suspended in a
methanol:water mixture (1:1, v/v), and the suspension
was subjected to two successive microwave irradiations
(30 s each) with a cooling step in between. No
degradation could be observed under these conditions,
but further irradiation was found to decrease the yield of
vicine and convicine. The yield obtained was 20% higher
than with the conventional Soxhlet extraction method.
Gossypol. As for faba beans, the presence of gossypol in
cotton seeds limits their use for human consumption or
animal feeding (Ganzler et al., 1986a; Ganzler and Salgo`,
1987). Extraction yields of gossypol were much higher
with three cycles of 30 s each of MAE than with the
traditional 4 h Soxhlet extraction, probably because the
compound is sensitive to high temperatures. This
phenomenon was conrmed by further MAE irradiations
which also lead to the degradation of gossypol.
Alkaloids. Sparteine, a lupine alkaloid, was extracted
from lupine seeds (Lupinus mutabilis) with methanol:a-
cetic acid (99:1, v/v) in a domestic microwave oven (see
caution above!), and the treatment (one to ve cycles of
30 s with a cooling step in between) gave 20% more
sparteine than was obtained with a shaken-ask extrac-
tion using the same solvent mixture for 20 min (Ganzler
et al., 1990).
Pyrrolizidine alkaloids were extracted from dried plant
material (Senecio sp.) by Bicchi et al. (1992) using closed
vessel technology with temperature feed-back control
(65100C). Qualitative and quantitative chromato-
graphic results were identical to those obtained with
traditional techniques, with a signicant reduction in
extraction time and solvent consumption with good
reproducibility.
Terpenes. Five terpenic compounds (linalool, a-terpi-
neol, citronellol, nerol and geraniol) associated with
grape (Vitis vinifera) aroma were extracted from must
samples by MAE (Carro et al., 1997). Four variables
(extracting solvent volume, extraction temperature,
amount of sample and extraction time) were optimised
by means of two- and three-level factorial designs.
Several conditions were xed, such as the extraction time
(10 min) and the applied power (475 W). The solvent
volume appeared to be the only statistically signicant
factor, but was limited to 15 mL by the cell size. The
highest extraction yield was obtained with both the
solvent volume and the temperature at their maximum
tested values. In contrast, the sample amount had to be
minimised in order to obtain the best recoveries. The nal
Figure 3. Schematic diagram of a closed-vessel microwave
system for extraction.
Figure 4. Schematic diagram of an open focused-microwave
system for extraction.
MICROWAVE-ASSISTED EXTRACTION 107
Copyright # 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 105113 (2002)
optimised extraction conditions were as follows: 5 mL
sample amounts extracted with 10 mL of dichloro-
methane at a temperature of 90C for 10 min with the
microwave power set at 50% (475 W).
Essential oils. MAE of essential oils has been patented
by Pare (1991). The samples were suspended in hexane
and the microwaves reached the inner glandular and
vascular systems of the plant material. Owing to the high
moisture content of these structures they were heated
almost specically and this promoted disruption of cell
membranes releasing the analytes into the solvent. The
same author also patented the extraction of volatiles from
biological material (Pare, 1990); the method involved the
exposure of oil-containing cellular matter or glandular
systems to microwaves and the dissolution of essential oil
in a suitable organic solvent.
Another patent has been deposited by Pare (1994)
concerning the extraction of volatile oils from plants and
biological material. This procedure was applied by Chen
and Spiro (1994) for the extraction of essential oils from
rosemary and peppermint leaves suspended in hexane,
ethanol or mixtures of the two solvents. Scanning
electron micrographs of the leaves showed structural
changes in the oil-containing glands after microwave
extraction: some of them were found to be collapsed and
others completely disintegrated. Two distinct extraction
mechanisms are plausible, one involving diffusion of the
essential oil across the gland wall and the other involving
rupture of the gland and liberation of the constituents into
the solvent. Either of the two phenomena may be
prominent according to the different maturation stages
of the glands.
MAE has also been compared to classical hydrodis-
tillation for the extraction of essential oils from 10
different plant species using a domestic microwave oven
(Collin et al., 1991). The yields were generally similar,
but the chromatographic proles varied dramatically,
especially with respect to the ratios between the different
substances.
The microwave technique was used to promote
desorption of essential oil from Lippia sidoides with a
modied commercial microwave oven (Craveiro et al.,
1989). Fresh leaves were irradiated in the oven and the
Table 2. Application of MAE to natural product extraction
Compounds Matrix System Extraction conditions Reference
Vicine, convicine
(pyrimidine glycosides)
Faba beans (Vicia faba) Domestic oven Methanol:water (1:1);
two successive
irradiations (30 s) with
an intermediate cooling
step
Ganzler et al. (1986a,
1986b); Ganzler and
Salgo (1987)
Gossypol Cotton seeds Domestic oven Three cycles of
irradiation (30 s) with
cooling steps in
between
Ganzler et al. (1986a);
Ganzler and Salgo
(1987)
Sparteine (alkaloid) Lupine seeds Domestic oven Four cycles (30 s) with
cooling steps in
between
Ganzler et al. (1986b,
1990)
Terpenes (linalool,
terpineol, citronellol,
nerol and geraniol)
Must (Vitis vinifera) Closed vessels 10 mL
dichloromethane;
475 W; 10 min; 90C
Carro et al. (1997)
Essential oils Monarda stulosa,
Allium sp.
Modied domestic
oven
Hexane Pare (1990)
Volatile oils Mentha piperita, Thuja
occidentalis
Modied domestic
oven
Hexane, alkanes
(transparent solvents)
Pare (1994)
Essential oils Rosemary and
peppermint leaves
Domestic oven Hexane, carbon
tetrachloride, toluene;
750 W; <60 s
Chen and Spiro (1994)
Essential oils Plant leaves Domestic oven Hexane; <60 s Collin et al. (1991)
Essential oils Fresh leaves of Lippia
sidoides
Domestic oven High cooking level; 5
min
Craveiro et al. (1989)
Carotenoids Paprika powder Closed vessels 50 W; 120 s; <60C Csiktunadi Kiss et al.
(2000)
Taxanes (paclitaxel) Needles of Taxus sp. Closed vessels 5 g fresh needles pre-
soaked with 5 mL water
prior to extraction with
10 mL of 95% ethanol;
100% power; 54 s; 85C
Incorvia Mattina et al.
(1997)
Ergosterol Fungal contaminations Domestic oven 375 W; 35 s Young (1995)
Withanolides Iochroma gesnerioides
leaves
Open cell, focused 100 mg material pre-
soaked with 0.6 mL
water prior to
extraction with 5 mL
methanol; 25 W; 40 s
Kaufmann et al. (2001a)
Cocaine and
benzoylecgonine
Erythroxylum coca
leaves
Open cell, focused Methanol; 125 W; 30 s Brachet et al. (2002)
Alkaloids Senecio sp. Closed vessels 65100C Bicchi et al. (1992)
108 R. KAUFMANN AND P. CHRISTEN
Copyright # 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 105113 (2002)
volatiles were extracted by an air current passing through
the system. The yields obtained after 5 min were
comparable to those obtained after 6090 min of steam
distillation, and both qualitative and quantitative perfor-
mances were found similar to the conventional method.
Carotenoids. The MAE of carotenoids from paprika has
been optimised by Csiktusnadi Kiss et al. (2000). Thirty
different water:organic solvent mixtures were evaluated,
and both extraction efciency and selectivity were
signicantly dependent on the dielectric constant of the
extracting solvent mixture. The extractions had to be
performed at temperatures lower than 60C because of
the possible rearrangement of molecules at higher
temperatures.
Steroids. Recently, a system has been developed
simultaneously to saponify and extract ergosterol by
MAE (Young, 1995). The determination of this com-
pound in ltered air or contaminated corn or dust can be
used as an indicator of fungal contamination. The
samples were placed in culture tubes containing 2 mL
methanol and 0.5 mL 2 M sodium hydroxide. Microwave
irradiation was applied at 375 W for 35 s and the samples
were cooled for 15 min before neutralisation with 1 M
hydrochloric acid followed by pentane extraction. It was
demonstrated that only 3040 s were sufcient to extract
ergosterol quantitatively and that the yield was similar to
or even higher than that obtained with the traditional
methanolic extraction followed by alkaline saponication
and pentane extraction.
Taxanes. The application of microwave energy to the
extraction of taxanes from Taxus biomass was reported
by Incorvia Mattina et al. (1997). Various parameters,
including temperature, extraction time, solvent choice
and water content were investigated in order to optimise
extraction efciency. Recoveries of taxane reached 100%
of the conventional method when the biomass was
freeze-dried to less than 10% moisture and pre-soaked
with water prior to extraction using 95% ethanol. As
some degradation of paclitaxel occurred at temperatures
above 115C, the temperature was set at 85C and MAE
was performed for 54 s. The extracts were quantitatively
and qualitatively equivalent to those obtained with the
conventional extraction method, but with considerable
reduction of both extraction time and solvent consump-
tion.
Open vessels
Very few papers have been published concerning the
focused microwave extraction of natural products.
Alkaloids. Cocaine and benzoylecgonine were extracted
from coca leaves (100 mg) by focused MAE (Brachet et
al., 2002). The extraction was optimised by taking into
account several parameters such as the nature of the
extracting solvent, particle size distribution, sample
moisture, applied microwave power and radiation time.
MAE was found to generate similar extracts to those
obtained by conventional SLE but in a more efcient
manner. Indeed, 30 s were sufcient to extract cocaine
quantitatively from leaves, using methanol as solvent and
a microwave power of 125 W.
Steroids. Focused MAE was applied to the extraction of
withaferin A, iochromolide and withacnistin from the
leaves of Iochroma gesnerioides (Kaufmann et al.,
2001a). Withanolides are steroidal lactones derived from
an ergostane-type skeleton. Six extraction variables, i.e.
the nature and volume of the extracting solvent, sample
moisture, extraction time, power of irradiation and
particle size of the matrix were investigated. The most
favourable conditions were obtained using powdered
plant material (<220 mm), previously moistened with
water for 15 min, and extracted with methanol for 40 s at
25 W. The extraction yield obtained with the optimised
method was comparable to that achieved with a Soxhlet
extraction.
PRESSURISED SOLVENT EXTRACTION
Pressurised solvent extraction (PSE) is a SLE technique
which has been developed as an alternative to current
extraction methods such as Soxhlet, maceration, percola-
tion or reux, offering advantages with respect to
extraction time, solvent consumption, extraction yields
and reproducibility. PSE uses organic solvents at elevated
pressure and temperature in order to increase the
efciency of the extraction process. Increased tempera-
ture accelerates the extraction kinetics and elevated
pressure keeps the solvent in the liquid state, thus
enabling safe and rapid extractions. Furthermore, high
pressure forces the solvent into the matrix pores and
hence should facilitate extraction of analytes. High
temperatures decrease the viscosity of the liquid solvent,
allowing a better penetration of the matrix and weakened
solutematrix interactions. In addition, elevated tempera-
tures enhance diffusivity of the solvent resulting in
increased extraction speed.
Instrumentation
ASE
2
(accelerated solvent extraction) is a form of PSE
and the rst instrument was commercialised by the
Dionex Corporation (Sunnyvale, CA, USA) in 1994
(Richter et al., 1996; Reighard and Olesik, 1996). More
recently, ESE (enhanced solvent extraction) or PSE
instruments have appeared (Bautz et al., 1998).
Accelerated solvent extraction. Figure 5 shows a
Figure 5. Scheme of an accelerated solvent extraction (ASE)
system (from Richter et al., 1996).
MICROWAVE-ASSISTED EXTRACTION 109
Copyright # 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 105113 (2002)
scheme of an ASE apparatus. A solid or semi-solid
sample is placed into a stainless steel extraction cell
which is lled with solvent and heated (50200C) in an
oven (Hoer et al., 1995a; Richter et al., 1996). The
heating process generates solvent expansion and thus
pressure in the extraction cell, typically in the region of
5003000 psi. In order to prevent over-pressurisation of
the cell, a static valve pulses open and closed auto-
matically when the cell pressure exceeds the set point.
The solvent that escapes during this venting is collected
in a vial. A static extraction stage of about 510 min is
followed by pumping fresh solvent through the system to
rinse the sample and the tubing. All the solvent present in
the system is then purged with a compressed gas,
generally nitrogen (Richter et al., 1995, 1996; Ezzel et
al., 1995; Levy, 1998), and the total solvent volume
(extractant and rinsings) is collected in the vial. The
Dionex apparatus offers the possibility of automation,
and up to 24 samples can be sequentially extracted. Cells
can be of different sizes: 1, 5, 11, 22 and 33 mL (Richter
et al., 1995; Morf et al., 1998; Richter, 1999).
Enhanced solvent extraction. An alternative approach
to the above is to perform accelerated solvent extractions
with a supercritical uid extraction (SFE) apparatus (see
Fig. 6; Li et al., 1998) in which the pressure is controlled
by the restriction device. The extraction can be static or
dynamic. In the dynamic mode, fresh solvent is
continuously pumped through the sample at elevated
temperature and pressure (Bautz et al., 1998). According
to Ficks rst law of diffusion, the transfer rates are
accelerated, leading to improved extraction efciencies
and reduced extraction times. Carbon dioxide can be used
to ush the remaining solvent out of the extraction
chamber (Ashraf-Khorassani et al., 1999). In this case, a
wide range of cell sizes is available, from 0.2 to a few
hundreds of millilitres. Furthermore, the possibility to
perform either PSE or SFE using the same apparatus has
obvious economical advantages.
Environmental applications
To date most of the published applications of ASE have
been in the area of environmental research. The tech-
nique was applied successfully to the extraction of
different pollutants from various environmental matrixes
(Hoer et al., 1995b). After comparison with the
corresponding reference methods, recoveries and preci-
sion were generally found equivalent, or even better.
Most papers stress the shorter extraction times and lower
solvent consumption of ASE in comparison to other
conventional preparation techniques of environmental
samples with the exception of SFE (Richter et al., 1995;
Jensen et al., 1996; Kreisselmeier and Durbeck, 1997;
Fisher et al., 1997). ASE and SFE can be considered as
complementary, the rst allowing the extraction of polar
compounds, while the second is more selective for
compounds of low or medium polarity. The ASE
methodology is accepted in the US EPA SW-846 Method
3545A for selected priority pollutants from environmen-
tal matrixes (Richter et al., 1996; Levy, 1998).
Applications to the extraction of natural products
Very few applications of ASE have been published in the
eld of natural product research and the major contribu-
tions are summarised in Table 3.
An evaluation of ASE has been made for the extraction
of various metabolites covering a large range of
structures and polarities (curcuminoids, saponins, avo-
nolignans, terpenes) present in different vegetal matrixes
such as leaves, roots, fruits, herbs and rhizomes (Benthin
et al., 1999). Performances were compared to corre-
sponding European Pharmacopoeia methods. Yields
were found to be equivalent or even higher with ASE,
with a reduction in the extraction time (especially when
consecutive extractions with solvents of increasing
polarity were made) and solvent consumption (by a
factor from two to ve), and with good reproducibility
probably occasioned by the minimal sample handling
required during the extraction procedure.
Curcuminoids. Three 6 min ASE cycles with methanol
at 80C were carried out on turmeric rhizomes (Curcuma
xanthorrhiza). Almost all of the curcuminoids present in
the sample were extracted during the rst cycle, and the
second and third extracts afforded only minimal
additional curcuminoids. As the amount determined by
the corresponding Pharmacopoeia method was much
lower, the authors assumed that the latter method was not
exhaustive.
Saponins. Horse chestnut (Aesculus hippocastanum)
contains a saponin mixture called aescin. The Pharma-
copoeia method for the extraction of this material
involves 3 h defatting followed by 3.5 h of maceration
in 65% methanol at room temperature and a 30 min
reux. A preliminary ASE defatting step (two cycles of
5 min each with dichloromethane at 100C) was followed
by extraction of saponins with 65% methanol at 100C in
two 6 min cycles. As previously mentioned, ASE
recovery was higher than the corresponding Pharmaco-
poeia method.
Flavonolignans. Silybin is a avonolignan contained in
milk thistle fruit (Silybum marianum) and it possesses
hepatoprotective properties. A single ASE treatment with
20 mL hexane (for defatting) followed by 5 min in
methanol lead to the quantitative extraction of the
avonolignans. The amounts extracted were similar to
those obtained using the traditional method which
Figure 6. Scheme of a supercritical uid extraction (SFE)
system slightly modied for application in pressurised
solvent extraction (PSE).
110 R. KAUFMANN AND P. CHRISTEN
Copyright # 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 105113 (2002)
consists of a 4 h defatting step (100 mL petroleum ether)
followed by Soxhlet extraction with 100 mL methanol.
With ASE, the solvent consumption was reduced by a
factor of 5.
Terpenes. Essential oil of thyme (Thymus vulgaris) was
obtained by hydrodistillation and, simultaneously, the
plant material was extracted by ASE. Thymol (the major
component) was quantied by GC-FID and comparable
results were demonstrated for both extraction methods.
The ASE conditions were one 5 min cycle with hexane at
50C, followed by one 5 min cycle with dichloromethane
at the same temperature. Exhaustive extraction of the
essential oil by hydrodistillation required 2 h.
Taxanes. ASE with methanol was used for the extraction
of taxanes from the bark of Taxus cuspidata (Kawamura
et al., 1999) and permitted the extraction of paclitaxel,
baccatin III and 10-deacetylbaccatin III (10-DAB) in
higher yields than could be obtained using classical SLE
(0.10 MPa; 2 24 h; 22C). For ASE various tempera-
tures, pressures, extraction times and solvent composition
were evaluated. The recoveries of taxanes were highest in
the temperature range 100150C for paclitaxel and 120
150C for 10-DAB. Degradation problems occurred at
temperatures greater than 160C. The yield hardly
changed within the pressure range 1.0120.27 MPa,
when extraction was performed at 150C for 15 min.
Particular attention has been given to ASE performed
with water. Paclitaxel, although reported to be poorly
soluble in this solvent, showed high recovery some 50
times higher than that obtained with cold water extraction
(22C; 48 h) and ve times higher than with hot water
extraction (100C; 1 h). Extraction at 10.13 MPa for
15 min at a temperature within the range 130140C for
paclitaxel, and 120130C for 10-DAB, gave the best
recoveries (Kawamura et al., 1999). Various methanol:
water ratios were also tested and it was shown that the
higher the proportion of water the greater the recovery of
overall extract from the bark: the highest yields of
taxanes were obtained with a methanol:water ratio of
90:10.
Naphthodianthrones. ASE was applied to the recovery
of naphthodianthrones (e.g. hypericin) from extracts and
pharmaceuticals containing St Johns wort (Hypericum
perforatum; Morf et al., 1998; Benthin et al., 1999).
These compounds are known to show poor solubility in
most organic solvents. The inuence of different par-
ameters on extraction efciency, such as solvent, time,
pressure, temperature and presence of lling material,
was studied. The results obtained were compared with
those of conventional methods (i.e. sonication and
Soxhlet extraction) which are time consuming and
require a preliminary pre-extraction step with dichloro-
methane followed by different treatments before a
colorimetric measurement. A rst ASE cycle with
dichloromethane at 100C was performed in order to
Table 3. Application of ASE or PSE to natural product extraction
Compounds Matrix System Extraction conditions Reference
Taxanes (paclitaxel,
baccatin III and 10-
deacetylbaccatin III)
Taxus cuspidata bark ASE Methanol:water (90:10); 10.13
MPa; 15 min; 150C
Kawamura et al. (1999)
Naphthodianthrones
(hypericin)
St John's wort herb,
extracts,
pharmaceuticals
ASE Methanol; 100 bar; two cycles of
5 min with Extrelut in the cell;
40C
Morf et al. (1998)
Naphthodianthrones St John's wort herb
(Hypericum
perforatum)
ASE Defatting with dichloromethane;
5 min; 100C; followed by
methanol extraction; three cycles
of 5 min; 50100C
Benthin et al. (1999)
Saponins Horse chestnut seeds
(Aesculus
hippocastanum)
ASE Defatting with dichloromethane;
two cycles of 5 min; 100C;
followed by extraction with 65%
methanol; two cycles of 6 min;
100C
Benthin et al. (1999)
Terpenes Thyme (Thymus
vulgaris)
ASE Hexane; one cycle of 5 min; 50C;
followed by dichloromethane;
one cycle of 5 min; 50C
Benthin et al. (1999)
Flavonolignans Milk thistle fruit
(Silybum marianum)
ASE Defatting with hexane (20 mL);
one cycle; 100C; followed by
extraction with methanol
(20 mL); one cycle of 5 min;
100C
Benthin et al. (1999)
Curcuminoids Turmeric rhizome
(Curcuma
xanthorrhiza)
ASE Methanol; one cycle of 6 min;
80C
Benthin et al. (1999)
Xanthones and avanones Osage orange tree
(bark) (Maclura
pomifera)
ASE Dichloromethane; 13.8 MPa;
three cycles of 5 min and 90 s
purge; 40, 80 or 100C
Da Costa et al. (1999)
Cocaine and
benzoylecgonine
Erythroxylum coca
leaves
PSE Methanol; 20 MPa; 1 mL/min;
10 min; 80C
Brachet et al. (2001)
Withanolides Iochroma
gesnerioides leaves
PSE Methanol:water (1 :1); 25 MPa;
0.5 mL/min; 10 min; 100C
Kaufmann et al. (2001b)
MICROWAVE-ASSISTED EXTRACTION 111
Copyright # 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 105113 (2002)
defat the plant material, and this was followed by three
cycles (5 min each) with methanol at a temperature
ranging from 50 to 100C. The total amounts extracted by
ASE were greater than those obtained using classical
methods, and the precision was around 1%.
Xanthones and avanones. SLE, SFE and ASE have
been compared with respect to the recovery of several
phenolic compounds from bark of the Osage orange tree
(Maclura pomifera; Da Costa et al., 1999). The ASE
involved three cycles of 5 min static extraction at 13.8
MPa with 5 min equilibration time in between and a 90 s
purge. Three temperatures (40, 80 and 100C) were
tested, and about 15 mL dichloromethane was used for
each extraction. The ASE required 35 min compared with
45 min for SFE (using carbon dioxide modied with 20%
methanol) or 48 h for solvent extraction. Both SFE and
ASE extracted xanthones and avanones from the plant
material at similar or higher yields than those obtained
with SLE.
Alkaloids. PSE was applied to the rapid extraction of
cocaine and benzoylecgonine from coca leaves (Brachet
et al., 2001). Several parameters, including the nature of
the extracting solvent, the pressure, temperature, extrac-
tion time, addition of alkaline substances and the granular
nature of the sample, were investigated in order to nd
the best extraction conditions. It was demonstrated that
10 min were sufcient to extract cocaine quantitatively at
80C and 20 MPa. Furthermore, addition of an alkaline
substance did not improve cocaine recovery, since
degradation occurred during the extraction process.
Steroids. A PSE method was developed for the recovery
of three withanolides (withaferin A, iochromolide, and
withacnistin) from the leaves of Iochroma gesnerioides
in the dynamic mode (Kaufmann et al., 2001b) and the
performance of the method was compared to traditional
Soxhlet extraction. The inuence of the nature of the
solvent, the ow rate, the pressure and the temperature of
the extracting solvent, as well as the particle size of the
plant material, on the recovery of analytes was
investigated. A 1:1 mixture of methanol:water at a ow
rate of 0.5 mL/min was found to be the most efcient
extracting solvent and allowed a quantitative extraction
in 10 min. PSE produced similar results to Soxhlet in
terms of recovery, repeatability and selectivity; however,
both total handling time and solvent consumption were
dramatically reduced with PSE.
CONCLUSIONS
MAE and PSE are emerging as attractive alternatives to
conventional extraction methods such as Soxhlet,
percolation, digestion, extraction under reux, sonication
and in some cases steam distillation. Initially employed
as a digestion method for different sample types such as
environmental, biological and geological matrixes, MAE
is now widely accepted in analytical laboratories. The
main advantage of MAE resides in the performance of the
heating source. The high temperatures reached by
microwave heating reduces dramatically both the extrac-
tion time and the volume of solvent required. PSE works
according to the principle of SLE with elevated
temperature and high pressures in order to keep the
solvent in a liquid state. Enhanced diffusivity of the
solvents leads to an increase in extraction speed and
efciency. With both MAE and PSE, recoveries of
analytes and reproducibilities are improved and, there-
fore, both methods should be considered as interesting
alternatives with the limitation that experimental condi-
tions must be chosen in order to avoid possible thermal
degradation. Finally the costs of the specialised equip-
ment (especially PSE) may also inuence the choice of
the extraction technique (Camel, 2001).
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