Food Chemistry 210 (2016) 172–181

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Extraction of a-humulene-enriched oil from clove using

ultrasound-assisted supercritical carbon dioxide extraction
and studies of its fictitious solubility
Ming-Chi Wei a, Jianbo Xiao b, Yu-Chiao Yang c,⇑
a
Department of Applied Geoinformatics, Chia Nan University of Pharmacy and Science, Tainan 71710, Taiwan
b
Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Taipa, Macau, China
c
Department and Graduate Institute of Pharmacology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan

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

Article history: Clove buds are used as a spice and food flavoring. In this study, clove oil and a-humulene was extracted
Received 18 November 2015 from cloves using supercritical carbon dioxide extraction with and without ultrasound assistance (USC–
Received in revised form 6 April 2016 CO2 and SC–CO2, respectively) at different temperatures (32–50 °C) and pressures (9.0–25.0 MPa). The
Accepted 17 April 2016
results of these extractions were compared with those of heat reflux extraction and steam distillation
Available online 19 April 2016
methods conducted in parallel. The extracts obtained using these four techniques were analyzed using
gas chromatography and gas chromatography/mass spectrometry (GC/MS). The results demonstrated
Keywords:
that the USC–CO2 extraction procedure may extract clove oil and a-humulene from clove buds with bet-
Clove
a-Humulene ter yields and shorter extraction times than conventional extraction techniques while utilizing less severe
Solubility operating parameters. Furthermore, the experimental fictitious solubility data obtained using the
Ultrasound-assisted supercritical CO2 dynamic method were well correlated with density-based models, including the Chrastil model, the
extraction Bartle model and the Kumar and Johnston model.
Gas chromatography/mass spectrometry Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction distillation, or solvent extraction (Bhuiyan, 2012; Guan, Li, Yan,

The dried unopened flower buds of Syzygium aromaticum (L.)
Merr. & Perry (Family Myrtaceae), called ‘‘clove” in Chinese, are
commonly used in folk medicines to treat dental disorders, respira-
tory disorders, headaches, digestive disorders, diarrhea and other
diseases (Issac, Gopakumar, Kuttan, Maliakel, & Krishnakumar,
2015). It is also used as a spice and food flavoring (Cortés-Rojas,
de Souza, & Oliveira, 2014). Clove is commonly known to be rich
in essential oils (approximately 15–20%), which are widely applied
in cosmetics and medicines, with many beneficial effects on
human health (Sebaaly, Jraij, Fessi, Charcosset, & Greige-Gerges,
2015; Xie et al., 2015). It is well known that a-humulene is an
important basic pharmaceutical constituent of clove oil, and it
exhibits various biological activities, such as anti-inflammatory
(Fernandes et al., 2007) and antitumor (Subhashini, Mahipal, &
Reddanna, 2005) effects. Therefore, a-humulene (Supplement S.
1) is an important anticancer constituent that contributes to the
pharmacological efficacy of clove oil. The extraction of oil from
clove is conventionally performed by hydrodistillation, steam
⇑ Corresponding author.
E-mail address: ycyang230@gmail.com (Y.-C. Yang).
Tang, & Quan, 2007), which are generally time consuming, require
large amounts of solvent, degrade thermolabile compounds and
cause hydrolysis and water solubilization of certain fragrance
constituents. Additionally, environmental safety regulations and
increased risks to public health are requiring industries to consider
alternatives to the traditional methods for clove oil extraction.
Recently, alternative extraction techniques, such as ultrasonic-
assisted extraction (UAE) (Alexandru, Cravotto, Giordana, Binello,
& Chemat, 2013; Xie et al., 2015), microwave-assisted extraction
(Yazdani et al., 2005) and supercritical carbon dioxide (SC–CO2)
extraction (Guan et al., 2007; Prado, Prado, & Meireles, 2011) have
increased the efficiency of the clove oil extraction processes and
have reduced the amount of organic solvents, operating tempera-
tures and procedure time. Among these techniques, SC–CO2 extrac-
tion is an environmentally friendly and efficient extraction
technique. By using this technique, it is possible to minimize the
environmental impact resulting from the use of volatile organic
solvents. Consequently, SC–CO2 extraction has been widely
employed for extracting oils from different materials due to its
facilitated mass transfer, solubility and selectivity (Rai, Mohanty,
& Bhargava, 2016; Tomita et al., 2014). It is faster than traditional
solvent extraction techniques, and it improves the extraction

http://dx.doi.org/10.1016/j.foodchem.2016.04.076
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
 M.-C. Wei et al. / Food Chemistry 210 (2016) 172–181 173

yields of desired compounds from complex matrices (Zhang, Zhou, 2. Experimental

& Fu, 2016). In addition, SC–CO2 can extract thermolabile com-
pounds under mild conditions due to its relatively low critical tem- 2.1. Materials
perature and pressure. More importantly, SC–CO2 can be removed
from the extracted material by simple depressurization into a suit- Dried clove buds (Syzygium aromaticum (L.) Merr. & Perry) were
able collection device, leaving no residue or contamination in the obtained from different local Chinese medicinal shops (Taiwan).
extract because it is a gas under ambient conditions, and it is con- The moisture content of the air-dried plant sample was deter-
sidered to be very safe for humans. Therefore, the extracts obtained mined to be 9.36% using Karl Fischer volumetric titration. Carbon
using SC–CO2 extraction are of superior quality compared to those dioxide with a purity of 99.8% was used as the solvent in this study,
obtained through conventional organic solvent extraction methods and it was purchased from the Yun-Shan Gas Co., Ltd. (Tainan, Tai-
(Rai et al., 2016). In addition to the advantages of possessing a rel- wan). Eugenol, b-caryophyllene, a-humulene and eugenyl acetate
atively low critical temperature and pressure and being neither were also procured as reference substances from the Sigma Chem-
toxic nor flammable, SC–CO2 is also an inert, non-explosive, inex- ical Co. (St. Louis, MO, USA) and were used without further
pensive, odorless, colorless, and environmentally friendly solvent purification.
with high purity, and it is generally considered to be a harmless
ingredient in pharmaceuticals and foods. Moreover, the solvent
2.2. Analytical methods
strength of SC–CO2 can be manipulated by changing the
temperature, pressure, static and dynamic times, flow rate and
Gas chromatography analysis of the extracts was conducted
the use of modifiers or cosolvents; therefore, it can achieve
using a GC-FID system (Shimadzu, model CG-14A, Kyoto, Japan)
remarkably high selectivity (Wei & Yang, 2015). Because of these
equipped with a DB-5 silica capillary column
characteristics, SC–CO2 extraction has become an excellent alter-
(30 m
0.25 mm
0.25 lm, J & W Scientific, Folsom, CA). The car-
native to replace conventional industrial methods for extracting
rier gas was helium (1.7 mL/min, 99.9% purity, Yun-Shan Gas Co.,
oils from various raw matrices. However, high-pressure equipment
Ltd., Tainan, Taiwan), and the split ratio was 1:20. The injector
is more expensive than that of conventional separation processes,
and detector temperatures were 220 and 240 °C, respectively,
it would result in an increase in capital and operating costs. Also,
and the column temperature was programmed to linearly increase
high-pressure equipment facilities create a potential safety hazard
from 60 to 246 °C at 3 °C/min and from 246 to 280 °C at 5 °C/min.
that has to be carefully operated and managed. Furthermore, when
Solutions of samples in chloroform were injected in an amount of
the SC–CO2 extraction at large scale uncontrolled release of large
1 lL. The peak identification of target compounds within the
quantities of CO2 can asphyxiate bystanders owing to air
extracts was performed by comparing the retention times and
displacement.
chromatographic behaviors to those of authentic standards. The
At present, SC–CO2 is widely applied as a solvent for extracting
quantity of the target compounds was calculated by comparing
the oil from clove (Darabi et al., 2011; Ivanovic, Dimitrijevic-
their peak area to that of the standards.
Brankovic, Misic, Ristic, & Zizovic, 2013), and clove has also been
Qualitative analysis of the extracts was performed by gas chro-
used as a model for studying the kinetics and thermodynamics of
matography/mass spectrometry (GC/MS). GC/MS was performed
SC–CO2 extraction systems (Hatami, Meireles, & Zahedi, 2010;
using a Thermo Finnigan Polaris Q Ion Trap with a TRACE GC/MS
Ivanovic, Zizovic, Ristic, Stamenic, & Skala, 2011) due to its oil-
equipped with a fused-silica capillary column (30 m
0.25 mm,
rich property (approximately 20%). However, this work is the first
0.25-lm film thickness, model HP-5MS, Agilent Technologies Co.,
time in which the combination of ultrasound-assisted static
Ltd., Palo Alto, USA) and a mass spectrometer from the same com-
extraction followed by SC–CO2 dynamic extraction has been used
pany, which was operated in the EI mode (energy voltage, 70 eV).
to obtain clove oil and a-humulene from clove. The application
The column temperature was initially set at 75 °C for 1 min, and
of ultrasonic energy to assist the SC–CO2 extraction process pro-
then it was programmed to heat from 75 to 275 °C at 10 °C/min
duced a relevant increase in the final yield of the oil, along with
and subsequently held at 275 °C for 60 min. The injector was main-
a noticeable reduction in energy consumption through an increase
tained at 250 °C, and helium was used as the carrier gas (1 mL/min;
in the extraction rate. Riera et al. (2010) reported that the yields of
1:10 split ratio). The ion source temperature was 200 °C. The ion-
almond oil and cocoa cake oil were increased by 90% and 43%,
ization energy was 70 eV with a scan time of 0.5 s and mass range
respectively, with the presence of ultrasound compared to tradi-
of 50–500 AMU. The samples were run in ethyl acetate with a dilu-
tional SC–CO2 extraction. Fan, Hou, Huang, Qiu, and Jiang (2015)
tion of 5% (v/v) (Yang, Wei, & Hong, 2014).
reported that under less severe operating conditions, the yield of
Eugenol, b-caryophyllene, a-humulene and eugenyl acetate in
lutein from Chlorella pyrenoidosa could increase by 34.5% when
the clove oil were identified by matching their mass spectra and
an ultrasonic field was integrated inside a supercritical extractor.
retention indices with those of pure compounds. Mass spectra cor-
The enhanced SC–CO2 extraction yields with ultrasound assistance
relations were performed using the NIST (National Institute of
were also consistent with previous findings (Riera et al., 2010;
Standards and Technologies) Mass Spectra Library and Wiley Mass
Yang & Wei, 2016). However, no reports have been found in the lit-
Spectra Library.
erature that consider the simultaneous application of SC–CO2 and
ultrasound (USC–CO2) for the extraction of clove oil and a-
humulene from clove buds. Therefore, the primary objective of this 2.3. Ultrasound-assisted SC–CO2 (USC–CO2) extraction
study was to test the possibility of using ultrasound-assisted SC–
CO2 for extracting clove oil and a-humulene from clove buds at a USC–CO2 extraction experiments were performed using a semi-
set pressure, temperature and dynamic extraction time. To com- batch flow extraction apparatus. The solvent was delivered into a
pare the USC–CO2 extraction results, clove was also extracted using surge tank where the temperature was maintained at 2.0 °C
traditional SC–CO2 extraction, steam distillation and heat-reflux (±0.1 °C) by a thermostatic bath (Haake F3-K, Haake, Karlsruhe,
extraction with n-hexane as the solvent. The a-humulenes Germany) to guarantee liquid CO2 in the pump head section. The
obtained using these four techniques were analyzed using gas solvent was then pressurized by a high-pressure pump (ISCO
chromatography and gas chromatography/mass spectrometry 260D, Lincoln, NE, USA) and pumped to fill the extraction vessel
(GC/MS). until the working pressure was reached. The herbal sample
174 M.-C. Wei et al. / Food Chemistry 210 (2016) 172–181
(20.0 g, 0.355 mm) was well mixed with 2-mm stainless steel balls
and was then charged into the 152-mL stainless steel extraction
vessel (SS304, i.d. of 2.2 cm and length of 40.0 cm). The extraction
vessel was then immersed into the ultrasonic bath (fixed at the
same position in the bath) to maintain a constant temperature
throughout the extraction process. More details about the SC–
CO2 extraction equipment (Fig. 1A) and its operation have recently
been described elsewhere (Yang & Wei, 2015a).
The process of ultrasound-assisted extraction (static stage) uti-
lized an ultrasonic bath with a working frequency of 40 kHz and
power of 185 W (Branson B-33810E-DTH, USA). Following the
dynamic extraction (with ultrasound assistance), CO2 loaded with
extract exited through a thermostated line into a temperature-
controlled micrometer valve, where it expanded to atmospheric
pressure, releasing the dissolved solute. The extracts were then
collected in two cold separators placed in series. In both cases,
optimum fractionation was achieved by operating at 9.0 MPa and
4 °C in the first separator and at 0.15 MPa and 5 °C in the second.
Finally, the expanded low-pressure CO2 gas released from the
extract passed through a wet-gas flow meter (type TG05, Ritter
Co., Germany) into the atmosphere. The gas flow rate was moni-
tored using a rotameter (model p23/10082-03G, Aalborg Co.),
and the total volume of gas was measured using a wet test meter.
This extraction process was repeated six times for the same
sample to ensure that accurate experimental data were obtained.
The standard error from replicate experiments was determined
to be ±6%. The data in this study are presented as the means ± stan-
dard deviation (SD).
2.4. Heat-reflux extraction (HRE)
The experimental apparatus and the extraction procedures have
been described in detail in the literature (Yang & Wei, 2015b).
Briefly, 5 g of dried powdered plant was placed in a flask, and
100 mL of hexane was added. To prevent solvent loss during
extraction, a condenser was connected to the flask. Then, 5 g of
dried clove buds (0.355 mm) was extracted using HRE for
240 min. After extraction, the solutions were carefully decanted,
and the plant material was re-extracted using the same solvent.
The solvent was removed via rotary vacuum distillation at
Fig. 1. Schematic flow diagram of the USC–CO2 extraction apparatus (A) and gas chromatograms of the extracted reference compound (B) and clove bud essential oil (C)
obtained using USC–CO2 extraction (75% duty cycle) at 44.0 °C, 18.5 MPa, particle size of 0.355 mm, CO2 flow rate of 1.4 g/min and extraction time of 115 min. V1, V2, V3, V4:
stopping valve (on-off valve); HPA, HPB: syringe pump; M: mixer; CB: circulation bath; CH: circulating heater; MV1, MV2: micro-metering valve; HC: heating coil; E:
extraction vessel; PG: pressure gauge; BR: backpressure regulator; FF: float flowmeter; T: thermocouple; TM: mercury-in-glass thermometer.
40–50 °C, and the residue was weighed. At least six replicates were
performed for each extraction condition tested. The yield (wt%)
was defined as the oil mass versus the material mass.
2.5. Steam distillation (SD)
One-hundred grams of dried and ground clove buds (0.355 mm)
in a 500-mL flask was subjected to steam distillation. The distilla-
tion was conducted until no more essential oil was obtained, and
the essential oil collected at the exit of the glass condenser was
separated by the action of gravity.
2.6. Solubility measurements
The solubility of the solute in SC–CO2 was measured using the
dynamic method following the description previously reported
by Rodrigues et al. (2002) and Sousa et al. (2002). To use the
dynamic method to assess the experimental solubility data, the
fixed bed was considered long enough and the solvent flow rate
sufficiently low to enable saturation/equilibrium conditions to be
obtained. For each experiment, a mixture of dried and ground clove
buds (20 g, 0.355 mm) and glass beads (1 mm diameter) was
placed in a 152-mL stainless steel extraction vessel (SS304, i.d. of
2.2 cm and length of 40.0 cm). The CO2 flow rate was maintained
at 0.09–0.65 g/min, and the extraction time was measured at 4–
10 min intervals for an overall time of 240 min. The equilibrium
solubilities of clove oil were measured from data obtained at tem-
peratures of 32, 37, 42 and 47 °C and pressures of 9.0, 17.0 and
25.0 MPa using particles with a size of 0.355 mm. The clove oil
was collected in the trap, which was a U-shaped glass tube with
a glass-wool filter inserted in the exit shoulder to prevent entrain-
ment of tiny oil droplets. The oil in the trap was weighed using an
analytical balance and analyzed for its eugenol, b-caryophyllene,
a-humulene and eugenyl acetate contents using GC and GC/MS.
The volume of CO2 consumed was measured under each extraction
condition using a wet flow meter and converted to mass. The mass
of CO2 was calculated from the SC–CO2 density in each condition.
The extraction yield of clove oil was expressed as the percent ratio
(% w/w) of the mass of oil per mass of clove buds. The extraction
yields of the compounds (eugenol, b-caryophyllene, a-humulene
 M.-C. Wei et al. / Food Chemistry 210 (2016) 172–181
and eugenyl acetate) were expressed as the percent ratio (% w/w)
of the mass of compound per mass of extracted oil.
Following studies of Rodrigues et al. (2002) and Sousa et al.
(2002), the overall extraction curves (OECs) of clove oil with SC–
CO2 were obtained using the dynamic method and were presented
as the total mass of clove oil (g clove oil/g clove buds) as a function
of extraction time (min) or as a function of the solvent/biomass
mass ratio (g CO2/g clove buds). The obtained OECs were character-
ized by three distinguishable regions or periods, namely, the con-
stant extraction rate (CER) period, the falling extraction rate
(FER) period and the diffusion-controlled period. The solubility of
clove oil can be obtained using the dynamic extraction method
and calculated from the slope of the linear portion of the OEC
obtained at equilibrium conditions, i.e., at a low solvent flow rate
to allow the SC–CO2 to saturate with solute. However, one diffi-
culty in measuring solubility using the dynamic method is deter-
mining an adequate CO2 flow rate. The CO2 flow rate should be
low enough such that the solvent is saturated at the extraction ves-
sel outlet, but at the same time, mass-transfer limitations should
be avoided. Using the experimental data, the OECs were fit to a
spline using two straight lines. The first line was identified as the
CER period. The rate of mass transfer (g clove oil/min) for the
CER period (MCER) and the time (min) corresponding to the inter-
ception of the two lines (tCER) were obtained from the spline fit.
The slope of the line in the CER period of the OEC corresponds to
the YCER value, i.e., the solvent phase solute mass ratio at the bed
outlet, evaluated during the CER period. YCER (g clove oil/g CO2)
was obtained by dividing MCER (g clove oil/min) by the mean sol-
vent flow rate (QCO2) (g CO2/min) for the CER period. At saturation,
the YCER should be maximum, and the corresponding CO2 flow rate
is suitable for measuring the solubility of clove oil. Thus, the max-
imum value of the YCER is identified as the solubility of clove oil, Y⁄
(g clove oil/g CO2), and the corresponding CO2 flow rate is denoted
by Q⁄. For CO2 flow rates that are larger than Q⁄, the solvent leaves
the supercritical fluid system unsaturated, whereas for CO2 flow
rates that are smaller than Q⁄, the axial dispersion may not be com-
pletely negligible. Thus, the solubility of clove oil in the supercrit-
ical CO2 was obtained from the CER period for the operating
conditions.
Because the obtained clove oil is a mixture of several interact-
ing solutes, solute-solute interactions affect the solubility behav-
ior of a constituent in the mixture compared with the solubility of
the pure counterpart. In this study, the maximum value of the
mass ratio of a constituent in the supercritical CO2 is used as
the fictitious solubility of the constituent (Yc⁄) (g a-humulene/g
CO2). The calculated fictitious solubility of the constituent (Yc⁄)
was similar to the solubility of clove oil; in addition, the curves
were constructed in terms of the cumulative yield of constituent
(g constituent/g clove oil) as a function of the extraction time
(min) or as a function of the solvent/clove oil ratio (g CO2/g clove
oil). Similarly, Yc⁄ (g a-humulene/g CO2) was obtained by dividing
Mc,CER (g a-humulene/min) by the mean solvent flow rate (QCO2)
(g CO2/min) for the CER period.
2.7. Statistical analysis
All yields and compositions were calculated on a moisture-free
basis. The mean and standard error of the mean were calculated
using six experiments. The results are expressed as the means ± SD.
Analysis of variance (ANOVA) was performed using Tukey’s
method with a significance level of P < 0.05 using Microsoft Office
Excel 2010 (Microsoft Co., USA) and Origin version 6.1 (Origin Lab
Co., Northampton, MA, USA). Furthermore, the concordance
between the experimental data and the calculated value was
established by the average absolute relative deviation (AARD) and
175
the coefficient of determination (R2), which were determined using
the following equations:
n  
100 X 
yp;i  ye;i 

AARD ¼ ð1Þ
n i¼1  ye;i 
Pn
i¼1 ðyp;i  ye;i Þ2
R2 ¼ ð2Þ
ðyp;i  ym Þ2
where yp,i and ye,i are the predicted and experimental solubili-
ties, respectively; ym is the mean solubility; and n is the number
of experimental runs.
3. Results and discussion
3.1. Identification and quantification
The knowledge of the oil characteristics is essential for identify-
ing the best process and extraction conditions. Therefore, the clove
oil analysis aimed to observe the changes in product quality as a
function of the extraction procedure used. The chemical composi-
tion of the clove oil was evaluated using GC–MS and GC. Eugenol,
b-caryophyllene, a-humulene and eugenyl acetate were the major
components identified. The contents of eugenol, b-caryophyllene,
a-humulene and eugenyl acetate in the clove oil were further iden-
tified and quantified using GC/MS and GC. The gas chromatograms
of the reference compound and of clove bud extracts obtained by
USC–CO2 extraction (75% duty cycle) at 44.0 °C, 18.5 MPa, particle
size of 0.355 mm, CO2 flow rate of 1.4 g/min and extraction time of
115 min are presented in Fig. 1B and C, respectively. Fig. 1C shows
that the compound of interest was well separated under the
optimal conditions described above. Furthermore, eugenol,
b-caryophyllene, a-humulene and eugenyl acetate were unequivo-
cally authenticated by their relative retention values and spectra.
Therefore, this GC system successfully separated and identified
the eugenol, b-caryophyllene, a-humulene and eugenyl acetate in
the extracted clove buds (Fig. 1C), and it can be used to quantify
the contents of eugenol, b-caryophyllene, a-humulene and eugenyl
acetate.
3.2. Effects of ultrasonic energy, extraction temperature and pressure
on a-humulene yield
Extraction pressure and temperature are undoubtedly two of
the most relevant process parameters of SC–CO2 extraction that
influence the extraction efficiency and selectivity of SC–CO2. To
obtain the best a-humulene extraction efficiency, the temperature
and pressure must be optimized. Fig. 2 shows the results obtained
using extraction pressures from 9.5 to 22.5 MPa and extraction
temperatures from 32 to 50 °C while the other parameters
remained fixed at their previously determined optimal levels: for
the USC–CO2 extraction, a static extraction time of 15 min, a
dynamic extraction time of 100 min, a mean plant particle size of
0.355 mm and a CO2 flow rate of 1.4 g/min at 40 kHz, 185 W and
a 75% duty cycle; and for the traditional SC–CO2 extraction, a static
extraction time of 30 min, a dynamic extraction time of 180 min, a
mean plant particle size of 0.355 mm and a CO2 flow rate of 1.8 g/
min. Fig. 2 reveals that at four different pressure levels (9.5, 14.5,
18.5 and 22.5 MPa) for SC–CO2 with or without ultrasound-
assisted extraction, the a-humulene yield decreased as the tem-
perature increased from 32 to 50 °C; this phenomenon is attributed
to the decreased density of the CO2 without compensation by the
increased diffusivity or the vapor pressure of the analytes to be
extracted. In addition, the decrease in extraction yield with
increasing temperature may be due to thermal degradation
176
(A) USC-CO2
α-humulene yield (mg/g)
4.0
3.5
1.0
0.0
10
P (MPa)
Fig. 2. Effects of extraction pressure and temperature on extraction yield of a-humulene using USC–CO2 (A) and conventional SC–CO2 (B) extractions.
occurring in the clove oil when the temperature is increased, as
a-humulene is considered to be a thermolabile compound due to
its physicochemical properties (Supplement S. 1). Therefore, there
was a downward trend when the temperature increased from 32 to
50 °C, implying that there was an optimal temperature for practical
applications. Consequently, the highest a-humulene yields were
obtained at 32 °C when the operation was conducted at the afore-
mentioned pressures for both the USC–CO2 and traditional SC–CO2
extractions. Similar phenomena were also reported when other oils
were extracted using traditional SC–CO2 extraction (Jia, Li, & Xiao,
2009) and when the oil and coixenolide from adlay seed were
extracted using USC–CO2 extraction (Hu, Zhao, Liang, Qiu, &
Chen, 2007). Additionally, the yield of a-humulene decreased
when the extraction pressure increased at a given temperature
within a certain pressure range (9.5, 14.5, 18.5 and 22.5 MPa), as
also presented in Fig. 2. Theoretically, increased pressure causes
the density of the SC–CO2 to increase, thereby enhancing its solvat-
ing power and therefore improving the extraction process. How-
ever, as the pressure increases, diffusivity decreases and the
molecules have more difficulty diffusing into pores and dissolving
the solute. In addition, increasing the SC–CO2 pressure causes the
solid matrix to be more tightly packed, thus decreasing the void
fraction and reducing the extraction efficiency. Moreover, the
decreased extraction yield due to sample compaction at a higher
pressure causes the SC–CO2 to channel and inhibits its diffusion
into the sample (Yang et al., 2014). Therefore, increasing the pres-
sure generates these three competing influences, resulting in a
reversed effect on the extraction efficiency; an optimal pressure
may exist. Furthermore, the reduction in the extraction yield at
higher pressures could be related to the higher vapor pressure of
a-humulene (Supplement S. 1), which eventually makes extraction
at high pressures unfavorable. Thus, the results presented in Fig. 2
revealed that for both USC–CO2 and traditional SC–CO2 extractions,
9.5 MPa is the best pressure to obtain the maximum yield of a-
humulene from clove extracts. Similar results were observed in
the SC–CO2 extraction of diterpenes from spent coffee grounds
(Barbosa, de Melo, Coimbra, Passos, & Silva, 2014) and of bioactive
compounds from Lavandula viridis L’Hér (Costa et al., 2012).
In this study, the USC–CO2 extraction conditions of 32 °C and
9.5 MPa may have been the optimal and most economical condi-
tions, for which the yield of a-humulene reached 4.0328 mg/g with
ultrasound at 25 kHz and 400 W. At each identical extraction tem-
perature and pressure (Fig. 2), ultrasound assistance enhanced the
M.-C. Wei et al. / Food Chemistry 210 (2016) 172–181
(B) SC-CO2 32 oC
38 oC
44 oC
3.5
α-humulene yield (mg/g)
50 oC
3.0
1.0
0.0
15 20 10 15 20
P (MPa)
effectiveness of a-humulene extraction by SC–CO2 such that the
yield of a-humulene obtained by USC–CO2 extraction for 100 min
was 1.1–1.2 times that obtained by SC–CO2 extraction for
180 min at various temperatures and pressures. Applying ultra-
sound clearly significantly accelerated the extraction procedure
and increased the yield of a-humulene. This finding can be
explained by the fact that ultrasound leads to the collapse of
cavitation bubbles near tissue surfaces, produces micro-jets, and
disrupts tissues and cells, thus allowing good penetration of the
SC–CO2 solvent into the tissue matrix and allowing the solvent to
access the soluble constituents. Such enhanced yields of the target
compound using USC–CO2 extraction under less severe operating
conditions are consistent with previous observations (Hu et al.,
2007). Therefore, this study revealed that the application of
ultrasound during the SC–CO2 extraction of clove buds results in
a significant rate acceleration and yield improvement.
3.3. The solubility of clove oil and fictitious solubility of a-humulene in
SC–CO2
Optimization of SC–CO2 extraction processes requires knowl-
edge of solubility data, which contributes to the selection of the
operating conditions. The clove solubility in SC–CO2 was deter-
mined using the dynamic method of USC–CO2 extraction (75% duty
cycle) using low CO2 flow rates (0.09–0.65 g CO2/min), assuming
that the solvent was saturated by the solute as it flows through
the bed of solids. The effect of the CO2 flow rate on the determina-
tion of the solubility of clove with SC–CO2 is presented in Fig. 3A
for assays performed at a pressure of 25.0 MPa and temperature
of 32 °C using a dried clove with a mean particle size of
0.355 mm. The results indicated that the value of the slope of the
straight line in the first portion (CER period) of the OEC (Fig. 3B,
Supplements S. 2 and S. 3) corresponds to the YCER (MCER/QCO2) val-
ues for USC–CO2 extractions performed at various CO2 flow rates.
Fig. 3A shows that the YCER initially increased when the CO2 flow
rate changed from 0.09 to 0.32 g CO2/min but slightly decreased
when the CO2 flow rate increased to 0.65 g CO2/min. This result
may have occurred because for the lower CO2 flow rates of 0.09
and 0.15 g CO2/min, the effects of axial dispersion were important,
resulting in a smaller value of YCER. In addition, at relatively lower
CO2 flow rates, the value of YCTE decreased, probably due to the
higher intra-particle solid diffusion, which may overcome the con-
vective effects. However, the higher CO2 flow rates (0.47 and 0.65 g
 M.-C. Wei et al. / Food Chemistry 210 (2016) 172–181 177

(C) 32 oC 37 oC 42 oC
24
47 oC
22 22

g clove oil / g clove (%)

g clove oil / g clove (%)

g clove oil / g clove (%)

22
g clove oil / g clove (%)

22
20 20
20 20

18 18
18 18

16 16 16 16

14 14 14 14

4 4 4 4
2 2 2 2
0 0 0 0
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
g CO2 / g clove g CO2 / g clove g CO2 / g clove g CO2 / g clove

(D)
g α-humulene / g clove oil (%)

32 oC

g α-humulene / g clove oil (%)

g α-humulene / g clove oil (%)

2.5 37 oC 2.5 42 oC 47 oC
g α-humulene / g clove oil (%)

2.5 2.0

2.0 2.0
2.0
25 MPa
17 MPa
1.5
9 MPa 1.5 1.5
1.5

0.0 0.0 0.0 0.0

0 5 10 15 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
g CO2 / g clove oil g CO2 / g clove oil g CO2 / g clove oil g CO2 / g clove oil

Fig. 3. The process for determining clove oil solubility and a-humulene fictitious solubility. (A) Effect of the CO2 flow rate on the YCER value (g clove oil/g CO2) for clove at
32 °C and 25.0 MPa using USC–CO2 dynamic extraction; (B) overall extraction curves (OECs) for the USC–CO2 dynamic extraction of clove at a CO2 flow rate of 0.09–0.65 g/
min, 32 °C and 25.0 MPa; (C) overall extraction curves (OECs) for the USC–CO2 dynamic extraction of clove at CO2 flow rate of 0.32 g/min, 32–47 °C and 9.0, 17.0 and
25.0 MPa; (D) cumulative yield of a-humulene for the USC–CO2 dynamic extraction of clove at a CO2 flow rate of 0.32 g/min, 32–47 °C and 9.0, 17.0 and 25.0 MPa.

CO2/min) did not have a positive effect on the values of YCER. This were selected to determine the CO2 flow rate values at which sat-
unforeseen effect of CO2 flow rates might be due to shorter resi- uration values become independent of the flow rate because in this
dence times, in which the contact time between the SC–CO2 and study, under such conditions, SC–CO2 reaches its highest density,
the solute will be shorter than the time required to saturate the which is 914.6 kg/m3 (Zuknik, Norulaini, Dalila, Ali, & Omar,
solvent, and thus, the YCER will be smaller than the saturation value 2016). Nonetheless, the effects of temperature and pressure on
(Sousa et al., 2002). Thus, to measure solubility using the dynamic the hydrodynamics of the extractor must be considered. In this
method evaluated in this study, the clove solubility (Y⁄) for each work, the variations in the thermophysical properties of the super-
operating condition was considered to be the highest value of YCTE. critical fluid system were relatively small due to the narrow inter-
Fig. 3A shows that the CO2 flow rate of 0.32 g/min can be used to val of both temperature and pressure, in which the effects of the
measure solubility using the dynamic method for the clove + CO2 flow rates on the values of YCER were not significant. Thus,
SC–CO2 system. Under this condition, it was assumed that when such flow rates are applied in other experiments for investi-
the SC–CO2 was saturated with the clove. The full saturation of gating clove solubility in SC–CO2, the solvent leaving the extractor
SC–CO2 for the CO2 flow rate of 0.32 g/min is considered to be under the saturation condition is assured, regardless of the other
evidence that equilibrium concentration levels were achieved conditions being applied. Therefore, all solubility measurements
under the corresponding pressure and temperature conditions. A for the clove + CO2 system (Fig. 3C) that were subsequently con-
temperature and pressure of 32 °C and 25.0 MPa, respectively, ducted in this study were performed at a CO2 flow rate of 0.32 g/
178 M.-C. Wei et al. / Food Chemistry 210 (2016) 172–181

Table 1 by the initial slope in the plot of the yield of the compound of inter-
Solubility measured by the dynamic USC–CO2 extraction method over the temper- est (g a-humulene/g clove oil) versus CO2 consumption (g CO2/ g
ature range of 32–47 °C and pressure range of 9.0–25.0 MPa for the pseudo-ternary
system.
clove oil) (Fig. 3D). The results of the fictitious solubility measure-
ments of a-humulene from clove extract are presented in Table 1.
T (°C) P (MPa) qa (kg/m3) Y*oilb
104 Y⁄a-humulenec
104 As shown in Table 1, the fictitious solubility of a-humulene
(g oil/g CO2) (g a-humulene/g CO2)
(49.3
104–70.5
104 g a-humulene/g CO2) decreases with
32 9.0 704.2 2432.4 ± 95.1 70.5 ± 2.8 increasing temperature, whereas the effect of pressure is not sig-
32 17.0 853.9 2560.7 ± 98.2 68.3 ± 2.7
32 25.0 914.6 2652.6 ± 95.3 68.2 ± 2.6
nificant. This effect of temperature on solubility is attributed to
37 9.0 506.7 2425.0 ± 92.9 62.4 ± 2.3 the fact that increasing temperature has a greater effect on reduc-
37 17.0 824.0 2611.0 ± 98.4 62.3 ± 2.4 ing the density of SC–CO2 than on increasing the vapor pressure of
37 25.0 893.3 2735.3 ± 93.7 62.2 ± 2.5 the analyte. In addition, thermal degradation of a-humulene may
42 9.0 423.7 2397.7 ± 86.9 57.5 ± 2.1
occur at higher temperatures (37, 42 and 47 °C). The phenomenon
42 17.0 791.9 2718.0 ± 94.8 60.3 ± 2.7
42 25.0 871.3 2804.2 ± 96.6 60.2 ± 2.4 of lower solubility at higher temperatures was also observed in
47 9.0 340.1 2298.1 ± 89.9 49.5 ± 1.9 previous studies (Barbosa et al., 2014; Chen et al., 2014). As shown
47 17.0 757.0 2755.9 ± 99.5 49.4 ± 1.8 in Table 1, the fictitious solubility of the compound of interest
47 25.0 848.7 2900.5 ± 107.6 49.3 ± 2.0 decreased slightly with increasing extraction pressure within the
a
q: density of the USC–CO2. selected pressure range. Although pressure has positive effects on
b
Y⁄oil: the solubility of clove oil in SC–CO2. Values are mean ± SD of 3 replications. the density of SC–CO2 and a negative effect on the diffusion coeffi-
c
Y⁄a-humulene: the fictitious solubility of a-humulene. Values are mean ± SD of 3 cient of the solute, the influence of decreasing solubility plays the
replications.
key role in the extraction process. In addition, an increase in pres-
sure causes the solid matrix to become more packed and the void
fraction to decrease, thereby leading to lower solubility because of
min. Table 1 presents the CO2 flow rate, the SC–CO2 density, the the decreased interaction between SC–CO2 and the sample. This is
YCTE values and the clove solubility for the investigated tempera- in agreement with earlier studies on the SC–CO2 extraction of var-
ture and pressure conditions. This table shows that the oil solubil- ious compounds from raw materials (Chen et al., 2014; Costa et al.,
ity for the clove buds/CO2 system varied from 2298.1
104 to 2012).
2900.5
104 g clove oil/g CO2 for the experimental conditions
in this study. As shown in Table 1, the solubility of clove increases 3.4. Correlation of the solubility data
with increasing temperature at pressures between 17.0 and
25.0 MPa. However, the inverse effect is observed for a lower pres- The experimental fictitious solubility results for a-humulene in
sure of 9.0 MPa, where the solubility of clove decreases with SC–CO2 were subsequently correlated with the following three
increasing temperature. It is known that an increase in tempera- density-based equations proposed by Bartle, Clifford, Jafar, and
ture at a constant pressure will result in a decrease in the density Shilstone (1991), Chrastil (1982) and Kumar and Johnston (1988).
of SC–CO2, which in turn lowers its solvating power. However, a Chrastil described one of the first density-based models, providing
temperature increase results in an increase in the solute vapor a linear relationship between the natural logarithms of the equilib-
pressure and in the solute diffusivity. At a lower pressure of rium mole fraction of the solute in SC–CO2, y2, the density of the
9.0 MPa, the density of SC–CO2 is considerably more sensitive to SC–CO2, q (g/L), and temperature that is given by the following
increases in temperature, which results in a large decrease in the relation:
density of SC–CO2 and, despite the increases in solute vapor pres-
a2
sure and diffusivity, in a net decrease in the solute solubility. In Lnðy2 Þ ¼ a0 þ a1 LnðqÞ þ ð3Þ
contrast, at higher pressures of 17.0 and 25.0 MPa, the density of T
SC–CO2 will not be substantially affected by an increase in temper- where T is the temperature (K), a1 is an average equilibrium associ-
ature, while the solute vapor pressure and solute diffusivity will ation number that also depends on the molar mass of the solute and
play a dominating role, resulting in a net increase in the solute sol- represents the average number of CO2 molecules in the solvated
ubility. Therefore, by examining the effect of temperature on the complex, and a0 is another association constant related to the
solubility of clove in Table 1, it can be concluded that retrograde molecular weight and the melting point of the solute and CO2. In
solubility behavior exists in the clove + SC–CO2 system. This retro- addition, the constant a2 depends on the total heat of solution by
grade solubility behavior is consistent with the extraction of rice
DHT
bran oil reported previously by Tomita et al. (2014), in which the a2 ¼ ð4Þ
solubility cross-over pressure for rice bran oil was approximately R
20.0 MPa. where DHT (kJ/mol) is the total heat of reaction or heat of solu-
For the USC–CO2 extraction of compounds of interest, the solu- tion, which is the sum of the enthalpies of vaporization (or subli-
bility of the solutes in the supercritical fluid phase is a key aspect mation), DHvap, and solvation of the solute, DHsolv, and R is the
for understanding the process and therefore for evaluating the universal gas constant (8.314 J/mol K).
characteristics of the obtained materials. Hence, fictitious solubil- The experimental fictitious solubility data for a-humulene in
ity measurements were performed using the overall extraction SC–CO2 are first correlated using the Chrastil model, which is
curves for the studied a-humulene in SC–CO2 under the pressure described as Eq. (3). The plots of Ln(y2) versus Ln(q) for each tem-
range of 9.0–25.0 MPa and at operating temperatures of 32– perature resulted in a straight line, which indicates that the model
47 °C, with a dynamic time ranging from 4 to 240 min under can accurately correlate the experimental data. Additionally, the
USC–CO2 extraction. The fictitious solubility of a-humulene in coefficients of the Chrastil model, a0, a1 and a2 (Eq. (3)), are
SC–CO2 was obtained from the CER period (Fig. 3D) for the condi- adjusted by fitting the experimental data to this model. The corre-
tions of 32–47 °C, 9.0–25.0 MPa and CO2 flow rate of 0.32 g CO2/ lation results and the parameters of the equation are shown in
min, which is considered to be a solvent flow rate that is low Table 2. As shown in this table, the Chrastil model provides a rather
enough to ensure the equilibrium condition. According to previous good fit, with an overall AARD(%) of 1.24%. In the Chrastil model,
reports (Rodrigues et al., 2002; Sousa et al., 2002; Zuknik et al., the total heat of solution was obtained according to Eq. (4)
2016), the fictitious solubility of a-humulene in SC–CO2 is given (Table 2).
 M.-C. Wei et al. / Food Chemistry 210 (2016) 172–181 179

Table 2
The fitted solubility parameters of three density-based models and the thermodynamic properties of a-humulene in SC–CO2.

Model Constant na AARD (%) Thermodynamic properties

Chrastil 12 1.24 DHT,C = 14.37 (kJ/mol)
a0 10.39
102a1 4.79
a2 1728.48
Kumar & Johnston 12 1.06 DHT,K–J = 12.67 (kJ/mol)
b0 10.03
106b1 7.32
b2 1523.95
Bartle 12 5.69 DHvap = 57.35 (kJ/mol)
c0 23.32
103c1 1.85
c2 6898.10
DHsolvb = 43.83 (kJ/mol)
a
Number of data points used in the correlation.
b
DHsolv ¼ 12 ðDHT;C þ DHT;KJ Þ  DHv ap .

The next model proposed by Kumar and Johnston (K–J) (1988) DHsolv ¼ DHT  DHv ap ð11Þ
correlates the solubility as
In the Bartle model, the constants c0, c1 and c2 are estimated
b2 from the experimental fictitious solubility data of a-humulene in
Lnðy2 Þ ¼ b0 þ b1 q þ ð5Þ SC–CO2. The resulting parameters for the Bartle equation (Eq. (9))
T
are listed in Table 2. The overall AARD% (5.69%) between the exper-
where y2 is the equilibrium mole fraction of the solute in SC–CO2, T
imental and correlated results shows that good agreement was
is the temperature (K), q is the density of SC–CO2 (g/L), and b0, b1
achieved using the Bartle model. A representation of Ln(y2P/Pref)
and b2 are adjustable parameters that can be obtained by regression
versus q-qref at different temperatures collapses into a single
analysis of the solubility data. In addition, the energy term (i.e., the
straight line. In the Bartle model, the heat of vaporization of the
coefficient of temperature term) parameter of b2 is related to the
solute in the supercritical fluid system was further approximated
total heat of solution, DHT, given by:
using parameter c2 (Eq. (10)). Based on the values of the total solu-
DH T tion heat and heat of vaporization, the heat of solvation can be cal-
b2 ¼  ð6Þ culated. The thermodynamic properties (DHsolv and DHvap) of a-
R
humulene in SC–CO2 are listed in Table 2.
Similarly, the Ln(y2) values were plotted against the density val-
ues, and the results were fitted as a straight line using a linear least
squares regression. The coefficients of the K–J model, b0, b1 and b2
3.5. Comparison of USC–CO2 and conventional extraction methods
(Eq. (5)), are also adjusted by fitting the experimental solubility
under the best conditions and other studies
data to this model. The K–J correlation results are reported in
Table 2. The results calculated using the K–J model are in satisfac-
The effects of the operating conditions were investigated, and
tory agreement with the experimental solubility data, with an
the best experimental conditions that provided the highest clove
overall AARD% value of 1.06%. Similarly, in the K–J model, the total
oil and a-humulene yields were recorded. A comprehensive com-
heat of solution was estimated using parameter b2 (Eq. (6)), as
parison of the yields of clove oil and a-humulene obtained using
shown in Table 2.
the different methods is presented in Table 3. The content of a-
Another semi-empirical density-based model has been pro-
humulene was determined using GC/MS and GC. As shown in
posed by Bartle et al. (1991), which starts from a linear semi-log
Table 3, the content of a-humulene in clove oil obtained using
relationship between solute solubility and solvent density, and
HRE is the lowest among the four extraction methods. Thermal
by adding a reference pressure and reference density, the following
degradation of a-humulene may occur during HRE. Additionally,
equations are obtained:
the crude extract obtained using HRE is a brown ointment, which
 
y2 P means that more undesired impurities and organic solvents may
Ln ¼ A þ c1 ðq  qref Þ ð7Þ be present. Furthermore, the values of all parameters were reduced
Pref
when ultrasound was applied, with USC–CO2 extraction offering
where the most important advantages over other methods. The yield
c2 obtained using USC–CO2 could be significantly increased by
A ¼ c0 þ ð8Þ approximately 31% and the extraction time decreased 45% com-
T
pared to conventional SC–CO2 extraction operating under its opti-
and then mal conditions. A comparison of SC–CO2 extraction and steam
  distillation was also performed. The a-humulene concentration
y2 P c2
Ln ¼ c0 þ c1 ðq  qref Þ þ ð9Þ in the clove extracted using steam distillation was 2.35%. The
Pref T
results show that the content of a-humulene in the extract
DHv ap ¼ c2 R ð10Þ obtained by USC–CO2 extraction was higher than that in the
extract obtained by steam distillation, whereas the extracting time
where P is the pressure (MPa), Pref is 0.1 MPa, T is the temperature of USC–CO2 extraction was considerably lower than that of steam
(K), q is the density of SC–CO2 (g/L), qref is a reference density with a distillation. Moreover, the clove oil obtained using USC–CO2 oper-
value of 700 g/L, and c0, c1 and c2 are constants. The solvation ating at the best conditions (44 °C/18.5 MPa) was 23.31% (Table 3),
enthalpy, DHsolv, can be approximated for each solute–CO2 pair sys- whereas the extraction with n-hexane resulted in a comparably
tem given by lower yield (17.2%). The steam distillation process unexpectedly
180 M.-C. Wei et al. / Food Chemistry 210 (2016) 172–181

Table 3
Comparison of the extraction conditions and extraction yields obtained by heat-reflux extraction (HRE), steam distillation (SD), supercritical carbon dioxide (SC–CO2) and
ultrasound-assisted supercritical carbon dioxide (USC–CO2) methods and other studies.

Extraction parameters Extraction mode

HRE UAE MAE SE SD SC–CO2 USC–CO2
Solvent n-hexane — CO2 CO2
Stirring rate (rpm) 300 — — —
Static extraction time (min) — — 30 15
Dynamic time (min) — — 180 100
Extraction time (min) 240 (4 cycles) 300 210 115
Extraction temperature (°C) 55 100 32 32
Extraction pressure (MPa) — — 9.5 9.5
Liquid/solid ratio (mL/g) 16 — 81.0 35.8
CO2 flow rate (g/min) — — 1.8 1.4
Duty cycle of ultrasound exposure (%) — — 75
a-Humulene yield (g/g oil, w/w%)a 1.71 ± 0.06 2.35 ± 0.09 1.89 ± 0.07 2.75 ± 0.09
a-Humulene yield (other studies) 2.03c 2.61c 1.93–2.37d
Clove oil yield (g/g clove, w/w%)b 17.20 ± 0.59 20.04 ± 0.78 20.97 ± 0.65 23.31 ± 0.77e
Clove oil yield (other studies) 22.43f 17.10g 17.00–20.59h 18.20–21.80i
a
a-Humulene yield (wt%) = weight of a-humulene in the clove oil/weight of the extracted clove oil. Values are written as the mean ± SD of six replications and are
calculated based on plant dry weight basis.
b
Clove oil yield (wt%) = weight of extracted clove oil/weight of the dry clove weight. Values are written as the mean ± SD of six replications.
c
Data obtained from other study using solvent extraction (SE) and SD reported by Guan et al. (2007).
d
Data obtained from other studies using SC–CO2 reported by Prado et al. (2011), Della Porta, Taddeo, D’Urso, and Reverchon (1998), Rodrigues et al. (2002) and Guan et al.
(2007).
e
Data obtained using USC–CO2 extraction (75% duty cycle) at 44.0 °C, 18.5 MPa, particle size of 0.355 mm, CO2 flow rate of 1.4 g/min and extraction time of 115 min.
f
Data obtained from other study using ultrasound-assisted extraction (UAE) reported by Tekin et al. (2015).
g
Data obtained from other study using microwave-assisted extraction (MAE) reported by Yazdani et al. (2005).
h
Data obtained from other studies using SD reported by Reverchon and Marrone (1997) and Guan et al. (2007).
i
Data obtained from other studies using SC–CO2 reported by Prado et al. (2011), Ivanovic et al. (2011), Hatami et al. (2010), Della Porta, Taddeo, D’Urso, and Reverchon
(1998), Reverchon and Marrone (1997), Rodrigues et al. (2002) and Guan et al. (2007).

had a higher yield (20.04%) than the n-hexane extraction process,
but the yield was lower than that typically reported for the SC–
CO2 process (20.97%).
Furthermore, the clove oil (23.31%) extracted with USC–CO2 in
this study is comparable with the extracted oil previously reported
by Tekin, Akalin, and Seker (2015), who obtained 22.43% total clove
oil using ultrasound bath-assisted extraction (Table 3). The amount
of clove oil extracted using USC–CO2 extraction in this study was
higher than that obtained using the microwave-assisted extraction
(MAE) method in n-hexane reported by Yazdani et al. (2005),
whereas it was lower than that previously reported by Huston
and Ji (1991) for the SC–CO2 process (24.0%). However, this clove
oil yield using USC–CO2 extraction in this study is considerably
higher compared to the yields of 20.0%, 18.2%, 19.25%, 20.7%,
20.8%, 21.8% and 19.56% obtained using SC–CO2 extraction
reported by Prado et al. (2011), Ivanovic et al. (2011), Hatami
et al. (2010), Della Porta, Taddeo, D’Urso, and Reverchon (1998),
Reverchon and Marrone (1997), Rodrigues et al. (2002) and Guan
et al. (2007), respectively (Table 3). In addition, the extraction yield
of a-humulene obtained using USC–CO2 extraction in this study
was higher than those obtained using the SC–CO2 extraction
method reported by Prado et al. (2011), Della Porta, Taddeo,
D’Urso, and Reverchon (1998), Rodrigues et al. (2002) and Guan
et al. (2007) (Table 3). The a-humulene extracted using USC–CO2
process in this study was also higher than that of steam distillation
and solvent extraction techniques reported by Guan et al. (2007)
(Table 3). However, this type of variation in the total oil content,
in addition to different extraction techniques and operating condi-
tions, may be due to geographical conditions, such as seasonal
temperature, nutrients, water availability and soil properties (Rai
et al., 2016).
4. Conclusion
A novel ultrasound-assisted supercritical carbon dioxide
(USC–CO2) extraction method and an efficient gas chromatography
analytical technique have been developed and optimized for the
rapid extraction and determination of clove oil and a-humulene
in clove buds. The best target compound extraction efficiency using
USC–CO2 was determined by varying the extraction time, extrac-
tion temperature and pressure. The results indicate that the com-
bined USC–CO2 and gas chromatography process is a rapid,
convenient and appropriate method for the quantitative analysis
of a-humulene in clove buds. However, a comparative study on a
pilot scale (applicability at industrial level) should be performed.
The dynamic method was also utilized to investigate the solubility
of clove oil in SC–CO2 at temperatures and pressures ranging from
32 to 47 °C and 9.0 to 25.0 MPa, respectively. To elucidate the
effects of temperature and pressure on the miscibility of a-
humulene in SC–CO2, fictitious solubility was evaluated under
pressure conditions ranging from 9.0 to 25.0 MPa and at tempera-
tures from 32 to 47 °C. The Chrastil, Bartle and Kumar and Johnston
models were found to fit the fictitious solubility of a-humulene
very well. By using the correlation results, the total heat of solu-
tion, heat of solvation and heat of vaporization for a-humulene
in SC–CO2 were estimated.
Acknowledgements
We gratefully acknowledge the financial support of the National
Science Council of Taiwan, Kaohsiung Medical University (KMU-
M104020) and Chuang Song Zong Pharmaceutical Co., Ltd. (S
102035 and S 102036) (Kaohsiung, Taiwan). We are indebted to
Professor Ing-Jun Chen, Hui-Fen Chiu, Show-Jen Hong, Jwu-Lai
Yeh, Bin-Nan Wu, Yi-Ching Lo and Ms. Li-Mei An (Kaohsiung Med-
ical University, Taiwan) for technical assistance. The authors would
like to thank Miss Hsiang-Hung Wei (National Hsinchu University
of Education, Taiwan), Fang-Yu Lin, Ying-Hsuan Chiu, Yi-Jing Jang,
Shao-Yuan Hao and Mei-Jing Tseng (Kaohsiung Medical University,
Taiwan) for the editorial assistance. Finally, the authors acknowl-
edge the editors and referees for their constructive comments
and encouragement.
 M.-C. Wei et al. / Food Chemistry 210 (2016) 172–181
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
04.076.
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