Analytica Chimica Acta 703 (2011) 8–18

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Analytica Chimica Acta

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Review

Pressurized liquid extraction as a green approach in food and herbal plants

extraction: A review
Arwa Mustafa ∗ , Charlotta Turner
Center for Analysis and Synthesis, Department of Chemistry, Lund University P.O. Box 124, SE-221 00, Lund, Sweden

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

Article history: Pressurized liquid extraction is a “green” technology for the extraction of nutraceuticals from foods
Received 3 February 2011 and herbal plants. This review discusses the extraction principles and the optimization of the extrac-
Received in revised form 1 July 2011 tion parameters that improves the extraction efficiency. The use of different solvent mixtures and other
Accepted 2 July 2011
extraction additives to enhance the efficiency of the extraction are discussed. Dynamic mode of extrac-
Available online 20 July 2011
tion in Pressurized liquid extraction, and the use of combined and hyphenated sample preparation and
analytical techniques are presented. This work discusses how different studies used Pressurized liquid
Keywords:
extraction to enrich phenolic compounds, lignans, carotenoids, oils and lipids, essential oils and other
Pressurized liquid extraction
Pressurized hot water extraction
nutraceuticals from foods and herbal plants.
Food © 2011 Elsevier B.V. All rights reserved.
Herbal plants
Nutraceuticals
Extraction parameters
Review article

Arwa Mustafa is a postdoctoral fellow in Analytical
Chemistry at Lund University in Sweden. She obtained
her PhD in food science (Acrylamide in bread, pre-
cursors; formation and reduction) from the Swedish
University of Agricultural Sciences in 2008 and her
M.Sc from the University of Leeds, UK in 2000. From
2008–2010, Arwa was a postdoctoral fellow at the
Analytical Chemistry Department, Uppsala Univer-
sity. Her research interest is extraction and particle
formulation of bioactive compounds and nutraceuti-
cals using Pressurized Fluid Extraction techniques and
Supercritical Carbon Dioxide technology.
Charlotta Turner is a senior lecturer in Analytical
Chemistry at Lund University in Sweden. She obtained
her PhD in analytical chemistry from Lund Univer-
sity in 2001. Turner went for a postdoc sejour at the
U.S. Department of Agriculture in Berkeley, CA, USA
(2001–2004), after which returned to Sweden and
Uppsala University (2004–2009), where she also initi-
ated her Green Technology Group. Charlotta Turner’s
research interests are focused on super- and sub-
critical fluid technology, antioxidant speciation in
plants and green analytical chemistry.

1. Introduction
The application of green technology aims to preserve the natural
environment and its resource, and to limit the negative influence
of human involvement. The philosophy of Green chemistry is to
develop and encourage the utilization of procedures that reduce
and/or eliminate the use or production of hazardous substances.
Anastas and Warner initially presented the principals of green
∗ Corresponding author. Tel.: +46 704350962; fax: +46 46 222 8209.
E-mail address: Arwa.Mustafa@organic.lu.se (A. Mustafa).
chemistry [1–4]. One way to adapt to the principles of green chem-
istry is to reduce the use of harsh organic solvents, and to facilitate
and encourage the use of novel extraction techniques that are
known to be more environmentally friendly. Traditional extrac-
tion techniques include: Soxhlet extraction, sonication, blending
and solid–liquid extraction. These techniques require long extrac-
tion times, and large amounts of samples, sorbents and organic
solvents, of which the later are often costly to purchase and dis-
pose, in addition to their negative environmental impact and other
human health issues [5,6]. The primary drawback of the traditional
extraction methods is that the obtained final extracts often require

0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2011.07.018
 A. Mustafa, C. Turner / Analytica Chimica Acta 703 (2011) 8–18
Fig. 1. Positioning of the analyte in the sample matrix.
subsequent concentration and clean-up prior to analysis. Further-
more, when considering the extraction of bioactive compounds that
are sensitive, thermolabile and are found in low concentrations,
traditional extraction techniques would not be the most suitable
option. This is due the fact that those compounds generally have
selectivity with a probable low extraction yields. To obtain those
bioactive compounds and natural food ingredients in an environ-
mentally friendly way, a green extraction approach is required.
Pressurized liquid extraction (PLE), especially using water as a sol-
vent, is an emerging greener technology compared to conventional
extraction techniques. The objective of this paper is to give an
overview of extraction fundamentals and in particular using PLE.
This paper explores how different studies have used and optimized
the main parameters of PLE to extract bioactive compounds and
nutraceuticals from foods and herbal plants.
2. Principle of extraction
The theory and principle of extraction have been reviewed in
comprehensive studies [6–12]. Briefly, the efficiency of the extrac-
tion depends on the nature of the sample matrix, the analyte to
be extracted and the location of the analyte within the matrix. The
process of extraction of heterogeneous samples was illustrated by
Pawliszyn in 2003 by a model that assumes that the sample particle
is porous and is surrounded by an organic layer. The extraction and
recovery of the analyte from the sample matrix can be expressed
in few steps. Initially, to be able to remove the analyte from the
extraction vessel, the compound is first desorbed from its site in
the sample matrix; then it is diffused through the organic part of
the matrix to be able to reach the matrix–fluid interface. At this
stage the analyte is distributed into the extraction phase, then it
diffuses through the extraction phase that is present within the
pore, thereafter it reaches the part of the extraction phase that is
affected by convection. The final stage of the extraction process
is collection of the extracted analyte [6,13]. A critical step in the
extraction process is the positioning and the status of the analyte
within the sample matrix; five different positions have been postu-
lated (Fig. 1); adsorbed to the surface of the matrix (1); dissolved in a
solvent pore and/or adsorbed at the surface (2); dissolved/adsorbed
in a matrix micro/nano-pore (3); chemically bonded to the matrix
(4); or dissolved in a bulk solution (5) [13,14]. The rate-limiting
step in the extraction process depends on the nature of the matrix
to be extracted. In environmental applications, desorption step is
usually the rate-limiting step, since solute–matrix interactions are
difficult to overcome, for example in natural sediments, soils and
sludge. In plant materials, the rate-limiting step is more commonly
the solubilization or the diffusion steps [6,11,15].
9
3. Pressurized liquid extraction (PLE)
PLE is a technique that involves extraction using liquid sol-
vents at elevated temperature and pressure, which enhance the
extraction performance as compared to those techniques carried
out at near room temperature and atmospheric pressure [6,16].
The merits of enabling the use of solvents at temperatures above
their atmospheric boiling point is the enhanced solubility and mass
transfer properties. Dionex Corporation first introduced PLE in 1995
at the Pittcon Conference, where it was introduced as Accelerated
Solvent Extraction Technology (ASE® ). This technique is also known
as pressurized liquid extraction; pressurized solvent extraction;
accelerated solvent extraction and enhanced solvent extraction.
In case when water is used as the extraction solvent, the tech-
nique is referred to as Pressurized Hot Water Extraction (PHWE),
sub-critical water extraction or superheated water extraction. In
this paper, these techniques will be referred to as PLE and PHWE,
respectively.
3.1. Equipment
Recently, [17] gave a comprehensive overview of the principles
and instrumentation of PLE. Briefly, there are two main set-ups
for PLE, static and dynamic instruments. In the dynamic setup, the
extraction solvent is continuously pumped through the sample ves-
sel. The dynamic system requires high-pressure pumps. As a matter
of fact there are no commercial dynamic PLE systems available
at the market. However, a new instrument (ASE-350® ) provides
both static and dynamic modes in the same run. This is featured by
the ability of the instrument to introduce fresh solvents during the
extraction process (Bruce Richter, personal communication 2011).
In the static set-up, the extraction process consists of one or several
extraction cycles with replacement of the solvent between cycles.
While in the dynamic (flow) mode of operation, the flow rate is
set during the static time, where the pump delivers solvent at a
constant flow rate. A wide range of extraction temperatures can be
applied in PLE, it usually ranges from room temperature to 200 ◦ C
and the pressure range is 35–200 bar. Depending on the nature of
the sample matrix, filter paper, dispersing or drying agents might be
used to facilitate the efficiency of the extraction. Hydromatrix is an
inert diatomaceous earth used both as dispersing and/or dehydrat-
ing agent during extraction [18]. The presence of moisture in the
sample matrix affects the efficiency of extraction, therefore hydro-
matrix is used to adsorb water from the matrix, which ultimately
enhances the extraction process. Glass beads could be used as dis-
persing agents when there is no need for moisture absorption from
the sample. Dispersion agents are also used to fill up the extrac-
tion cell and reduce the consumption of solvents by reducing cell
volume. In most PLE equipments, when the set parameters of the
extraction temperature and pressure are reached, the extraction is
then performed in a static mode for a predetermined time, a com-
mon range is 5–15 min that is done in different cycles. At the end
of the last extraction cycle and to avoid any loss or memory effects,
the sample cell is purged with inert gas to wash off the solvent from
the cell and the tubing into the collection vial. Most of the extrac-
tion cells are made of 316L-stainless steel, which restrict extraction
at extreme pH, however, the new PLE extraction instrument has
extraction vessels that are made of dionium and the tubing down-
stream the extraction chamber are pH hardened, i.e. can withstand
a wide pH-range. This new feature may allow in-vessel treatment
during the extraction, e.g. saponification during lipid extraction.
In the static extraction mode, the critical factors are the tem-
perature and time of the extraction. The efficiency of the extraction
depends on the solubility of the analyte in the extraction solvent
and on the partitioning of the target-compound between the water
and the extraction solvent. A suggested drawback of the static
10 A. Mustafa, C. Turner / Analytica Chimica Acta 703 (2011) 8–18
Fig. 2. Extraction yield (%) vs extraction time (min).
extraction mode is that complete extraction might not be achieved
due to the limited volume of the extraction fluid [15,19]. How-
ever, using more extraction cycles could be a strategy to surmount
this limitation. The extraction process could be explained in two
stages as is commonly explained in SFE, first it starts as solubility-
controlled then followed by a diffusion-controlled phase [20]. In
dispersion-controlled sample matrices usually there is strong inter-
actions between matrix and analytes, or long diffusion paths for the
analytes to pass through the sample matrix. In this case the tem-
perature of the solvent and particle size might be critical factors
to enhance the extraction efficiency. Increasing the temperature
and/or reducing the particle size will most likely prevent extraor-
dinary long extraction times. While in solubility-controlled sample
matrices, the analyte–matrix interactions are quite weak and the
extraction rate mainly depends on the partitioning of the analyte
between the matrix and the extraction fluid. In this case the yield
is enhanced by using more frequent replacement with the fresh
extraction solvent (Fig. 2).
In a snapshot, PLE is suitable for a wide range of solutes, polar
to nonpolar, with the factors affecting the extraction being: type
of solvent, extraction time, temperature, particle size and water
content of the sample. Optimization strategies involving these fac-
tors have been discussed in Ref. [6], are illustrated in Fig. 3 and are
discussed below (Fig. 3).
3.2. Solvents
The rule of the thumb in solvent choice is “like dissolves like”,
which indicates the use of polar solvents for polar analytes and
likewise nonpolar analytes are dissolved in nonpolar solvents. An
elementary aspect in the choice of solvent is the solubility char-
acteristic of the desired analyte, their diffusivity in the solvent
and the characteristics of the sample [17,21]. Ideally, an extract of
high purity and high selectivity should be achieved, which implies
that the analyte of interest should have high solubility in the sol-
vent while other compounds should have no or minimal solubility.
When extracting analytes at low concentrations, the rate of extrac-
tion is not affected by the analyte concentration but rather by the
rate of mass transfer, therefore the right solvent chemical prop-
erties should be chosen to ensure solvation and the release of the
analyte. There are models describing the solubility of solutes in a
solvent, for instance the Hansen solubility parameter [22]. How-
ever, since so many other factors than just the solubility some into
play during an extraction process, a theoretical consideration of
solubility of solute in different solvents is only a good first approx-
imation.
Economy, safety, and sustainability aspects should be taken in
consideration when the choice of solvent is made. Less toxic and
non-harmful solvents that are easy to remove or recover should
be preferred. The novel concept of “green” solvents reflects the
goal to diminish the environmental impact resulting from the
use of solvent in chemical process [23]. In a study that defines
“green” solvents, it was shown that simple alcohols (ethanol,
methanol) or alkanes (heptane, hexane) are more environmen-
tally preferable solvents compared to dioxane, acetonitrile, acids,
formaledyde and tetrahydrofuran. Furthermore, when compar-
ing solvent mixtures it was shown that methanol–water or
ethanol–water mixtures are environmentally favorable compared
to pure alcohol or propanol–water mixtures. In PLE, a wide range
of organic solvents has been used for the extraction of bioactive
compounds from foods and herbs, and those solvents are pre-
sented in a number of studies [24–31]. For example, two solvent
mixtures (chloroform–methanol 2:1, v/v, and hexane–isopropanol
3:2, v/v) were used for extraction of oxysterols from spray-dried
whole egg, vanilla cake and egg noodles [26]. Methanol–water
mixture was used for the extraction of capsaicinoids from pep-
pers [25]. Methanol was used for the extraction of tocopherols
and tocotrienols in cereals [32]. Acetone–water mixture was used
for the extraction of phenolic compounds from parsley [33]. And
dimethyl sulphoxide: ethanol: water solvent mixture was used for
the extraction of isoflavones from soybean [34].
Hawthorne and Miller first introduced the application of water
as a solvent in PLE. A more common name for this technique is
PHWE [35]. In their study they showed that by applying high tem-
perature during the extraction, the dielectric constant (polarity)
of water decreases substantially. Increasing the temperature of
water also results in a steady decrease in its viscosity and its sur-
face tension, as well as faster diffusivity characteristics [36–45], as
described further below. This fact makes water a suitable solvent to
extract polar, moderately polar and non-polar organic compounds
by increasing the temperature during the extraction. Hawthorne
and Miller [35] have shown that the extraction efficiency mainly
depends on the temperature while pressure has lesser important
effect. Pressurized hot water usually refers to liquid water at tem-
perature between 100 ◦ C and 374 ◦ C (water’s critical point). This
temperature range is sufficient to produce physicochemical prop-
erties of water enabling the extraction of many compounds without
risking degradation [46]. However, for thermolabile compounds
such as antioxidants, there is always a risk of degradation during
PHWE. This fact has been studied deeper for PHWE of anthocyanins
from red onion [47] and antioxidants in general from birch bark
[48].
Nevertheless, at higher temperature, some pressure would be
needed to keep the condensed phase of water, for example 15 bar
are needed at a temperature of 200 ◦ C and 85 bar at 300 ◦ C. If the
pressure decreases to below the boiling point of water, super-
heated steam will be formed [49]. Some factors including the
temperature and the pressure of the extraction, mode of extraction
(static or dynamic) and the mixing with other solvents (modi-
fiers) and additives can influence the efficiency during extraction.
These factors were reviewed exclusively in numerous reviews
[11,15,19,46,50–52].
Furthermore, the use of solvent mixtures was found to enhance
the extraction yields by improving the solubility and increasing
interaction of the targeted analyte with the extraction solvent
[53,54]. Using a solvent mixture during the extraction could be
important to improve the extraction efficiency. For example, in
a dual mixture, one solvent could improve the solubility of the
analyte, while the other would enhance the analyte desorption. In
this context, water is usually important to aid breaking matrix and
matrix–analyte (hydrogen) bonding. Using a mixture of organic sol-
vent and water improved the recoveries of catechins from tealeaves
and grapes seeds and phenolic compounds from grapes in PLE
[55,56]. Furthermore, using surfactant assisted PHWE was found to
 A. Mustafa, C. Turner / Analytica Chimica Acta 703 (2011) 8–18
Fig. 3. Factors affecting pressurized liquid extraction (PLE) and optimization parameters.
enhance the extraction of thermally labile and more hydrophobic
species in medicinal plants at a lower temperature [15].
3.3. Extraction parameters and effects
3.3.1. Effect of temperature
Temperature during the extraction is one of the critical fac-
tors that affect the efficiency and selectivity in PLE. The use
of high temperatures improves the efficiency of the extraction
as it helps the disruption of analyte-sample matrix interactions
caused by van der Waals forces, hydrogen bonding and dipole
attraction [16]. The use of thermal energy helps to overcome cohe-
sive (molecule–molecule) interactions and adhesive interactions
between dissimilar molecules, in this case the analyte and the sam-
ple matrix, by decreasing the activation energy required for the
desorption process. Furthermore, elevated temperature decreases
the surface tension of the solvent, solutes and matrix and there-
fore enhances the solvent wetting of the sample. A decrease in
solvent surface tension will allow solvent cavities to be formed
more easily, thus permitting analytes to faster dissolve in the sol-
vent [57]. An increased temperature decreases the viscosity of a
liquid solvent, thereby enhancing its penetration inside the matrix
particle, which results in an improved extraction process [58].
A final important advantage of using higher temperature of the
solvent is the improved diffusion rate, i.e. mass transfer of the
molecule in the solvent, which allows faster extractions especially
in diffusion controlled samples. On the other hand, the amount of
co-extracted analytes might be greater at higher temperatures, i.e.
decreased selectivity of extraction. In addition, high temperatures
might affect thermo-labile compounds that are subject to disinte-
gration and hydrolytic degradation [59,60]. The main feature of PLE
is that it employs high diffusion fluids that improve the rate of the
extraction process, while the amount of needed solvent is consid-
erably decreased [16,61]. The principle of how high diffusion rate
improves extraction is for instance discussed in Ref. [58].
11
3.3.2. Effect of pressure
The main advantage of applying pressure during the extraction
is that a temperature above the boiling point can be used while
the solvent maintains its liquid state. The use of elevated pressure
at high temperature and reduced solvent surface tension helps to
force the solvent within the matrix pore to contact the analyte and
extract them. Using pressure during extraction could exert pres-
sure on the matrix resulting in disruption, which could enhance
the mass transfer of the analyte from the sample to the solvent.
High pressure during the extraction controls problems related to
air bubbles found within the matrix that hinder the solvent from
reaching the analyte. These conditions boost the analyte solubility
and desorption kinetics from the sample matrix [62,63]. However,
the effect of pressure on recovery of most substances is usually neg-
ligible, e.g. as reported for essential oil extraction from some herbal
plants [42,64,65].
3.3.3. Addition of additives
It is reported that the use of additives such as micellar media
could be used as an alternative to organic solvents in PLE to extract
organic pollutants from liquid and environmental samples [66,67].
It has recently been demonstrated that non-ionic surfactants solu-
tions can be used as an alternative solvent system in PLE [64].
In a quantitative comparative analysis of marker compounds in
R. glycyrrhizae and E. sinica surfactant assisted PLE with sodium
dodecyl sulfate and Triton X-100 proved to be at least equivalent
or better compared to sonication with organic solvent [68]. It was
reported that using micelle-mediated extraction with some sur-
factants controlled the degradation of compounds when compared
to regular PHWE [69]. Other additives of interest in PLE are the
addition of protective antioxidants, such as ascorbic acid or buty-
lated hydroxtoluene (BHT), especially for oxidation prone analytes.
CO2 is some times added to lower the pH of water. Other com-
mon additive is drying agents and that has been discussed above
[69].
12 A. Mustafa, C. Turner / Analytica Chimica Acta 703 (2011) 8–18
Table 1
Studies on the application of pressurized liquid extraction in food and nutraceuticals.
Compounds group Research work references
Phenolic compounds in general [34], [39], [51], [76], [56], [90–96]
Polyphenols [97], [98]
Isoflavones [34], [63], [99–102]
Lignans [103–111]
Carotenoids [10], [24], [25], [125–133]
Oils and lipids [134–139]
Essential oils [29], [42], [65], [140–150]
Antioxidants in general [39], [48], [127], [130], [153–161]
Medicinal compounds and herbal [15], [37], [40], [65], [153], [163–166]
nutraceuticals
4. Hyphenated and combined techniques
Recently, there has been a new breakthrough in the devel-
opment and the improvement of the extraction and analytical
instrumentations and the integration of different steps in one step.
This approach has added a new level of complexity and diversity by
multiple on-line coupling and combination of different analysis and
detection techniques and multidimensional approaches, also called
“hyphenation” [70,71]. The prospect of hyphenation and/or combi-
nation of different sample preparation and analytical techniques in
one single process is one of the most recent strategies in analytical
chemistry. These combinatory techniques were for instance used
for the extraction of phenolic compounds [72,73]. It was demon-
strated to be useful in the extraction of aliphatic and polycyclic
aromatic hydrocarbons or polychlorinated biphenyls from plants
and biological and samples [74,75]. Other hyphenation methods
used for the extraction of bioactive compounds from food mate-
rials included pressurized solid-phase extraction for the isolation
of trans-resveratrol from grapes [76]. Combining PLE with son-
ication was used for the extraction of isoflavone from soybean,
this combination gave better recoveries compared to FEE alone,
sonication alone and Soxhlet [77]. In-line coupling of PLE with
solid-phase extraction has been used for the extraction of phenolic
compounds from grapes [55]. Using this procedure it was possible
to extract cinnamic esters, and improve the selectivity of the pheno-
lic compounds between the methanolic and waters phases. A recent
review article has been published that mainly discuss hyphenated
and combinatory techniques in food analysis [78].
5. Food and nutraceuticals applications in PLE
The term “nutraceutical” was first coined in 1989 by the Founda-
tion for Innovation in Medicine and it is described as “any substance
that may be considered a food or part of a food, and provides
medical or health benefits, including the prevention and treatment
of disease”. Nutraceuticals are pharmaceutics with physiological
or metabolic function. Furthermore, a nutraceutical is “a nutrient
that not only maintains, supports, and normalizes any physio-
logic or metabolic function, but can also potentiate, antagonize,
or otherwise modify physiologic or metabolic functions” [79,80].
Nutraceuticals may include dietary fibers, different types of phe-
nolic compounds and antioxidants, polyunsaturated fatty acids,
amino acids, proteins and minerals. Recently, PLE is becoming more
and more popular for extraction of nutraceuticals and other bioac-
tive compounds (Table 1). This increasing interest is mainly due to
the fact that PLE is automated with reduced extraction time and sol-
vent consumption and its set-up suits analytes that are oxygen and
light sensitive. PLE requires minimal sample pre-treatment, espe-
cially for non-fatty sample – only homogenization and/or drying.
Fig. 4. Recoveries for catechin (mg g− ) from grape seeds; non-fermented tea leaves;
medium-fermented tea leaves and fermented tea leaves using pressurized liquid
extraction (PLE); stirring-assisted extraction (SAE); ultrasound-assisted extraction
(UAE) and static extraction (SE). Results in this figure are from data reproduced from
(Pineiro, Palma and co-workers 2004) [56].
5.1. Phenolic compounds
Phenolic compounds are one of the four major secondary
metabolites found in plants and they are well known for their
importance in plant physiology [12,81–85]. The main source of
phenolic compounds in human diet is plant-derived foods, namely
fruits, vegetables, cereals, legumes and nuts. Diets rich in phe-
nolic compounds has been attributed to many health prompting
effects, such as reducing the risk of developing coronary heart
diseases, cancer, hypertension, diabetes and inflammatory pro-
cesses [86–89]. Determination of phenolic compounds in plants
becomes pivotal to be able to facilitate research and development
in relation to nutrition and health effects. Extraction process rep-
resents a critical factor in this development. The extraction of
phenolic compounds using PLE has been demonstrated in numer-
ous studies. The analytical procedures for the determination of
phenolic compounds in different categories of plant food mate-
rials and herbs were recently reviewed in Ref. [90,91]. A number
of studies have presented systematic approaches for optimization
of the extraction procedure, PLE or PHWE, and comparing yield
to that obtained from traditional methods [34,51,76,92–96]. The
common conclusion in these studies is that PLE methods were pre-
ferred, giving comparable and/or better recoveries yet being very
time efficient and economic when it comes it solvents and other
laboratory facilities utilization compared to the traditional meth-
ods. For instance, the extraction of catechin and epicatechin was
carried out in a comparative study employing magnetic stirring,
ultrasound-assisted extraction, and extraction with pressurized
solvents [56]. Solvents utilized in this study were water, methanol,
ethanol, and ethyl acetate. Fig. 4 shows the recoveries of catechin
from different matrices using different extraction technologies. The
optimization results revealed that PLE was a better methodology in
terms of recovery (RSD was 3%; n = 5), reproducibility and time effi-
ciency. Different extraction parameters in PHWE were optimized to
extract naringenin and other major flavonoids (dihydrokaempferol,
naringin) from knotwood of aspen [92]. Recoveries were then
compared to those from other extraction procedures, namely, ultra-
sonic extraction and reflux in methanol. Results proved that PHWE
was fast and effective for the isolation of biofunctional flavonoids
from aspen knotwood, producing higher recoveries than Soxhlet
extraction, sonication or reflux. The total phenolic content and the
antioxidant capacity of extracts from canola meal was determined
in different extraction procedures, PLE at 110 and 160 ◦ C, hot water
extraction (80 ◦ C) and ethanolic (95%, v/v) extraction. The highest
 A. Mustafa, C. Turner / Analytica Chimica Acta 703 (2011) 8–18
total phenolic content and antioxidant capacities were achieved in
ethanolic extracts when expressed on per gram of extract basis.
However, when expressed on a per gram of meal basis, the highest
content of total phenolics and antioxidant capacities were found
in extracts PHWE at 160 ◦ C [39]. This indicates that discrepancies
in the results are not only related to the extraction procedure, or
the way results are expressed, but majorly points out that there are
different selectivity using different extractions methodologies.
The effect of extraction conditions on the yield of polyphenols
from apple and peach pomaces was determined using PLE with
carbon dioxide to improve the solubility of the polyphenols. The
total phenolic content of the extracts were 0.47 and 0.26 mg gallic
acid equivalent/g sample and the antiradical efficiencies (1/EC50 ) of
the extracts were 3.30 and 1.5 mg DPPH/mg sample for apple and
peach pomaces, respectively [97]. PLE was as well used to extract
polyphenols from the needles of Pinus taiwanensis and Pinus mor-
risonicola by involving enzymatic hydrolysis using mixed enzyme
of cellulase, hemicellulase, pectinase and protease. In this study, a
comparison between the static and the dynamic modes was inves-
tigated, revealing that the content of polyphenols expressed as
catechin equivalents obtained from the dynamic mixed enzymatic
hydrolysis mode were higher than those obtained from the static
enzymatic hydrolysis mode [98].
Isoflavones are important bioactive compounds that are present
in plants, known to exist in nature as free aglycones or as conjugates
with sugars and/or acids. The effect of extraction parameters and/or
techniques from different soybeans and soy foods on the yield of
isoflavones has been discussed in some studies [34,63,99–101].
A systematic study was carried out to evaluate variation in
solvents and extraction procedures used for the extraction of
isoflavones from soybeans. In this study seven different solvent
mixtures were evaluated: acetonitrile–water; ethanol–water;
methanol–water; dimethylsulphoxide–acetonitrile–water;
dimethylsulphoxide–ethanol–water and Genapol–water. The
assessment was done using six different extraction techniques:
shaking, vortexing, sonication, stirring, Soxhlet and PLE. The opti-
mum isoflavones recoveries from soybean samples were obtained
with dimethylsulphoxide–ethanol–water (5:70:25, v/v/v) solvent
mixture using a PLE [34]. Recently, the extraction of quercetin from
yellow onions and converted to its aglycone using a combination
of PHWE and enzymatic hydrolysis [102], results in this study
showed that hyphenation of sub-critical water extraction/enzyme
hydrolysis is both a fast and sustainable method to obtain quercetin
from onion by-products.
5.2. Lignans
Lignans are phenolic compounds of plant origin, derived from
phenylalanine, and one of the major classes of phytoestrogens
[103–105]. The major sources of lignans are flax seeds and sesame
seeds, other sources include rye, wheat and barely. They has also
been found in many plants of oriental medicine and reported
to have pharmacological activities. Extraction, chromatographic,
electromigration and hyphenated methods for the separation and
Lignans metabolites in biological matrices and food samples is
reviewed in Ref. [106]. Extraction of lignans using ethanol–water
mixture was optimized, the best yield was obtained with 70%
ethanol concentration at 40 ◦ C and 28 h extraction time [107]. How-
ever, PLE was developed for the extraction and determination of
nine types of bioactive lignans in Schisandra chinensis in compari-
son to conventional reflux and sonication extractions [108]. Results
from this study showed that the yield from PLE was comparable
with higher extraction yields at temperature of 125 ◦ C and 5 min
one static cycle compared to 3 h of extraction for the reflux and
the sonication (Fig. 5). The total phenolic compounds were 17.2,
14.9 and 15.3 mg g− for the PLE, reflux and sonication extractions,
13
Fig. 5. Extraction of different types of lignans from Schisandra chinensis using pres-
surized liquid extraction (PLE), reflux and sonication extraction. Results in this figure
are from data reproduced from (Lee and Kim 2010) [108].
respectively. Further studies has presented the extraction of lignans
and other phenolic compounds from flaxseed using pressurized hot
water, giving a range of yield was in the order of 14–21 mL g− of
extract, with quite similar extraction parameters. In some cases
alkaline pH was used [109–111].
5.3. Carotenoids
Carotenoids are naturally occurring fat-soluble pigments. There
are about 600 known carotenoids, which are divided into two
main classes, xanthophylls and carotenes. They are naturally
found macromolecules and their major sources for human health
are fruits and vegetables [112–114]. Consuming a diet rich in
carotenoids are said to be correlated to lowering the risk for degen-
erative diseases such as diabetes, range of cancers, cardiovascular
diseases and osteoporosis [88,115–120]. The conventional methods
for the extraction of carotenoids involves a range of organic solvent
and solvent mixtures, as were reviewed in Ref. [121,122]. In food
samples, solvents used to extract carotenoids included acetone,
petroleum ether, diethylether, tetrahydrofuran, methanol, hex-
ane, dichloromethane and methanol chloroform mixture [123,124].
When extracting food samples with high water content, it is more
suitable to use organic compounds that have high water miscibility.
To avoid the aforementioned adverse-effect of using organic sol-
vent extractions, PLE has become more popular for the extraction
of carotenoids. One of the earlier studies presenting the use of PLE in
the extraction of carotenoids was in different types of green algae.
In this study it was shown that PLE has equal extraction efficiency
compared to conventional organic extraction methods, utilizing
less volumes of conventional solvents and less time of extraction
[125]. The extraction of carotenoids from different types of micro
and macro algae was studied in Ref. [10,24,126–130]. Pressurized
hot ethanol was used for the extraction of astaxanthin from shrimp
waste [131]. Recently, carotenoids were qualitatively and quantita-
tively determined in herbal teas and vegetables, and bioavailability
of different carotenoid species was also investigated [132,133]. PLE
has also been used for the extraction of capsaicinoids from peppers
[25].
5.4. Oils and lipids
PLE was used to extract polar and nonpolar lipids from corn
and oats. In general, increasing the extraction temperature and
the polarity of the extraction solvent resulted in more yield of
14 A. Mustafa, C. Turner / Analytica Chimica Acta 703 (2011) 8–18
polar lipids. In corn, total phytosterols was found to be about 0.6
and 2.1 wt% in the hot ethanol extracts and the hot methylene
chloride extracts, respectively. While in the oat extracts digalac-
tosyldiacylglycerol was the most abundant polar lipid with levels
ranging from 1.6 wt% in the cold hexane extracts to 4.3 wt% in the
hot ethanol extracts [134]. Furthermore, the extraction of polar
lipids from the leaves of Lochroma gesnerioides was carried using
PLE [135]. The extraction of grape seed oil rich in vitamin E was
carried out using conventional methods (Soxhlet and mechanical
press extraction) and compared to PLE with hexane as solvent, of
which the later technique gave better yield of the vitamin E rich
extract [136]. The extraction of antioxidants, emulsifying agents
and emulsion-stabilizers from rice bran and black rice was done
using PHWE. The bran extracts treated at 260 ◦ C for 5 min had a
higher activity toward autoxidation of linoleic acid. Furthermore,
the bran extracts prepared at 40–200 ◦ C for 5 min showed the emul-
sifying and emulsion-stabilizing activities, compared to extracts
from lower temperatures [137–139].
5.5. Essential oils
Essential oils are volatile aroma compounds that are found
in herbal plants. They are well known for their use in food,
pharmaceutical and cosmetic products. Some essential oils are
known for their antimicrobial, antioxidant and antifungal activities.
Additionally, they have been widely used for their aroma-active
constituents and flavor properties [140–143]. A PHWE method
was optimized for the extraction of essential oils from differ-
ent types of herbs [144–149] and other Chinese medicinal plants
[65,144]. The extraction of essential oils from coriander seeds
was done in a comparative study using PHWE, hydrodistillation
and Soxhlet extraction. The results showed that PHWE produced
essential oils are more concentrated in valuable oxygenated com-
ponents, while hydrodistillation and Soxhlet extraction had higher
efficiencies [147]. Essential oils in Acorus tatarinowii Schott were
determined by PHWE followed by solid-phase micro-extraction
and gas chromatography–mass spectrometry. Results showed that
this hyphenated method gave a recovery of 92% of the active
compound asarone with good repeatability (RSD less than 13%)
[42,65]. Furthermore, the extraction yield and antioxidant potency
of essential oils from boldo leaves was determined using differ-
ent types of extraction methods, Soxhlet extraction, supercritical
and PHWE. The highest yield and antioxidant potency was found
in the PHWE extracts [150]. Extraction of pharmacological essen-
tial oils from Cyperus rotundus was done using three methods,
hydro-distillation, PLE and supercritical fluid extraction. Among
these 3 methods, PLE exhibited the highest extraction efficiency for
alpha-copaene, cyperene, beta-selinene, beta-cyperone and alpha-
cyperone, nevertheless SFE had the best selectivity for extraction
of beta-cyperone and alpha-cyperone [29].
5.6. Other bioactive compounds
Natural antioxidants are commonly phenolic compounds,
including sub-classes of flavonoids, tocopherols and phenolic acids
[82,122,151]. Also other classes of compounds are represented, for
instance, carotenoids. Common for the studies referred to here
is that they use antioxidant assays to describe the efficiency of
extraction. The traditional method for extraction of antioxidants
from plants is carried out by techniques based on organic sol-
vents [151,152]. However these techniques are less favored due
to reason discussed earlier in this paper. In a number of com-
parative studies, water and other solvents and solvent mixtures
were used for the extraction of antioxidants from plant materials,
usually PLE was preferred to the traditional extraction methods
[39,48,130,153–156].
To maximize the antioxidant capacity of the extracts from three
spices of Lamiaceae family, rosemary, oregano and marjoram, PLE
parameters were optimized. The optimized temperature was found
to be 129 ◦ C and the optimal methanol concentrations with respect
to the antioxidant activity of rosemary and marjoram extracts were
56% and 57% respectively. Oregano showed a different response to
the effect of methanol, while a lower concentration of 33% was
found to be optimal for oregano. Comparing to solid–liquid extrac-
tion, the antioxidant capacity from PLE extracts were significantly
higher (p < 0.05) [157]. Different extraction techniques were studies
for the extraction of antioxidants from rosemary; they included PLE
using water and ethanol as solvents, and supercritical fluid extrac-
tion under different conditions and solvents combination. The PLE
method produced extracts with the highest antioxidant activity
[127,158,159]. In a recent study, the use of PHWE was presented as
a promising technique to obtain antioxidants, mainly from natural
sources. The use of high extraction temperatures in the process
was shown to result in the generation of new bioactive com-
pounds/antioxidants during the extraction process via Maillard,
caramelization and thermoxidation reactions. The results suggest
that the antioxidant capacity of the neoformed compounds from
those processes during the extraction depends on the nature of the
sample [160]. PHWE was used to obtain and characterize nutraceu-
ticals from oregano leaves (Origanum vulgare L.) [153]. The highest
antioxidant activity was observed at the highest extraction tem-
perature, 200 ◦ C, while the total phenolic content was not affected
by the extraction temperature. Antioxidant or radical-scavenging
ability of grape skin extracts was characterized in a novel combi-
nation of PLE and electron paramagnetic resonance spectroscopy,
using different extraction solvents and temperatures. Other inves-
tigated parameters included composition of anthocyanins, total
phenolic compounds content, tristimulus color values, and pH val-
ues. Results showed that extracts retained their antioxidant and/or
radical-scavenging properties, serving as a source of functional food
supplements or color enhancers [161].
Benthin et al. 1999 [162] presents one of the early studies on
herbal nutraceuticals. In this study the extraction yield of cur-
cuminoids, saponins flaconolignans and terpenes were compared
using PLE and the traditional European Pharmacopoeia. Essential
oil in Acorus tatarinowii Schott was determined by pressurized
hot water extraction followed by solid-phase microextraction and
gas chromatography–mass spectrometric determination. Results
showed that this methodology gave a recovery of 92% of the active
compound asarone with good repeatability (RSD less than 13%)
[65]. Yields were found to be equivalent or higher when using PLE
with substantial decrease in the extraction time and solvent con-
sumption. Xanthones and flavanones from bark of Psage orange
tree Maclura pomifera were extracted using solvent liquid extrac-
tion, supercritical fluid extraction and PLE, of which the later two
gave better yield [163]. PLE was as well used for the extraction of
stevioside from Stevia rebaudiana Bertoni [164]. The anti-microbial
activity of PLE extracts from the microalga Haemotococcus pluvialis
were determined, of which extracts in ethanol from the red phase
of the microalgae showed higher antimicrobial activity compared
to other solvents used [165]. Additionally, two bioactive com-
pounds stevioside and rebaudioside A were extracted from Stevia
rebaudiana Bertoni using heating under water with reflux, PHWE
and microwave-assisted extraction. The two later techniques gave
better efficiency [37]. In a comparative study, Gastrodin and vanil-
lyl alcohol from Gastrodia elata Blume, showed better stability
when extracted with PHWE as compared to heating under reflux
using water. Extraction method using PHWE had a good preci-
sion, showing that this method is a better alternative for thermally
labile compounds present in medicinal plants [15,40]. The effect of
extraction technique on the bioactive hydrolysable tannins from
Phyllanthus niruri Linn was done by comparing the recovery of
 A. Mustafa, C. Turner / Analytica Chimica Acta 703 (2011) 8–18 15

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