FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2001; 16: 64–73

Selective extraction of oxygenates from savory and

peppermint using subcritical water
Alena Kubátová, Arnaud J. M. Lagadec, David J. Miller and Steven B. Hawthorne∗
Energy and Environmental Research Center, University of North Dakota, Campus Box 9018, Grand Forks, ND 58202, USA
Received 26 April 2000
Revised 2 August 2000
Accepted 12 August 2000

ABSTRACT: The yields of oxygenated and non-oxygenated flavour and fragrance compounds from savory (Sat-
ureja hortensis) and peppermint (Mentha piperita) were compared using subcritical water extraction, supercritical
carbon dioxide extraction (SFE) and hydrodistillation. Extraction rates with subcritical water increased with tem-
perature (100–175 ° C), but some desired organics (linalool and -terpinene) showed substantial degradation at
temperatures >150 ° C. However, subcritical water did not expose extracted compounds to atmospheric oxygen
(as occurs in hydrodistillation) and thus may avoid the degradation of compounds like thymoquinone. Extraction
of savory with subcritical water at 100 ° C for 40 min gave ca. 100% recoveries (compared to hydrodistillation)
for thymol and carvacrol, and >150% recoveries of borneol and linalool. Recoveries with 60 min of SFE (pure
CO2 at 400 bar and 50 ° C) were similar to hydrodistillation for borneol and linalool, but only ca. 50% for thymol
and carvacrol. For peppermint, 30 min (at 150 ° C) or 12 min (at 175 ° C) of subcritical water extraction and 1 h of
SFE gave good quantitative agreement with 4 h of hydrodistillation for carvone, pulegone, piperitone, eucalyptol,
menthone, neomenthol and menthol, but the short subcritical water extractions only recovered ca. 40% of the less
polar menthyl acetate. Subcritical water preferentially extracts more polar (oxygenated) flavour compounds, and
ca. 80% extraction of oxygenated flavour compounds could be achieved under conditions which only extracted
ca. 10–15% of the monoterpenes and <5% of the sesquiterpenes. In contrast, SFE had the lowest degree of
selectivity and SFE extracts included plant alkane waxes as well as the same flavour compounds recovered by
hydrodistillation. Copyright  2001 John Wiley & Sons, Ltd.
KEY WORDS: selective extraction; subcritical water extraction; green solvents; supercritical fluid extraction
(SFE); savory; Satureja hortensis; peppermint; Mentha piperita; flavour and fragrance compounds

Introduction the potential to yield rapid recoveries.1,3 – 7 Early work

Flavour and fragrance compounds are normally extracted
using hydrodistillation or organic solvent extraction,
although the desire to reduce extraction time and organic
solvent use has recently led to new extraction techni-
ques.1 Supercritical fluid extraction (SFE) with non-toxic
carbon dioxide (CO2 ) is becoming more common for the
extraction of flavours and fragrances, and can often yield
more rapid extractions than hydrodistillation, as well
as recovering some species that are not recovered by
hydrodistillation.2 However, high recoveries with SFE
can require an organic solvent as a modifier.2
Most recently, the use of subcritical water (hot water
under pressure sufficient to maintain water in the liq-
uid state) for the extraction of flavours has shown
*Correspondence to: S. B. Hawthorne, Energy and Environmental
Research Center, University of North Dakota, Campus Box 9018,
Grand Forks, ND 58202, USA. E-mail: shawthorne@eerc.und.no-
dak.edu
Contract/grant sponsor: US Environmental Protection Agency, Office
of Research and Development, NCERQA; Contract/grant number:
R825394-1.
with subcritical water demonstrated its ability to selec-
tively extract different classes of compounds, with the
more polar organics being extracted at lower temper-
atures and the less polar organics being extracted at
higher temperatures. For example, phenols, polycyclic
aromatic hydrocarbons, and alkanes were sequentially
extracted from a petroleum waste sludge by stepwise
increases in subcritical water temperatures.8 Thus, the
selectivity of subcritical water extraction (a preference
for more polar organics at milder conditions) is opposite
to that of supercritical CO2 (a preference for non-polar
organics before polar organics). Recently, the solubil-
ities of oxygenated flavour and fragrance components
were measured and found to be substantially higher
than non-oxygenates in subcritical water.9 For exam-
ple, the oxygenates carvone and eugenol have solu-
bilities at 100 ° C (ca. 65 bar) two orders of magni-
tude higher than the non-oxygenated d-limonene.9 These
trends in solubility correspond to studies demonstrat-
ing high recoveries of oxygenated flavour and fragrance
components. 3 – 7

Copyright  2001 John Wiley & Sons, Ltd.

 OXYGENATES EXTRACTION FROM SAVORY AND PEPPERMINT
In the present study, we compared the use of sub-
critical water at several temperatures to conventional
hydrodistillation and SFE with pure CO2 for the extrac-
tion of flavours from savory and peppermint. Subcritical
water conditions were optimized for speed and selectiv-
ity for oxygenates and the potential for degradation of
extracted compounds was investigated.
Experimental
Summer savory (Satureja hortensis) and peppermint
(Mentha piperita), obtained from Penn Herb Co. Ltd.
(Philadelphia, PA), were used as received (air-dried and
coarsely ground). Sample sizes were 0.8, 1.0 and 25 g for
subcritical water extraction, SFE and hydrodistillation,
respectively. All extractions were performed in triplicate.
After each extraction, the extraction efficiencies of each
method were confirmed by extracting the plant tissue
residues using 18 h of sonication in 15 ml acetone.
Subcritical Extraction with Water
Subcritical water extraction was performed in a home-
built apparatus previously described in detail.9,10 The
extraction system consists of two ISCO Model 100D
syringe pumps (ISCO, Lincoln, NE) delivering water and
methylene chloride at a constant flow rate to an HP 5890
gas chromatograph oven (Hewlett-Packard, Wilmington,
DE), where the extraction cell was mounted. The water
(HPLC Grade, Fisher Scientific, Pittsburgh, PA) was
purged with nitrogen to remove dissolved oxygen prior
to the extraction, and supplied to the system with the
pump at a constant flow of 1 ml/min. To preheat the
extractant water to the required temperature, a 3 m
preheating coil was installed in the oven before the
extraction cell. A second pump delivered methylene
chloride at a flow of 1 ml/min to a mixing ‘tee’ installed
in the oven after the outlet of the extraction cell to collect
the extracted compounds as the water cooled after exiting
the extraction oven. Finally, a miniature back-pressure
regulator (Upchurch Scientific, Oak Harbor, WA) was
placed at the outlet of the extraction system (outside of
the oven) to maintain the system pressure at 60–70 bar,
thus ensuring that the water was in the liquid state at all
temperatures tested.
All extractions were carried out in a 3.47 ml SFE
cell (9.4 mm i.d., 50 mm long, Keystone Scientific,
Bellefonte, PA), equipped with a 0.5 µm frit at the inlet,
and a 2 µm frit at the outlet. The larger pore frit was
installed at the outlet to prevent plugging with plant
material. In addition, seven layers of glass microfibre
filter (1 µm, Whatman International Ltd., Maidstone)
were placed inside of the cell to protect the outlet frit.
The extraction cell was completely filled with plant
Copyright  2001 John Wiley & Sons, Ltd.
65
material (ca. 0.8 g) and mounted vertically in the oven,
with water flowing from top to bottom.
The extraction procedure started by pressurizing the
system with water to ca. 60 bar at a flow of 1 ml/min and
heating the oven to the required temperature, a process
which required ca. 3 min. At this time, the back-pressure
regulator (set to 60 bar) opened, the methylene chloride
collection solvent flow was started, and collection of the
eluent began (extraction time D 0). For the temperature
dependence experiments, each temperature was held for
15 min, then the collection vial was replaced and the sys-
tem heated to the next higher temperature (requiring ca.
30 s). For the extraction rate experiments, the collection
vial was replaced at frequent time intervals.
After each extraction, the methylene chloride frac-
tion was collected and the water fraction rinsed once
more with an additional aliquot of methylene chloride.
The peppermint extracts, because of the formation of an
emulsion, were rinsed three times with methylene chlo-
ride and centrifuged each time. Combined fractions of
the methylene chloride extracts were analysed to deter-
mine extraction efficiencies.
The stability of flavour compounds in subcritical water
was tested using a static (no flow) subcritical water
cell (4.1 ml) constructed from a threaded (npt) stainless
steel pipe fitting with end caps (64 mm long, 7 mm i.d.,
12 mm o.d.; Minnesota Valve and Fitting, Eden Prairie,
MN). For the static experiments, the cell was filled with
water, leaving a 1 ml headspace (to ensure that the max-
imum safety pressure was not exceeded11 ) and spiked
with tested standards and internal standards. It was then
placed in the oven and heated to the required tempera-
ture. After cooling, the cell was emptied and the contents
washed with several rinses of methylene chloride. The
methylene chloride extract was analysed by GC and
results were compared to a parallel experiment, where
spiked water was not exposed to higher temperatures.
Hydrodistillation
All extractions were performed with 25 g portions of
each sample for 4 h using 600 ml water per extraction.2
Following hydrodistillation, the extracted essential oils
were quantitatively transferred into a volumetric flask,
using three rinses of methylene chloride and diluted as
appropriate for gas chromatographic analyses.
SFE with CO2
SFE was performed on 1 g portions of each sample,
using ‘SFE-grade’ CO2 (Scott Specialty Gases, Plum-
steadville, PA) supplied to an ISCO SFX 210 extraction
unit with a Model 260D syringe pump (ISCO, Lin-
coln, NE). All extractions were performed at 400 bar
Flavour Fragr. J. 2001; 16: 64–73
66 A. KUBÁTOVÁ ET AL.
and a temperature of 50 ° C using 10 ml cells supplied
with the unit. SFE flow rates were maintained at ca.
1 ml/min (measured as liquid CO2 at the pump), using
a variable flow restrictor (ISCO, Lincoln, NE) heated to
80 ° C. Extracted analytes were collected in 15 ml methy-
lene chloride placed in a 22 ml glass screw-top vial.
All extractions were performed with pure CO2 in the
dynamic (continual flow) mode and extracts were col-
lected in two 30 min fractions.
Gas Chromatographic Analysis
Quantitative analyses were performed using GC with
flame ionization detection (GC–FID) on a Hewlett-
Packard Model 5890 Series II gas chromatograph equip-
ped with an autosampler. Chromatographic separations
were accomplished with a 30 m DB-5 column with
0.25 mm i.d. and 0.25 µm film thickness (J&W Sci-
entific, Rancho Cordova, CA), with injections in the
splitless mode. The oven temperature was held at 35 ° C
for 0.2 min and was then increased to 60 ° C with a gradi-
ent of 30 ° C/ min, followed by 6 ° C/ min to 200 ° C and
then by 30 ° C/ min to the final temperature of 320 ° C.
Quantitations were based on the addition of n-dodecane
as the internal standard to each extract and on standard
Figure 1. Effect of temperature on the extraction efficiency of savory (S. hortensis) flavour and fragrance compounds
using subcritical water. Each temperature was held for 15 min. Percentage removals are based on sonication of the
residue after water extraction
Copyright  2001 John Wiley & Sons, Ltd.
calibration curves generated from pure standard com-
pounds. When the pure compounds were not available,
the FID relative response factors were estimated, based
on the FID responses of the pure standards having the
same molecular formula.
Except as otherwise noted, all identifications were
based on comparisons of mass spectra and retention
data of pure standards with those of the sample species.
GC–MS analyses were performed using the same GC
conditions and a Hewlett-Packard Model 6890 GC with
a Hewlett-Packard Model 5973 MS.
Results and Discussion
The development of subcritical water extraction con-
ditions consisted of four main steps. First, appropri-
ate water temperatures were determined by sequen-
tially extracting savory and peppermint with stepwise
increases in temperature. Second, the extract rate curves
were determined at the selected temperatures. Third, the
potential for compound degradation in subcritical water
at higher temperatures was evaluated. Finally, the recov-
eries and selectivity of the subcritical water extractions
were compared using hydrodistillation and SFE.
Flavour Fragr. J. 2001; 16: 64–73
 OXYGENATES EXTRACTION FROM SAVORY AND PEPPERMINT
Water Temperature
The effect of extraction temperatures on the recovery
of flavour compounds from savory and peppermint are
shown in Figures 1 and 2. Each sample was sequen-
tially extracted for 15 min at 50 ° C, followed by 100 ° C,
125 ° C, 150 ° C, 175 ° C and 200 ° C (each temperature for
15 min). Following this extraction sequence, the plant
residues were further extracted by 18 h of sonication
in acetone to determine unextracted organics and to
allow determination of overall extraction efficiencies (i.e.
100% extracted for Figures 1 and 2 correspond to the
-terpinene)
total mass of each compound extracted by the water and
acetone residue extractions).
As shown for savory in Figure 1, oxygenated com-
pounds extract at substantially lower water tempera-
tures than non-oxygenated compounds. For example, at
100 ° C ca. 55–75% of most oxygenated compounds are
extracted in only 15 min, while the monoterpenes (e.g.
p-cymene) and sesquiterpenes (ˇ-caryophyllene) show
less than ca. 5% extracted. Even after an additional
15 min of extraction at 125 ° C (sequentially after the
100 ° C extraction), only a small percentage of the non-
oxygenated organics are extracted, while the extraction
of the oxygenated compounds is nearly complete (i.e.
typically >85%).
Figure 2. Effect of temperature on the extraction efficiency of peppermint (M. piperita) flavour and fragrance
compounds using subcritical water. Each temperature was held for 15 min. Percentage removals are based on
sonication of the residues after water extraction
Copyright  2001 John Wiley & Sons, Ltd.
67
The effect of water temperature on the extraction of
flavours and fragrances from peppermint was similar to
that found for savory, i.e. substantial extraction of the
oxygenates occurs in only 15 min at 100 ° C, while nearly
complete extraction of these oxygenates is achieved after
an additional 15 min extraction at 125 ° C (Figure 2). The
behaviour of the sesquiterpene ˇ-caryophyllene was also
similar in savory and peppermint, i.e. water temperatures
greater than 175 ° C were required to achieve significant
extraction. However, one oxygenate, menthyl acetate
from peppermint, shows extraction behaviour more sim-
ilar to the monoterpenes (p-cymene and
extracted from savory, rather than the rapid extraction
behaviour shown by the other oxygenates. This might be
explained by its lower polarity in comparison to other
oxygenates.
Extraction Rates
Based on the temperature behaviour of savory shown in
Figure 1, three temperatures, 100 ° C, 150 ° C and 175 ° C
were selected to determine the effect of extraction time
on the selective extraction of savory’s oxygenated com-
pounds (Figure 3). As shown in Figure 3a, subcriti-
cal water extraction at 100 ° C yields good selectivity
Flavour Fragr. J. 2001; 16: 64–73
68 A. KUBÁTOVÁ ET AL.

Figure 3. Effect of extraction time on the extraction efficiency of savory (S. hortensis) flavour and fragrance
compounds using subcritical water at (a) 100 ° C, (b) 150 ° C and (c) 175 ° C. Percentage removals are based on sonication
of the residue after water extraction

towards oxygenates, especially during the first 20 min,
when at least 70% of all oxygenated compounds are
extracted and less than 10% of the non-oxygenated com-
pounds are extracted. After 40 min at 100 ° C, recover-
ies of oxygenated compounds increase to nearly 100%,
but at the expense of less selectivity against the non-
oxygenated compounds.
When the extraction of savory was performed at
150 ° C, the extraction rates of both oxygenated and
non-oxygenated compounds were approximately twice
as fast (Figure 3b) as their rates at 100 ° C (Figure 3a),
which agrees with the increase in water solubilities by
a factor of ca. 2–3-fold previously reported to occur for
150 ° C vs. 100 ° C water.8 Similarly, the solubility data8

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 64–73
 OXYGENATES EXTRACTION FROM SAVORY AND PEPPERMINT 69

Figure 4. Effect of extraction time on the extraction efficiency of peppermint (M. piperita) flavour and fragrance
compounds using subcritical water at (a) 150 ° C and (b) 175 ° C. Percentage removals are based on sonication of the
residue after water extraction

correspond to an increase in the extraction rates by a
factor of two when comparing temperatures of 175 ° C
and 150 ° C (Figure 3b,c). Based on the results shown in
Figures 3a, b and c, similar recoveries of the oxygenated
compounds (and selectivities against the non-oxygenated
organics) can be achieved in ca. 5 min at 175 ° C, 10 min
at 150 ° C and ca. 20 minutes at 100 ° C.
The extractions of peppermint at 150 ° C and 175 ° C
are shown in Figure 4. As for savory, the oxygenates
(except menthyl acetate) show substantially higher extra-
ction rates than non-oxygenates such as ˇ-caryophyllene.
As would be expected, based on the results of the
extractions conducted at sequentially higher tempera-
tures (Figure 2), menthyl acetate was extracted at a
much lower rate than the other peppermint oxygenates
(Figure 4). It is interesting to note that several of the
peppermint oxygenates (piperitone, pulegone and car-
vone) and one of the savory oxygenates (thymoquinone)
show significantly higher extraction rates than the other
oxygenates in each sample (Figures 3 and 4). All four
of these species have ketones conjugated with carbon-
to-carbon double bonds, while the other oxygenates in
savory and peppermint do not. While little solubility data
in subcritical water is available for these compounds, at
100 ° C the solubility of carvone (which has the conju-
gated double bond and ketone) is ca. 50 times higher
than eugenol and 100 times higher than eucalyptol (both
compounds which do not have the conjugated double
bond and ketone).9
Extraction Efficiencies of Subcritical Water
Extraction vs. Hydrodistillation and SFE
Savory
Based on the results discussed above, subcritical water
extractions were performed on savory at 100, 150 and
175 ° C. As indicated by the extraction rate experiments
(Figure 3), two fractions were collected at each selected
temperature; i.e. two 20 min fractions at 100 ° C, two
10 min fractions at 150 ° C and two 6 min fractions
at 175 ° C.
The extraction efficiencies of the flavour compounds
from savory are shown in Table 1. Subcritical water at

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 64–73
70 A. KUBÁTOVÁ ET AL.

Table 1. Concentrations of flavour and fragrance components in savory (Satureja hortensis) based on subcritical
water extraction, hydrodistillation and SFE

Concentrations µg/g (% RSD)a

Subcritical water extraction SFE

tr 100 ° C 150 ° C 175 ° C Hydrodi- SFE plus

stillation residued
Species (min) 20 min 40 minc 10 min 20 minc 6 min 12 minc 240 min 30 min 60 minc
p-Cymene 6.5 59 (19) 126 (18) 61 (23) 104 (24) 59 (14) 154 (10) 407 (3) 219 (19) 267 (16) 551 (3)
-Terpinene 7.2 11 (2) 22 (2) 12 (27) 17 (26) 10 (9) 25 (4) 97 (4) 42 (16) 57 (13) 112 (7)
Thymol 12.6 672 (5) 864 (2) 573 (19) 690 (13) 448 (2) 665 (1) 830 (3) 400 (13) 464 (11) 862 (4)
Carvacrol 12.9 980 (5) 1200 (3) 980 (17) 1140 (12) 740 (5) 1050 (2) 1190 (2) 550 (13) 630 (11) 1180 (4)
C10 H18 Ob 7.5 116 (2) 116 (2) 2 (0) 2 (0) 1 (0) 1 (0) 0 123 (11) 189 (13) 239 (43)
Linalool 8.1 166 (7) 239 (7) 75 (21) 87 (19) 55 (11) 70 (9) 139 (2) 110 (9) 172 (7) 276 (5)
Borneol 9.7 182 (1) 252 (2) 98 (19) 110 (14) 82 (3) 114 (1) 156 (3) 109 (11) 171 (9) 289 (7)
C10 H18 Ob 9.9 144 (1) 216 (1) 97 (17) 118 (12) 64 (11) 95 (8) 154 (3) 67 (8) 82 (27) 152 (11)
C11 H16 Ob 11.4 20 (6) 40 (8) 18 (42) 29 (28) 19 (5) 47 (2) 154 (2) 55 (14) 68 (11) 147 (0)
Thymoquinone 11.6 494 (11) 506 (11) 269 (24) 277 (23) 149 (2) 176 (1) 22 (33) 251 (20) 291 (18) 498 (43)
C9 H10 O3 b 18.5 174 (56) 174 (56) 770 (21) 793 (20) 778 (2) 838 (2) 38 (15) 99 (26) 99 (26) 402 (2)
ˇ-Caryophyllene 15.5 0 (0) 0 (0) 4 (28) 4 (25) 4 (4) 6 (7) 146 (10) 50 (17) 59 (15) 144 (2)
a
Concentration and percentage relative standard deviations (RSD) were based on triplicate extractions performed on the same day.
b Tentative identification based on MS.
c
Includes the sum extracted in both time periods.
d
Includes the sum of the amounts found in the SFE and residues extracts.

100 ° C extracted most (ca. 70–95%) of the oxygenates in
the first 20 min, <15% of the monoterpenes p-cymene
-terpinene and ˇ-caryophyllene
and
-terpinene, and less than 4% of sesquiterpenes
(compared to hydrodistillation). When the subcritical
water extraction was extended to 40 min, the recover-
ies of non-oxygenates doubled, while the increase in
oxygenates was only between 20–30%. Notably, both
borneol and linalool reached 100% recovery of the
hydrodistillation recoveries in the first 20 min, while car-
vacrol and thymol (both aromatic compounds) were fully
extracted after 40 min. Interestingly, 20 times higher
yields of thymoquinone were found in the subcritical
water extract compared to hydrodistillation. While the
possibility exists that thymoquinone was formed dur-
ing the subcritical water extraction, high concentrations
of thymoquinone were also found by SFE with pure
CO2 at 50 ° C (Table 1), thus indicating that thymo-
quinone is lost during hydrodistillation. When the savory
residue was extracted with acetone (after hydrodistilla-
tion), ca. 50% of the thymoquinone (compared to the
amount extracted by subcritical water) was recovered.
This recovery indicates that both poor extraction and
degradation are responsible for low thymoquinone recov-
eries using hydrodistillation. Similarly, an unidentified
compound (C9 H10 O3 in Table 1) shows much higher
recoveries in the subcritical water and SFE (plus residue)
extracts.
When the subcritical water extraction temperature was
increased to 150 and 175 ° C, the recoveries of thymol
and carvacrol achieved in 20 min (150 ° C) and 12 min
(175 ° C) were similar to those achieved in 40 min at
100 ° C, and agreed well with those achieved by 4 h
of hydrodistillation. Also, the selectivities for oxy-
genates vs. p-cymene,
remained similar to those achieved with 100 ° C extrac-
tions (although the extraction of p-cymene increased
somewhat at 175 ° C). However, the yield of the oxy-
genates borneol and linalool (and the unidentified C11
H16 O compound) dropped substantially at the higher
temperatures. Since neither compound was found in
residue extracts, it appears that degradation of borneol
and linalool occurred in subcritical water at the higher
temperatures (discussed below).
Thus, for savory the best overall yield of oxygenates
was achieved with subcritical water at 100 ° C (but
required longer times than at the higher temperatures).
With 40 min of subcritical water extraction at 100 ° C,
the yields of the major oxygenates were the same as
4 h of hydrodistillation for thymol and carvacrol. How-
ever, subcritical water extraction at 100 ° C yielded nearly
twice as much borneol and linalool (as well as ca.
20 times more thymoquinone, as discussed above) as
hydrodistillation.
SFE with pure CO2 was also able to extract flavour
and fragrance compounds from savory (Table 1), but
with somewhat lower recoveries than hydrodistillation,
especially for thymol and carvacrol (although with much
higher recoveries of thymoquinone). The presence of
unextracted compounds in the SFE residues confirms
that lower recoveries were limited by solubility in pure
CO2 . These results are in agreement with previous work2
which demonstrated that high extraction efficiencies of
flavour compounds from savory required the addition of
organic modifiers to CO2 , rather than only the use of
pure CO2 .

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 64–73
 OXYGENATES EXTRACTION FROM SAVORY AND PEPPERMINT 71

Peppermint

Based on the results shown in Figure 2, the extrac-

tion of peppermint was performed at 150 ° C and 175 ° C
(Table 2). Extraction with subcritical water at 150 ° C and
175 ° C, hydrodistillation and SFE all gave comparable
results and reproducibilities for oxygenated monoter-
penes with the exception of methyl acetate. The presence
of the remaining menthyl acetate in the subcritical water
residues demonstrated that poor extraction (rather than
degradation) was responsible for the lower recoveries. In
addition, the amount of menthyl acetate found in the first
and second subcritical water fractions (at both 150 ° C
and 175 ° C) was the same, which demonstrates that its
extraction is limited by solubility in the subcritical water,
and that either longer extraction times or higher temper-
atures would be required to achieve yields similar to
hydrodistillation. Also, stability tests (discussed below)
demonstrated that degradation was not significant, there-
fore it was not necessary to use lower temperatures for
the peppermint extraction.
As was the case for savory, the selectivity of subcrit-
ical water extraction was high for the peppermint oxy-
genates over the only significant non-oxygenated com-
pound (ˇ-caryophyllene), with <3% of ˇ-caryophyllene
being extracted by subcritical water (Table 2). Sonica-
tion of the peppermint residue (after subcritical water
extraction) recovered the ˇ-caryophyllene, thus demon- Figure 5. GC–FID chromatograms of savory (S. hortensis)
extracts obtained using: (a) subcritical water extraction;
strating that subcritical water did not degrade the ˇ-
(b) supercritical fluid extraction; (c) hydrodistillation
caryophyllene.
Compared to the savory extractions (Table 1), in
which SFE recoveries were often lower than hydrodistil-
lation, SFE with pure CO2 for 1 h gave quite good recov- Selectivity of the Methods
eries of the peppermint flavour compounds, compared to
hydrodistillation (Table 2). SFE yields compared to 4 h The relative selectivities of subcritical water, hydrodis-
hydrodistillation ranged from ca. 80% (e.g. carvone) to tillation and SFE with pure CO2 are illustrated in
ca. 135% (e.g. eucalyptol). Figure 5, which shows the FID chromatograms resulting

Table 2. Concentrations of flavour and fragrance components in peppermint (Mentha piperita) based on subcritical
water extraction, hydrodistillation and SFE

Concentrations µg/g (% RSD)a

Subcritical water extraction SFE

tr 150 ° C 175 ° C Hydrodi-

stillation
Species (min) 15 min 30 minb 6 min 12 minb 240 min 30 min 60 minb
Carvone 11.5 287 (9) 295 (8) 256 (7) 271 (7) 307 (6) 209 (6) 241 (6)
Pulegone 11.4 61 (9) 62 (8) 47 (5) 53 (2) 58 (7) 41 (4) 50 (4)
Piperitone 11.8 55 (5) 55 (5) 46 (4) 48 (5) 56 (3) 36 (1) 41 (11)
Eucalyptol 6.6 221 (4) 268 (18) 142 (11) 212 (4) 233 (8) 258 (2) 316 (2)
Menthone 9.4 690 (3) 807 (3) 456 (12) 706 (6) 869 (8) 731 (3) 829 (2)
Neomenthol 9.7 323 (3) 378 (2) 222 (12) 369 (3) 377 (7) 287 (1) 317 (2)
Menthol 9.9 2290 (3) 2560 (2) 1470 (11) 2280 (4) 2280 (2) 1780 (2) 1780 (2)
Menthyl acetate 12.7 106 (4) 220 (2) 78 (14) 194 (8) 513 (5) 430 (0) 509 (1)
ˇ-Caryophyllene 15.5 0 (0) 1 (89) 1 (0) 5 (77) 195 (3) 183 (2) 195 (3)
a
Concentration and percentage relative standard deviations (RSD) were based on triplicate extractions performed on the same day.
b Includes the sum extracted in both time periods.

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 64–73
72 A. KUBÁTOVÁ ET AL.
from the extraction of savory with the three extraction
methods.
As discussed above, subcritical water selectively extr-
acts oxygenated (more polar) flavour compounds over
non-oxygenated (less polar) monoterpenes and sesquiter-
penes. Thus, the subcritical water extract of savory at
100 ° C shows primarily oxygenated organics (Figure 5).
Such selectivity would be expected based on the reported
solubilities of flavour compounds in subcritical water.9
However, both subcritical water and hydrodistillation
use water as the extraction solvent, so it is interesting
that subcritical water shows compound class selectiv-
ity while hydrodistillation does not. The selectivity of
subcritical water exists because extraction depends on
solvation of the target compounds in the liquid state
of the water. As a polar fluid, water naturally sol-
vates more polar compounds more readily than non-polar
compounds. Higher temperatures reduce the polarity of
water, and thus increase its ability to solvate non-polar
compounds.
SFE also depends on solvation of the target com-
pounds, but has exactly opposite polarity considerations
from those of water, i.e. supercritical CO2 prefers non-
polar organics over more polar organics. Since (under
the SFE conditions used in this study), both the non-
oxygenates and oxygenated flavour compounds have
reasonable solubility, little or no selectivity between
these compound classes is shown in the SFE extracts
(Tables 1 and 2; Figure 5). Also, since SFE used non-
polar CO2 as the solvent, it is the only method that yields
detectable levels of the plant wax alkanes (Table 3;
Figure 5). Subcritical water fails to extract larger alka-
-terpinene. Thus, the lower
nes, even at temperatures as high as 300 ° C, simply
because it is still too polar of a solvent to solvate such
alkanes.8
In contrast to subcritical water and SFE (where sol-
vation controls the extraction), hydrodistillation depends
heavily on vaporization of the target compounds, and
-terpinene (which par-
thus recovers non-polar (e.g. monoterpenes) and more
polar compounds with little selectivity (Tables 1 and 2;
Figure 5). Hydrodistillation does not extract the plant
Table 3. Concentrations of n-alkanes extracted by SFE
from savory (Satureja hortensis) and peppermint
(Mentha piperita)
Concentrations µg/g (% RSD)
n-Alkane Savory Peppermint
Heptacosane (C27 ) 34 (10) 139
Nonacosane (C29 ) 187 (5) 213
Untriancontane (C31 ) 198 (11) 162
Tritriacontane (C33 ) 96 (21) 134
-terpinene, occurs at temperatures above 150 ° C.
Pentatriacontane (C35 ) 82 (4) 96
Total 596 745
a
Concentrations and percentage relative standard deviations (RSD) were
based on triplicate extractions performed on the same day.
Copyright  2001 John Wiley & Sons, Ltd.
wax alkanes shown in the SFE extracts (Figure 5), pre-
sumably because of their relatively low volatility.
Effect of Temperature on Compound Degradation
As shown in Figures 1–4, subcritical water extrac-
tion of flavour compounds at higher water tempera-
tures greatly increases their extraction rates and may
still allow reasonable selectivity against non-oxygenated
compounds. However, higher temperatures may also
lead to degradation of desirable compounds. Oxida-
tion of organics seems unlikely, since the water used
for subcritical water extraction is deoxygenated and no
exposure to air occurs during extraction (in contrast
to hydrodistillation, where the flavour compounds are
exposed to atmospheric oxygen at 100 ° C). However,
degradation from hydrolysis and/or thermal instability
may cause loss of some compounds at higher water
temperatures. 5,9
Therefore, we tested the stability of several major
savory and peppermint compounds at 100–175 ° C in
static subcritical water (as described above) for 10 min.
At 175 ° C, borneol, thymol, carvacrol, menthol, men-
thone, pulegone and menthyl acetate were stable, i.e.
their recoveries were ca. 100% (within analytical error).
These results, together with the compounds presence
in the residues, demonstrate that the lower recover-
ies of (for example) menthyl acetate from peppermint
(Table 2), were a result of slower extraction in subcriti-
cal water than degradation during extraction. However,
exposure to 175 ° C water led to nearly complete decom-
position of linalool and
recovery of linalool from savory (Table 1) at 175 ° C
compared to 100 ° C is probably a result of degrada-
tion at the higher temperature. GC–MS analysis showed
p-cymene as the major degradation product, although
small amounts of other monoterpenes were present. At
150 ° C, all compounds except
tially degrades to p-cymene) were stable. All of the test
compounds were stable at 100 ° C and 125 ° C.
Conclusions
Subcritical water extraction yields quantitative extrac-
tion and recovery of oxygenated flavour and fragrance
a
compounds in as little as 12 min, compared to 4 h of
hydrodistillation. Faster extractions are achieved with
(2) higher water temperatures but must be used with caution,
(9) since degradation of some compounds, such as linalool
(8)
(13)
and
(2) However, since subcritical water depends on solvation
in degassed water rather than vaporization (which results
in exposure to atmospheric oxygen during hydrodistil-
lation), other compounds, such as thymoquinone, may
Flavour Fragr. J. 2001; 16: 64–73
 OXYGENATES EXTRACTION FROM SAVORY AND PEPPERMINT
be protected from degradation by the use of subcritical
water. Subcritical water selectively extracts oxygenated
compounds (under conditions which extract only a few
per cent of non-oxygenated mono- and sesquiterpenes),
while hydrodistillation shows no selectivity among oxy-
genated and non-oxygenated compounds. SFE with pure
CO2 yielded lower recoveries than either technique for
savory but comparable recoveries for peppermint. How-
ever, SFE shows the least selectivity by extracting plant
wax alkanes as well as the flavour compounds.
Acknowledgements—The financial support of the US Environmental
Protection Agency, Office of Research and Development, NCERQA,
is gratefully acknowledged. Although the research described in this
article has been wholly or in part by the US Environmental Protection
Agency through Grant No. R825394-1 to the University of North
Dakota Energy and Environmental Research Center, it has not been
subjected to the Agency’s required peer and policy review and,
therefore, does not necessarily reflect the views of the Agency, and
no official endorsement should be inferred.
Copyright  2001 John Wiley & Sons, Ltd.
73
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