Separation and Purification Technology 144 (2015) 37–45

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Enhancement of microwave-assisted extraction of bioactive compounds

from cabbage outer leaves via the application of ultrasonic pretreatment
Patsaporn Pongmalai a, Sakamon Devahastin a,b,⇑, Naphaporn Chiewchan a, Somchart Soponronnarit c
a
Department of Food Engineering, Faculty of Engineering, King Mongkut’s University of Technology Thonburi, 126 Pracha u-tid Road, Tungkru, Bangkok 10140, Thailand
b
PTT Group Frontier Research Center, PTT Public Company Limited, 555 Vibhavadi Rangsit Road, Chatuchak, Bangkok 10900, Thailand
c
Division of Energy Technology, School of Energy, Environment and Materials, King Mongkut’s University of Technology Thonburi, 126 Pracha u-tid Road, Tungkru, Bangkok
10140, Thailand

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

Article history: Although microwave-assisted extraction (MAE) has proved to be a rapid alternative to extract bioactive
Received 3 October 2014 compounds from plant materials, it might be possible to enhance MAE further through structural
Received in revised form 5 February 2015 modification of the plant matrix. In this study, the effect of plant structure modification via ultrasonic
Accepted 12 February 2015
pretreatment prior to MAE was investigated. The evolutions of selected bioactive compounds, namely,
Available online 21 February 2015
glucosinolates, sulforaphane, vitamin C and phenolics, and antioxidant activity of the extract from cab-
bage outer leaves during sonication, which is indeed equivalent to ultrasonic-assisted extraction (UAE),
Keywords:
were first monitored to identify a suitable time for the pretreatment prior to subsequent MAE. For
Antioxidant activity
Energy consumption
comparison MAE and Soxhlet extraction were conducted and their results compared with those belong-
Soxhlet extraction ing to UAE and UAE + MAE. Microstructural changes of cabbages, as observed via confocal laser scanning
Structural modification microscopy (CLSM) and quantified via the use of the fractal dimension, undergone different extraction
Ultrasonic-assisted extraction methods were observed and used to explain the extraction results. Energy consumption of different
extraction methods was also evaluated. UAE + MAE led to higher contents of extractable bioactive com-
pounds due to the effects of acoustic cavitation and subsequent internal heating within the plant cells by
microwave irradiation, which resulted in more structural damage and hence better release of the
compounds. The contents of the extractable bioactive compounds from UAE, MAE and UAE + MAE were
significantly lower than those from Soxhlet extraction in almost all cases; Soxhlet extraction time was
much longer, however. MAE exhibited the highest energy efficiency compared with UAE, UAE + MAE
and Soxhlet extraction.
Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction to plant materials prior to extraction to help enhance the extracta-

Microwave-assisted extraction (MAE) has recently received
much attention as an alternative means to extract bioactive
compounds from plant materials [3]. Although MAE has noted to
possess a number of advantages over a more traditional solvent
extraction, including rapid extraction and less consumption of an
extraction solvent, the extraction yield achievable through MAE
is still generally low [13]. Any means that can help enhance MAE
and hence to obtain a higher yield is desirable.
It is possible to enhance the extractability of any extraction
methods, including MAE, by modifying the structure of a material
to be extracted. Several pretreatment methods have been applied
⇑ Corresponding author at: Department of Food Engineering, Faculty of Engi-
neering, King Mongkut’s University of Technology Thonburi, 126 Pracha u-tid Road,
Tungkru, Bangkok 10140, Thailand. Tel.: +66 2 470 9244; fax: +66 2 470 9240.
E-mail address: sakamon.dev@kmutt.ac.th (S. Devahastin).
bility of bioactive compounds through plant structure modifica-
tion. Tanongkankit et al. [24], for example, investigated the effect
of blanching pretreatment on the extraction yield of glucosinolates
from cabbage outer leaves. Use of steam blanching helped enhance
glucosinolates extraction since blanching led to softening of the
cabbage structure, leading to easier extraction. Hiranvarachat
et al. [7] investigated the effects of selected pretreatment methods
on the bioaccessibility, which is closely related to the extractabi-
lity, of b-carotene in carrots. Blanching led to carrot cell wall dis-
ruption, leading to more extensive release of b-carotene from the
carrot matrix, resulting in higher bioaccessibility.
Despite its advantage in terms of structural modification capa-
bility, thermal pretreatment may lead to thermal degradation of
bioactive compounds. Non-thermal pretreatments are therefore
of interest. Among many alternatives, ultrasonic pretreatment
has widely been applied to help modify the structure of many

http://dx.doi.org/10.1016/j.seppur.2015.02.010
1383-5866/Ó 2015 Elsevier B.V. All rights reserved.
38 P. Pongmalai et al. / Separation and Purification Technology 144 (2015) 37–45
materials without posing a significant problem to heat-sensitive
bioactive compounds [15]. Ultrasonic pretreatment relies on
acoustic cavitation, which causes disruption of cell walls of plant
materials, leading to a more extensive release of internal cell com-
pounds [14]. Bagherian et al. [1], among others, investigated the
effect of ultrasonic pretreatment prior to MAE on the yield of pect-
in from grapefruit. Ultrasonic pretreatment led to higher extraction
yield of pectin when comparing with the values obtained from
both conventional solvent extraction and MAE without pretreat-
ment. Pananun et al. [19] and Izadifar [9] also observed a similar
effect of ultrasonic pretreatment on the extraction yields of isofla-
vones from soybean and phenolics from wheat dried distiller’s
grain (DDG), respectively. More recently, Pongmalai et al. [20]
studied the effect of ultrasonic pretreatment prior to MAE on the
extraction yield of glucosinolates from cabbage outer leaves and
found that ultrasonic pretreatment at a frequency of 37 kHz and
a power of 320 W prior to MAE led to more extensive breakdown
of cabbage structure than at other tested conditions. This in turn
resulted in a higher extraction yield of glucosinolates. Neverthe-
less, the evolution of glucosinolates (and other important com-
pounds in cabbages) during sonication was not reported
although monitoring the changes of compositional profiles is of
importance if an effective ultrasonic pretreatment protocol is to
be designed.
Despite some prior studies on the use of ultrasonic pretreat-
ment to enhance the extractability of bioactive compounds during
subsequent extraction, including MAE, limited information is avail-
able on how a suitable condition, especially in terms of the
treatment time, for ultrasonic pretreatment could be determined.
The first aim of this study was therefore to determine a suitable
ultrasonic pretreatment time prior to MAE; cabbage outer leaves
were used as the test material. The evolutions of selected bioactive
compounds, namely, glucosinolates, sulforaphane, vitamin C and
phenolics, as well as antioxidant activity of the extract from cab-
bages were monitored along with the change in the temperature
during ultrasonic pretreatment, which is indeed equivalent to
ultrasonic-assisted extraction (UAE). The yields of all the interested
bioactive compounds achievable upon subsequent MAE (or in fact
UAE + MAE) were then determined. For comparison MAE and Soxh-
let extraction were conducted and their results compared with
those belonging to UAE and UAE + MAE. Microstructrural changes
of cabbages, as observed via confocal laser scanning microscopy
(CLSM) and quantified via the use of the fractal dimension, under-
gone different extraction methods were observed and used to
explain the extraction results. Energy consumption of UAE, MAE,
UAE + MAE and Soxhlet extraction was also evaluated and
compared.
2. Materials and methods
2.1. Materials and chemicals
Outer leaves of cabbage (Brassica oleracea L. var. capitata) were
obtained from Pakklong Talad market in Bangkok; the leaves were
kept at 4 °C until the time of an experiment. Before starting of each
experiment, the leaves were washed with tap water and drained
on a screen to get rid of excess water. The leaves were then
chopped with an electric chopper (Moulinex, DPA141, Ecully,
France) for 2 min to obtain an average size of cabbages of 1.7–
2.5 mm.
Ascorbic acid standard, gallic acid as well as 2,2-diphenyl-1-
picryl-hydrazyl (DPPH) were obtained from Sigma–Aldrich
(Steinheim, Germany), Folin–Ciocalteu reagent was purchased
from Carlo Erba (Milan, Italy), while sinigrin and sulforaphane
standards were obtained from Sigma–Aldrich (St. Louis, MO).
2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammo-
nuim salt (ABTS) was purchased from Sigma–Aldrich (Oakville,
Canada) and 6-hydoxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox) was purchased from Sigma–Aldrich (Buchs,
Switzerland).
2.2. Methods
2.2.1. Ultrasonic pretreatment or UAE
Experiments were first performed to determine a suitable time
for ultrasonic pretreatment by monitoring the evolutions of select-
ed bioactive compounds of the extract from cabbages. The results
are indeed representative of the UAE of the compounds.
Five g of chopped cabbages was dispersed in 50 mL of 99.9% (v/
v) ethanol, which was used as an extraction solvent, in a 250-mL
beaker, which was placed in an ultrasonic bath (Elma, Elmasonic
P, Singen, Germany) containing 1 L of distilled water and sonicated
at a frequency of 37 kHz and a set power of 320 W (or absorbed
ultrasonic power of 0.03 W/g of the mixture of chopped cabbages
and ethanol). This condition was selected based on the results of
Pongmalai et al. [20] who reported that sonication at a frequency
of 37 kHz and a power of 320 W led to a higher extractable glucosi-
nolates content than at other conditions. The sonication time was,
on the other hand, varied from 0 to 40 min.
After sonication an ethanolic extract was filtered through a fil-
ter paper. The filtrate was concentrated using a rotary evaporator
(Buchi, R-215, Flawil, Switzerland) at 50 °C for 7 min before being
diluted with another solvent; the type and amount of the added
solvent depended on the required subsequent chemical analysis.
Five mL of 99.9% (v/v) ethanol was added if glucosinolates content,
total phenolics content (TPC) and antioxidant activity, both by the
DPPH and ABTS assays, were to be determined. For sulforaphane
analysis, 2 mL of acetonitrile was added, while for vitamin C analy-
sis 2 mL of 3% (w/v) metaphosphoric acid was added. A diluted
sample was kept at 18 °C in a vial until further analysis.
2.2.2. MAE
A domestic microwave oven (Samsung, GE-872D, Port Klang,
Malaysia), which is capable of operating at a maximum input pow-
er of 850 W at a frequency of 2450 MHz, was modified for MAE as
described by Hiranvarachat et al. [8]. The whole content of a sam-
ple (chopped leaves and 99.9% (v/v) ethanol) was subject to micro-
wave irradiation at 100 W (or absorbed microwave power of
0.63 W/g) for 2 min; the reasons for the selected conditions will
be given in Section 3.2. The extract was filtered through a filter
paper and the filtrate was concentrated using the rotary evaporator
at 50 °C for 7 min to produce a crude extract. The content was
diluted with different extraction solvent, which was again added
depending on the type of the required chemical analysis. A diluted
sample was kept at 18 °C in a vial until further analysis.
In the case of UAE + MAE, a sample was ultrasonically pretreat-
ed as described in Section 2.2.1. The sonication time for the pre-
treatment was 30 min; this time selection decision will be
explained in Section 3.1. After sonication the whole content of
the pretreated sample was subject to microwave irradiation at
100 W (or absorbed microwave power of 0.63 W/g) for 2 min as
mentioned earlier.
2.2.3. Soxhlet extraction
A Soxtec System HT (Soxtec Extraction Unit 1043 and Service
Unit 1046, Tecator, Höganäs, Sweden) was used for Soxhlet extrac-
tion. Five gram of chopped cabbages was placed in a thimble,
which was placed in an extraction chamber and dipped into a
cup containing 50 mL of 99.9% (v/v) ethanol. The cup was heated
to 50 ± 2 °C for 3 h. An extract was filtered through a filter paper
and the filtrate was concentrated using the rotary evaporator at
 P. Pongmalai et al. / Separation and Purification Technology 144 (2015) 37–45
50 °C for 7 min to produce a crude extract. The content was again
diluted with different extraction solvent depending on the type of
the required chemical analysis. A diluted sample was kept at
18 °C in a vial until further analysis.
2.2.4. Determination of total glucosinolates content
The determination of the total glucosinolates content was per-
formed following the method of Tanongkankit et al. [24] with some
modification as follows:
2.2.4.1. Preparation of Sephadex pyridine-acetate column. Glucosi-
nolates were separated using an ion-exchange column. The column
was prepared by adding 25 mg of dry DEAE-Sephadex A-25 (Sig-
ma–Aldrich, Steinheim, Germany) into a syringe (0.8  4 cm),
which was subsequently filled with deionized water. The syringe
was left overnight at room temperature and kept at 4 °C until use.
Before loading an extract into the column, the pH of the eluate
was adjusted to neutral by passing 0.5 N of NaOH (5 mL) and water
(10 mL) through the column. The column was converted into an
acetate form by adding 5 mL of 0.5 M pyridine acetate solution
and 10 mL of water. Three mL of an extract was then added into
the prepared column. The column was washed twice with 2 mL
of water, 2 mL of 30% (v/v) formic acid and 2 mL of water in order
to discard the eluate. Finally, the column was eluted twice with
4.75 mL of 0.3 M potassium sulfate; the content was adjusted by
adding 0.3 M potassium sulfate to 10 mL. The extract eluate from
the column was kept at 18 °C until further analysis.
2.2.4.2. Quantification of glucosinolates. One mL of an extract eluate
was transferred to a tube and 7 mL of 80% (v/v) sulfuric acid and
1 mL of 1% (w/v) thymole solution were added. The solution was
mixed by a vortex mixer (Vortex-Genie 2, G560E, Bohemia, NY)
for 30 s and placed in a water bath (Heto, AT 110, Allerod, Den-
mark) at 100 °C for 60 min. The tube was cooled with tap water
and mixed again by the vortex mixer for 30 s. Spectrophotometer
(Thermo Fisher Scientific, 4001/4 Genesys 20, Fair Lawn, NJ) was
used to measure the absorbance of glucosinolates at 505 nm;
0.3 M of potassium sulfate was used as a blank. Glucosinolates
were quantified using sinigrin as a standard; the total glucosino-
lates content was calculated from a standard curve of sinigrin,
which was prepared at the concentrations of 0–0.5 lmol/mL.
2.2.5. Determination of sulforaphane content
Two mL of an extract was introduced to Oasis HLB, 3 cc column
(Waters, Milford, MA). The eluate was purged with N2 for 10 min
and dissolved with 0.5 mL of 1% (v/v) acetic acid. The whole con-
tent was then filtered through a 0.2-lm nylon filter. Ten lL of
the filtrate was injected into Xselect CSH C18 HPLC column
(4.6 mm  250 mm) (Waters, Milford, MA) with 15% acetonitrile
and 85% of 1% (v/v) acetic acid as a mobile phase; the flow rate
was set at 1.2 mL/min. A UV detector at a wavelength of 254 nm
was used for detecting sulforaphane. The sulforaphane content
was calculated from a standard curve of sulforaphane, which was
prepared at the concentrations of 0–50 lg/mL.
2.2.6. Determination of vitamin C content
The vitamin C analysis method was modified from that of Nil-
nakara et al. [16]. Two mL of an extract was filtered through a
0.2-lm nylon filter. Ten lL of the filtrate was injected into Atlantis
C18 HPLC column (3.9 mm  150 mm) (Waters, Milford, MA). The
mobile phase was 0.2 M KH2PO4 (pH 2.4) at a flow rate of 0.5 mL/
min. A UV detector at a wavelength of 254 nm was used for the
quantification of vitamin C. Vitamin C was quantified using ascor-
bic acid as a standard; the vitamin C content was calculated from a
standard curve of ascorbic acid, which was prepared at the concen-
trations of 0–400 lg/mL.
39
2.2.7. Determination of TPC
Total phenolics content (TPC) of an extract was evaluated fol-
lowing the method described by Nilnakara et al. [16]. Fifty lL of
an extract was diluted with 3 mL of distilled water, 0.25 mL of
Folin–Ciocalteu reagent and 0.75 mL of 20 g/mL sodium carbonate
solution. After 2 h of reaction at ambient temperature, the absor-
bance at 765 nm was measured using the spectrophotometer.
The absorbance was used to calculate the TPC using gallic acid as
a standard; the TPC was calculated from a standard curve of gallic
acid, which was prepared at the concentrations of 0–1 mg/mL.
2.2.8. Determination of antioxidant activity
2.2.8.1. DPPH assay. The DPPH assay was conducted according to
the method of Tanongkankit et al. [22]. The DPPH solution
(1.5 mL of 0.2 mM DPPH radicals in methanol) was added to
0.5 mL of an extract. The reaction mixture was vortex-mixed for
30 s and left to stand in dark at room temperature for 30 min.
The absorbance was then measured at 517 nm using the spec-
trophotometer. Pure ethanol was used to calibrate the spectropho-
tometer. The antioxidant activity was quantified using Trolox as a
standard.
2.2.8.2. ABTS assay. The ABTS assay was conducted following the
method of Hiranvarachat et al. [8]. ABTS+ radicals were prepared
by mixing ABTS stock solution (7 mM) with 2.45 mM potassium
persulfate (at a ratio of 2:1). The mixture was kept in dark at room
temperature for 16 h. The solution was diluted with 99.9% (v/v)
ethanol until its absorbance reached 0.70 ± 0.02 at 734 nm. One
mL of ABTS+ solution was mixed with 100 lL of an extract. The
absorbance was then measured at 734 nm using the spectropho-
tometer exactly 1 min after the mixing. The antioxidant activity
was again quantified using Trolox as a standard.
2.2.9. Confocal laser scanning microscopy (CLSM)
A sample was prepared for CLSM by being chopped into a 1.5-
mL Eppendorf tube containing 500 lL of 4% (w/v) paraformaldehy-
de (Electron Microscopy Sciences, Hatfield, PA). The sample was
then incubated at room temperature for 30 min. The whole content
was centrifuged at 1500 rpm for 1 min via the use of a microliter
centrifuge (Hettich, MIKRO 120, Tuttlingen, Germany). The super-
natant of 4% (w/v) paraformaldehyde was discarded; the sample
residue was mixed with 500 lL of 25 mM phosphate buffered sal-
ine (pH 7.4) (Sigma, St. Louis, MO) and centrifuged again at
1500 rpm for 1 min. The whole process was repeated 3 times.
The sample residue was placed into a microscope slide and 5 lL
of DAPI (40 ,6-diamidino-2-phenylindole; Invitrogen Molecular
Probes, Eugene, OR) was dropped into the sample. A glass coverslip
was placed onto the microscope slide. The slide was left to stand in
dark at room temperature for 24 h prior to CLSM.
Microstructural image of each cabbage sample was obtained via
a confocal laser scanning microscope (Olympus, FV1000, Tokyo,
Japan) at 400 magnification. Argon laser exciting wavelength at
405 nm was applied, while the emission wavelength was set at
450 nm.
2.2.10. Determination of fractal dimension
Fractal dimension (FD) of each microstructural image of cab-
bages was calculated using a box counting method via the use of
ImageJ software (version 1.47v, National Institutes of Health,
Bethesda, MD). An image was equally divided into four sub-images
and FD of each sub-image was determined; the final FD value of
each image is then reported in terms of the average value and
standard deviation. This was performed to assign each image a
numerical value, so that a statistical analysis could be performed
to determine whether FDs of different images were statistically dif-
ferent [17].
40 P. Pongmalai et al. / Separation and Purification Technology 144 (2015) 37–45

2.2.11. Determination of energy consumption

The absorbed microwave and ultrasonic powers were evaluated
according to the method of Khraisheh et al. [12] with some
modification. Absorbed power of a mixture (cabbages and ethanol
solution) can be calculated by:
DT
Q abs ¼ mC p ð1Þ
Dt
where Qabs, m, Cp, DT and Dt are the absorbed power (W/g), mass of
the mixture (g), specific heat of the mixture (kJ/kg °C), temperature
increase of the mixture (°C) and period of time during the increase
of the temperature (s), respectively.
On the other hand, the input power (Qin, W/g) of each extraction
method was simply calculated from the electric power rating (W)
of each equipment used for each extraction process, i.e., ultrasonic
bath, microwave oven and Soxtec extraction unit, and the mass of
the mixture (g). Energy consumption (kW h/g) of each extraction
method was then calculated by:
Q in  t
Q con ¼ ð2Þ
1000
where Qcon, Qin and t are the energy consumption (kW h/g), input
power (W/g) and extraction time (h), respectively.

2.2.12. Statistical analysis

The experimental data were subject to the analysis of variance
(ANOVA) and are presented as mean values with standard devia-
tions. Differences between the mean values were established using
Duncan’s new multiple range tests; the differences were
considered at a confidence level of 95%. All statistical analyses
were performed using SPSSÒ software (version 17, SPSS Inc.,
Chicago, IL). All experiments were performed at least in duplicate. Fig. 2. Evolutions of bioactive compounds and antioxidant activity of the extract
from cabbages during UAE. (a) ( ) Total glucosinolates content and ( )
Sulforaphane content; (b) ( ) Vitamin C content and ( ) TPC; (c) Normalized
3. Results and discussion antioxidant activity by ( ) DPPH assay and ( ) ABTS assay.

3.1. UAE
Preliminary experiments were conducted to determine a suit-
able sonication time. This was done by monitoring the evolution
of the temperature of a mixture of chopped cabbages and ethanol
as well as the changes of selected bioactive compounds and antiox-
idant activity of an extract during sonication.
Fig. 1 illustrates the temperature evolution of the mixture of
cabbages and ethanol during sonication. The initial temperature
of the mixture was around 25 °C. The temperature of the mixture
increased with the sonication time until reaching approximately
57 °C when the sonication was conducted for 40 min. The increase
in the temperature was due to the energy released from collapsing
bubbles during sonication [10].
The evolution of glucosinolates of the extract during sonication
is shown in Fig. 2a. The glucosinolates content of the extract from
the fresh cabbages was 53.43 lmol/100 g dry mass. Upon sonica-
tion the total glucosinolates content continuously increased
Fig. 1. Temperature evolution of the mixture of cabbages and ethanol during UAE.
toward the end of the treatment. This is probably due to the effect
of cavitation bubbles that caused damage to the cabbage structure
[4], resulting in more glucosinolates release during sonication. The
microstructural results will later be discussed in Section 3.5.
Since glucosinolates can be converted into sulforaphane, which
possesses anticarcinogenic activity, through hydrolysis reaction by
myrosinase when the cabbage structure is damaged, the evolution
of sulforaphane is of interest. Note that although the activity of
myrosinase might be reduced in ethanol, it was reported that the
reduced activity should still be sufficient to allow the hydrolysis
reaction to take place [2].
The sulforaphane content evolution is also presented in Fig. 2a.
The sulforaphane content of the extract from the fresh cabbages
was around 0.21 mg/100 g dry mass. Upon sonication the sul-
foraphane content increased until reaching its maximum value
(1.71 mg/100 g dry mass) at 30 min, when the sample temperature
was around 52 °C (Fig. 1). The sulforaphane content subsequently
decreased toward the end of the treatment. This was expected
since sulforaphane is a heat-sensitive compound and generally
degrades if the cabbage temperature is higher than around 50 °C
[26]. This result is similar to that of Tanongkankit et al. [23] who
reported that the formation of sulforaphane occurred within the
temperature range of 25–53.5 °C, while thermal degradation of sul-
foraphane occurred when the cabbage temperature exceeded this
range. In addition, the decrease of sulforaphane content during
prolonged sonication might be due to the oxidation of sul-
foraphane [6].
Fig. 2b shows the evolution of vitamin C content of the extract
during sonication. The vitamin C content of the extract from the
fresh cabbages was 76.33 mg/100 g dry mass. Vitamin C content
 P. Pongmalai et al. / Separation and Purification Technology 144 (2015) 37–45
first increased until reaching its maximum value (278.87 mg/
100 g dry mass) at 15 min; the content then remained almost
unchanged until the end of the treatment. This is probably because
the vitamin C that was released into ethanol might become
saturated; at the same time the mixture temperature, which was
always lower than 57 °C, did not lead to vitamin C degradation.
Note that if the sonication time was extended to 60 min, leading
to the mixture temperature of around 63 °C, the content of vitamin
C would start to decrease, indicating the degradation of vitamin C
(data not shown).
The evolution of the total phenolics content (TPC) during
sonication is shown in Fig. 2b. The TPC of the extract from the fresh
cabbages was around 515.15 mg GAE/100 g dry mass. An increase
in the TPC during sonication was observed; phenolics seemed to
be quite insensitive to heat and oxygen, which had been exposed
to the sample due to sonication. The trend is similar to that
observed by Golmohamadi et al. [5] who investigated the effects
of ultrasonic time and acoustic cavitation on the TPC of red rasp-
berry puree.
Since it is well known that antioxidant activity of an extract is a
function of the concentration of bioactive compounds contained in
such an extract, it is necessary to apply an appropriate normaliza-
tion procedure to the antioxidant results. In this case, the normal-
ization was done by dividing the antioxidant activity values at
different time by the initial value (value belonging to the extract
from the fresh cabbages). The normalized antioxidant activity of
the extract, as assessed by both the DPPH and ABTS assays,
significantly increased upon sonication as shown in Fig. 2c. The
increased antioxidant activity was thought to be related to
the higher extractable contents of the bioactive compounds of
the extract.
It can be seen from the above-mentioned results that sonication
for 30 min resulted in the highest contents of sulforaphane and
other bioactive compounds and their antioxidant activity. This
time was then chosen for ultrasonic pretreatment prior to MAE
to help enhance compounds release from cabbages.
3.2. MAE
For MAE the set microwave power of 100 W, which is equiva-
lent to the specific absorbed power of 0.63 W/g, was selected from
the preliminary experiments as the value that led to the highest
release of extractable sulforaphane content (data not shown).
Fig. 3 illustrates the temperature evolution of the mixture of cab-
bages and ethanol during MAE. The initial temperature of the mix-
ture was around 25 °C. The temperature of the mixture increased
until reaching the value of around 61 °C when the mixture was
subject to microwave irradiation for 3 min. To allow a fair compar-
ison of the extractability of the MAE to that of the UAE, the maxi-
mum mixture temperature should nevertheless be kept at 50 °C,
which was the temperature of the mixture subjected to UAE for
Fig. 3. Temperature evolution of the mixture of cabbages and ethanol during MAE.
41
30 min (see Fig. 1). MAE was therefore conducted only up to
2 min in subsequent experiments.
The evolutions of bioactive compounds contents and antioxi-
dant activity of the extract during MAE are shown in Fig. 4. The
content of glucosinolates extractable from the fresh cabbages
was 68.34 lmol/100 g dry mass. When the mixture was subject
to microwave irradiation for 2 min, the extractable content of glu-
cosinolates increased to 362.19 lmol/100 g dry mass, which was
higher than that in the case of UAE at 30 min. Subjecting the mix-
ture of cabbages and ethanol to microwave irradiation might cause
disruption of cabbage cellular structure via dipole rotation of the
polar molecules. In fact, two modes of action might be at play:
(i) ethanol in the mixture absorbed microwave energy during
MAE, leading to rapid heating and disruption of the cells and (ii)
water within the cabbage cells also absorbed microwave energy,
promoting internal heating, inducing cell rupture [21,25] and more
release of glucosinolates from the cabbage matrix. The microstruc-
tural results of the cabbages undergone MAE will also be illustrat-
ed and discussed in Section 3.5. Since thermal degradation of
glucosinolates was identified at temperatures higher than 100 °C
[18], only 2-min heating during MAE, which led to the mixture
temperature of only 50 °C, did not result in glucosinolates
degradation.
The evolution of the sulforaphane content during MAE is also
shown in Fig. 4a. The content of sulforaphane extractable from
the fresh cabbages was around 0.29 mg/100 g dry mass. Upon
MAE for 2 min, the sulforaphane content increased to 1.72 mg/
100 g dry mass; the value was not significantly different from that
obtained via UAE at 30 min. Although the rapid temperature rise
during the 2 min of MAE might have caused disruption of the
Fig. 4. Evolutions of bioactive compounds and antioxidant activity of the extract
from cabbages during MAE. (a) ( ) Total glucosinolates content and ( )
Sulforaphane content; (b) ( ) Vitamin C content and ( ) TPC; (c) Normalized
antioxidant activity by ( ) DPPH assay and ( ) ABTS assay.
42 P. Pongmalai et al. / Separation and Purification Technology 144 (2015) 37–45

cabbage cell structure, resulting in more release of glucosinolates,

less hydrolysis reaction of glucosinolates into sulforaphane might
have taken place since the period of conversion was rather short.
On the other hand, during the 30 min of UAE, more extensive
hydrolysis reaction of glucosinolates might have taken place. How-
ever, due to the less extensive cell structure disruption in the case
of UAE (see Section 3.5), the formed sulforaphane might be more
slowly released from the cabbage matrix to reach the same level
to that extractable via 2-min MAE. For this reason, the sul-
foraphane contents of both extracts were not significantly
different.
Fig. 4b shows the evolution of the vitamin C content of the
extract. The content of vitamin C extractable from the fresh cab-
bages was 81.79 mg/100 g dry mass. MAE led to an increase in
the vitamin C content to around 286.47 mg/100 g dry mass, which
was higher than that obtained via UAE. This might be because of a
rapid temperature increase due to MAE, which resulted in the rup-
ture of the cabbage cell structure and more release of vitamin C.
The mixture temperature was also lower than the vitamin C degra-
dation temperature of 63 °C, so most released vitamin C did not
thermally degrade.
TPC of the extract from the fresh cabbages was 522.44 mg GAE/
100 g dry mass. When the cabbages were subject to MAE for 2 min,
the extractable TPC increased to 862.44 mg GAE/100 g dry mass as
shown Fig. 4b. The TPC extractable via MAE was higher than that
extractable via UAE. Since microwaves induced rapid heating and
increased internal pressure gradients within the cabbage matrix,
more rupture of the cabbage structure took place, resulting in
enhanced release of phenolic compounds even over a short period
of time.
The normalized antioxidant activity of the extract obtained via
MAE as assessed by the DPPH and ABTS assays was not significant
different from that obtained via UAE. Nevertheless, it is important
Fig. 6. Evolutions of bioactive compounds and antioxidant activity of the extract
to note again that MAE involved much shorter extraction time than from cabbages during UAE + MAE. (a) ( ) Total glucosinolates content and ( )
that required by UAE to reach the same level of bioactive com- Sulforaphane content; (b) ( ) Vitamin C content and ( ) TPC; (c) Normalized
pounds concentrations and antioxidant activity. antioxidant activity by ( ) DPPH assay and ( ) ABTS assay.

3.3. UAE + MAE

The temperature evolution of the mixture of cabbages and etha-
nol during UAE + MAE is demonstrated in Fig. 5. As mentioned in
Section 3.1, the temperature of the mixture after UAE for 30 min
was 50 °C. Although MAE started almost immediately after
UAE, the mixture temperature dropped to 38 °C; this temperature
was the initial temperature of UAE + MAE. The temperature of the
mixture increased until reaching the maximum value of around
60 °C when the mixture was subject to the same 2 min of micro-
wave irradiation.
The contents of the bioactive compounds (and their antioxidant
activity) extractable from cabbages that had undergone UAE + MAE
are shown in Fig. 6. The extractable total glucosinolates content
was significant higher compared with that obtained via UAE or
Fig. 5. Temperature evolution of the mixture of cabbages and ethanol during
UAE + MAE.
MAE alone. This is most probably because ultrasounds induced dis-
ruption of cabbage cellular structure and created a favorable con-
dition for glucosinolates release. During subsequent MAE rapid
heating and vaporization of internal moisture might also lead to
further cellular disruption, leading to enhanced compound release
from the matrix and improving the extraction yield [21].
In terms of sulforaphane, subjecting the cabbages to 30-min
ultrasounds might have induced some cellular disruption, leading
to the release of glucosinolates and myrosinase and hence hydro-
lysis conversion of glucosinolates into sulforaphane. In comparison
with UAE on MAE, the content of sulforaphane extractable via
UAE + MAE was expectedly significantly higher. Although the mix-
ture temperature of UAE + MAE increased beyond the degradation
temperature of sulforaphane, the degraded portion of the com-
pound might be smaller than the initially formed and released por-
tion since the latter period where the temperature of the mixture
undergoing UAE + MAE was higher than the degradation tem-
perature of 50 °C was rather short (only 1 min). This conse-
quently led to the higher sulforaphane content.
The content of vitamin C of the extract via UAE + MAE was
significantly higher compared with that obtained via UAE or MAE
alone due to the combined effect of UAE and MAE mentioned ear-
lier. TPC of the extract obtained via UAE + MAE was also higher
than that achievable only by UAE or MAE. The normalized antiox-
idant activity of the extract obtained via UAE + MAE was not sig-
nificantly different from the values obtained via UAE or MAE alone.
To determine if the time lapse between UAE and MAE would
pose any effect on the extractability of the MAE, experiments were
 P. Pongmalai et al. / Separation and Purification Technology 144 (2015) 37–45
performed by varying the time lapse between 0 and 6 h; the results
are shown in Fig. 7. The contents of all bioactive compounds of the
extract as well as its normalized antioxidant activity decreased
with an increase in the lapse time. This might be due to the
oxidation of bioactive compounds during the lapse period. For this
reason, MAE was conducted immediately after UAE (i.e., at time
lapse of 0 h) as mentioned earlier.
3.4. Comparison between UAE, MAE, UAE + MAE and Soxhlet
extraction
For a fair comparison between the different extraction methods,
the Soxhlet extraction temperature was set at 50 ± 2 °C. The extrac-
tion was conducted for up to 3 h as this time was noted to yield the
largest extractable content of sulforaphane, which is the most
desirable compound from cabbages.
Table 1 presents the results on bioactive compounds and anti-
oxidant activity of the extract obtained via Soxhlet extraction.
The contents of the extractable bioactive compounds (and their
normalized antioxidant activity) from UAE, MAE and UAE + MAE
were significantly lower than those from Soxhlet extraction in
almost all cases. However, if the extraction rate was taken into
consideration, Soxhlet extraction possessed a much lower extrac-
tion rate (0.0634 lg/min) than UAE and UAE + MAE (0.1057 and
0.2200 lg/min, respectively).
The content of vitamin C extractable via Soxhlet extraction was
significantly lower than those via UAE, MAE and UAE + MAE. This
Fig. 7. Effect of time lapse between UAE and MAE on bioactive compounds and
antioxidant activity of the extract from cabbages. (a) ( ) Total glucosinolates
content and ( ) Sulforaphane content; (b) ( ) Vitamin C content and ( ) TPC; (c)
Normalized antioxidant activity by ( ) DPPH assay and ( ) ABTS assay.
43
Table 1
Bioactive compositions and normalized antioxidant activity of extract from cabbage
outer leaves via Soxhlet extraction.
Composition Value
Total glucosinolates content 494.20 ± 8.17 lmol/100 g dry mass
Sulforaphane content 4.64 ± 0.20 mg/100 g dry mass
Vitamin C content 148.03 ± 3.59 mg/100 g dry mass
TPC 1,081.17 ± 42.96 mg GAE/100 g dry mass
Normalized antioxidant activity 5.35 ± 0.15
by DPPH
Normalized antioxidant activity 3.01 ± 0.08
by ABTS
might be because of the long extraction time of Soxhlet extraction,
which led to the long exposure of vitamin C to heat and oxygen and
hence extensive vitamin C degradation.
3.5. Microstructural changes of cabbages
Fig. 8 shows the microstructure of the fresh cabbages and those
of the cabbages after UAE, MAE as well as UAE + MAE. Fresh cab-
bages exhibited clear cell periphery and cell integrity (Fig. 8a).
After UAE intercellular spaces and collapse of cell structure were
observed as shown in Fig. 8b. These structural modifications were
most probably due to the effect of acoustic cavitation [14]. After
MAE (see Fig. 8c) the sample structures suffered even more dam-
age when compared with the structure of the sample undergone
UAE. This is probably because of the rapid internal heating induced
by microwaves within the cabbage cells, which led subsequently to
cell rupture and release of cellular compounds from the cabbage
matrix. In the case of cabbages subjected to UAE + MAE, the struc-
ture suffered even more extensive damage as can be seen in
Fig. 8d; combined effect of cavitation and rapid heating seemed
to be significant.
To quantitatively evaluate the microstructural changes of cab-
bages undergone different extraction methods, FD of each
microstructural image was calculated. The corresponding binary
images of the original images of Fig. 8a–d are shown, respectively,
in Fig. 8e–h; these binary images were used for the FD calculation.
The FD of the fresh sample image was noted to be 1.875 ± 0.008,
while the values of the images belonging to the samples undergone
UAE, MAE and UAE + MAE significantly increased to 1.894 ± 0.007,
1.912 ± 0.004 and 1.934 ± 0.003, respectively. The significant
increase (p < 0.05) in the FD of the images of the samples under-
gone UAE, MAE and UAE + MAE (as compared with that of the fresh
sample image) corresponded to the more severe damage suffered
by the samples [11]. The microstructural results corresponded to
the extraction results reported in Figs. 2, 4 and 6. The increase in
the FD of the sample images corresponded to the increase in the
extractable bioactive compounds contents, implying that the
increased extractability was due to the more damaged structure
of the cabbages.
3.6. Energy consumption of different extraction processes
Table 2 lists the input powers and energy consumption values
of various extraction processes. MAE exhibited the highest energy
efficiency compared with UAE, UAE + MAE and Soxhlet extraction
due to its high absorbed power at less input power and shorter
extraction time; the energy consumption was, as a result, also
the smallest. Although Soxhlet extraction provided higher extrac-
tion yields of the bioactive compounds than UAE, MAE and UAE + -
MAE, Soxhlet extraction required much longer extraction time and
higher energy consumption than other extraction methods.
44 P. Pongmalai et al. / Separation and Purification Technology 144 (2015) 37–45

(a) (e)

(b) (f)

(c) (g)

(d) (h)

Fig. 8. CLSM images of cabbage microstructures (a–d) and their corresponding binary images (e–h). (a) Fresh cabbages; (b) Cabbages undergone UAE; (c) Cabbages undergone
MAE; (d) Cabbages undergone UAE + MAE. FDs of images (e) to (h) were 1.875 ± 0.008a, 1.894 ± 0.007b, 1.912 ± 0.004c and 1.934 ± 0.003d, respectively. Different letters over
FD values indicate that the values are significantly different (p < 0.05).
 P. Pongmalai et al. / Separation and Purification Technology 144 (2015) 37–45
Table 2
Energy analysis results of UAE, MAE, UAE + MAE and Soxhlet extraction.
Input Extraction Energy
power (W/g) time (min) consumption (kW h/g)
UAE 7.19 30 3.60  103
MAE 2.25 2 7.48  105
UAE + MAE 9.44 32 5.04  103
Soxhlet extraction 33.71 180 101.12  103
4. Conclusions
The evolutions of selected bioactive compounds (i.e., glucosino-
lates, sulforaphane, vitamin C and phenolics) and antioxidant
activity as assessed by the DPPH and ABTS assays of the extract
from cabbage outer leaves undergoing sonication were first inves-
tigated to determine a suitable ultrasonic pretreatment time prior
to MAE; sonication for 30 min was chosen as a suitable time.
UAE + MAE led to higher contents of extractable bioactive
compounds than UAE or MAE alone. The structure of the cabbages
undergone UAE + MAE suffered more extensive damage than those
of the cabbages undergone only UAE or MAE. FD of the microstruc-
tural images corresponded well with the extractability results. The
contents of the bioactive compounds (and their normalized
antioxidant activity) extractable via UAE, MAE and UAE + MAE
were significantly lower than those from Soxhlet extraction in
almost all cases. However, if the extraction rate was taken into
consideration, Soxhlet extraction possessed a much lower extrac-
tion rate (0.0634 lg/min) than UAE and UAE + MAE (0.1057 and
0.2200 lg/min, respectively).
MAE exhibited the highest energy efficiency compared with
UAE, UAE + MAE and Soxhlet extraction. Although Soxhlet
extraction led to higher extraction yields of the bioactive
compounds than UAE, MAE and UAE + MAE, Soxhlet extraction
required much longer extraction time and higher energy
consumption than other extraction methods.
Acknowledgements
The authors express their sincere appreciation to the Thailand
Research Fund (TRF), the Research Chair Grant of the National
Science and Technology Development Agency (NSTDA), Thailand,
the Rayong Institute of Science and Technology Foundation, the
Higher Education Research Promotion and the National Research
University Project of Thailand as well as the Office of the Higher
Education Commission for supporting the study financially.
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