food and bioproducts processing 8 9 ( 2 0 1 1 ) 67–72

Contents lists available at ScienceDirect

Food and Bioproducts Processing

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

Comparison of different extraction methods for the

extraction of major bioactive flavonoid compounds from
spearmint (Mentha spicata L.) leaves

Mandana Bimakr a,∗ , Russly Abdul Rahman a,b , Farah Saleena Taip a , Ali Ganjloo b ,
Liza Md Salleh a,d , Jinap Selamat c , Azizah Hamid c , I.S.M. Zaidul c
a Department of Process and Food Engineering, Faculty of Engineering, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
b Department of Food Technology, Faculty of Food Science and Technology, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
c Department of Food Science, Faculty of Food Science and Technology, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
d Faculty of Chemical and Natural Resources Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia

a b s t r a c t

Different bioactive flavonoid compounds including catechin, epicatechin, rutin, myricetin, luteolin, apigenin and
naringenin were obtained from spearmint (Mentha spicata L.) leaves by using conventional soxhlet extraction (CSE)
and supercritical carbon dioxide (SC-CO2 ) extraction at different extraction schemes and parameters. The effect of
different parameters such as temperature (40, 50 and 60 ◦ C), pressure (100, 200 and 300 bar) and dynamic extrac-
tion time (30, 60 and 90 min) on the supercritical carbon dioxide (SC-CO2 ) extraction of spearmint flavonoids was
investigated using full factorial arrangement in a completely randomized design (CRD). The extracts of spearmint
leaves obtained by CSE and optimal SC-CO2 extraction conditions were further analyzed by high performance liquid
chromatography (HPLC) to identify and quantify major bioactive flavonoid compounds profile. Comparable results
were obtained by optimum SC-CO2 extraction condition (60 ◦ C, 200 bar, 60 min) and 70% ethanol soxhlet extraction.
As revealed by the results, soxhlet extraction had a higher crude extract yield (257.67 mg/g) comparing to the SC-
CO2 extraction (60.57 mg/g). Supercritical carbon dioxide extract (optimum condition) was found to have more main
flavonoid compounds (seven bioactive flavonoids) with high concentration comparing to the 70% ethanol soxhlet
extraction (five bioactive flavonoids). Therefore, SC-CO2 extraction is considered as an alternative process compared
to the CSE for obtaining the bioactive flavonoid compounds with high concentration from spearmint leaves.
© 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Spearmint (Mentha spicata L.); Bioactive flavonoid; Supercritical carbon dioxide (SC-CO2 ) extraction; Con-
ventional soxhlet extraction (CSE); High performance liquid chromatography (HPLC)

1. Introduction in Britain and other European countries (Wang et al., 2004;

Paranjpe, 2001).
Spearmint belongs to the genus Mentha in the family Labi- Different surveys have shown that herb extracts are use-
ateae (Lamiaceae) (Sweetie et al., 2007). A number of studies ful as stabilizers of edible oils. Most studies on antioxidant
have found that herbs of the Lamiaceae family are a potential compounds in the Lamiaceae family are directed to phenolic
source of natural antioxidants (Choudhury et al., 2006). Rose- diterpenes, flavonoids and phenolic acids (Kivilompolo and
mary (Rosmarinus officinalis L.), sage (Salvia officinalis L.), thyme Hyotylainen, 2007). Flavonoids which are widely distributed
(Thymus vulgaris L.) and lavender (Lavendula angustifolia Mill.) in the leaves, seeds, bark and flowers of plants are a broad
are native to the Mediterranean region; balm (Melissa offici- class of low molecular weight compounds. Flavonoids are a
nalis L.) and spearmint (Mentha spicata L.) are common plants kind of highly effective antioxidant and less toxic than syn-

∗
Corresponding author. Tel.: +60 142679858; fax: +60 3 89423552.
E-mail addresses: [email protected] (M. Bimakr), [email protected] (R.A. Rahman).
Received 23 December 2008; Received in revised form 2 March 2010; Accepted 3 March 2010
0960-3085/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.fbp.2010.03.002
68 food and bioproducts processing 8 9 ( 2 0 1 1 ) 67–72

thetic antioxidants such as BHA and BHT. Therefore, these 2. Materials and methods
secondary plant phenolics have received the greatest atten-
tion and have been studied extensively (Heim et al., 2002; 2.1. Materials
Naczk, 2004; Syed and Sharma, 2001). Effective separation of
antioxidants (high extraction yield and concentration of bioac- The leaves of spearmint (M. spicata L.) were obtained from
tive compounds) from a complex plant matrix is a difficult Cameron Highland in Pahang, Malaysia. After harvesting, the
procedure due to co-extraction of other various compounds, leaves were separated and washed under tap water. Leaves
which are undesirable in antioxidant extract. Different extrac- were dried at 40 ◦ C in a ventilated drying oven (1350FX, USA)
tion techniques such as percolation, soxhlet and supercritical for 24 h and then stored at ambient temperature (22 ◦ C) in
fluid extraction have been used to isolate antioxidants from the dark. The samples were ground in grinding mill (MX-335,
plants. Conventional soxhlet extraction (CSE) is a standard Panasonic, Malaysia) for 10 s to produce a powder with an
technique which has been used for a long time. The main approximate size of 0.525 mm.
disadvantages of conventional soxhlet extraction are long
extraction time and consumption of large amounts of used 2.2. Reagents
solvents. This extraction method is also not suitable for the
extraction of thermo-sensitive compounds due to the pos- Carbon dioxide (CO2 , SFE grade), contained in a diptube
sibility of thermal decomposition of target compounds as cylinder, was purchased from MOX Company in Malaysia.
extraction usually occurs at the boiling point of used sol- Methanol (MeOH, analytical grade), ethanol (EtOH, 99.5%,
vent for a long time. However, conventional soxhlet extraction analytical grade) and petroleum ether (analytical grade)
comparing to supercritical fluid extraction (SFE) is still widely were purchased from Scharlau chemical, European Union.
used due to its simplicity (Grigonis et al., 2005). Supercriti- Methanol (MeOH, HPLC grade) was purchased from Fisher sci-
cal fluid extraction with CO2 is an attractive method for food entific chemical, USA. TFA (trifluoroacetic acid, ≥98%) was
industry applications due to its unique properties (Lin et al., obtained from Sigma–Aldrich, Germany. All flavonoid stan-
1999). dards including (+)-catechin, (−)-epicatechin, apigenin, rutin,
The unique characteristic of this system is application of luteolin, myricetin and naringenin were purchased from
gases above their critical points to extract selective soluble Sigma–Aldrich, Germany.
components from a raw material (Cavero et al., 2006). How-
ever, CO2 is not a suitable solvent for the extraction of polar 2.3. Conventional soxhlet extraction (CSE)
compounds because it behaves as non-polar fluid for certain
conditions of temperature and pressure. Therefore, this is Three grams (3 g) of dried and ground spearmint leaves were
the main limiting step in its use for the separation of polar placed in a soxhlet apparatus. Extraction was performed with
phenolic compounds. But, in order to increase the polarity 150 ml of an appropriate solvent for 6 h. After extraction, a
of extraction solvent some food grade modifiers like ethanol rotary vacuum evaporator (Eyela, A-1000S, Japan) at 40 ◦ C was
can be used (Qingyong Lang, 2001). The objectives of the used in order to remove solvent. In this experiment four sol-
present work were to investigate the effect of different param- vents were used: pure ethanol, methanol, petroleum ether and
eters, such as pressure, temperature and dynamic extraction 70% ethanol. All extractions were performed in duplicate.
time on the supercritical fluid extraction of spearmint leaves
flavonoids and to compare the extraction yield and concentra- 2.4. Supercritical carbon dioxide (SC-CO2 ) extraction
tion of flavonoids in the extracts obtained under conventional
soxhlet and supercritical fluid extraction. To the best of our Supercritical CO2 (SC-CO2 ) extraction was performed on a
knowledge, no report has been yet appeared on the super- supercritical fluid extractor (ABRP200, Pittsburgh, PA, USA)
critical fluid extraction (SFE) of Malaysian spearmint leaves with the extractor volume 500 ml (Fig. 1). The flow rate of CO2 ,
flavonoid compounds. the extraction temperature and pressure were adjusted by the

Fig. 1 – Schematic diagram of supercritical fluid extractor.

 food and bioproducts processing 8 9 ( 2 0 1 1 ) 67–72 69

ICE software, and the extraction time was measured by the process of extraction was optimized with complete random-
stopwatch. Liquid CO2 was supplied from a gas cylinder. Before ized design (CRD) full factorial for a higher extraction yield
liquid CO2 passed into the extraction vessel, filled with the from spearmint (M. spicata L.) leaves. Independent variables for
samples, by the means of a pump (P-50, Thar designs, Inc. SC-CO2 extraction were temperature (40, 50 and 60 ◦ C), pres-
Pittsburg, PA, USA), it was pressurized to the desired pressure sure (100, 200 and 300 bar) and dynamic extraction time (30,
and heated to the specified temperature in order to reach the 60 and 90 min). Data were subjected to analyses of variance
supercritical state. Absolute ethanol (EtOH) acted as the co- (ANOVA) and multiple comparison tests were performed using
solvent and the flow rate was maintained at 3 g/min. In this LSD’s test at the 95% of confidence level. All the analyses were
study, extractions were performed at three different temper- carried out using the statistical software, Minitab v.14. A prob-
atures (40, 50 and 60 ◦ C), three different pressure levels (100, ability value of p < 0.05 was considered significant. The factors
200 and 300 bar) and three different dynamic extraction time and levels investigated are reported in Table 1.
(30, 60 and 90 min). The supercritical CO2 flow rate was main-
tained at 15 g/min and the duration of static extraction time 3. Results and discussion
was fixed to 30 min. The powdered plant material (30 g) was
mixed with 90 g glass beads (2.0 mm in diameter), placed into 3.1. Optimization of the experimental conditions
the extractor vessel. The extractions were performed in dupli-
cate. In this study, the SC-CO2 extraction of spearmint leaves Optimization of the experimental conditions is a critical step
was planned according to the full factorial with complete ran- in the development of SC-CO2 extraction method due to the
domized design (CRD) for the highest crude extract yield. effect of various parameters on the extraction yield. Generally,
extraction pressure, temperature and dynamic time are con-
2.4.1. Further processes sidered as important factors. The extraction yields obtained
After extraction, the extract was collected and the co-solvent under studied conditions were 30.12–60.57 mg/g. In general,
(ethanol) was removed with vacuum rotary evaporator (Eyela, a full evaluation of the effect of three selected factors from
A-1000 S, Japan) under reduced pressure at 40 ◦ C (water-bath three levels and duplicate experiments on the extraction yield
temperature). Then a gravimetric measurement was used needed 54 (33 × 2) experiments.
to obtain the amount of total crude extract weight. After- The mean values of the extraction yields for the corre-
ward, to destruct glycosides bounds each sample was acid sponding parameters at each level were calculated according
hydrolysed as follows: 0.05 g sample + 16 ml H2 O + 24 ml pure to the assignment of the experiment. The mean values of
methanol + 5 ml HCl (6 M) (Perva-Uzunalic et al., 2006). the three levels of each parameter show how the extraction
After 2 min manual homogenizing in order to complete the yield changes when the level of that parameter is changed.
effect of acid hydrolysis the sample was refluxed at 90 ◦ C for All the three studied parameters had significant effect on the
2 h and finally cooled at room temperature. Then, the sam- extraction yield (p < 0.05). In Table 2 the average response of
ple was filtered through filter paper 541, 70 mm. By using filter each level about extraction yield was presented. Also, R-value
syringe set, sample was filtered through 0.45-␮m nylon mem- which is mentioned in Table 2 means range between three
brane filter. At the end, 20 ␮l of extract obtained using SFE was average responses of each level about extraction yield. From
quantitatively analyzed by HPLC for the flavonoid compounds the R-value it can be concluded that temperature (R = 10.58)
detection. The extractions were performed in duplicate. had a dominant effect on the extraction yield followed by the
pressure (R = 10.38) and dynamic time (R = 8.12).
2.5. High performance liquid chromatography (HPLC)
analysis 3.1.1. Effect of temperature
Fig. 2 shows the effect of temperature on the extraction yield
The extracts obtained from optimum supercritical CO2 (SC- of spearmint (M. spicata L.) leaves in SC-CO2 extraction at three
CO2 ) extraction condition and conventional soxhlet extraction temperature levels of 40, 50 and 60 ◦ C. The density of CO2 at
(CSE) were analyzed using a HPLC which was composed of constant pressure is reduced with increasing temperature and
a Water 600 pump Controller, 9486 tuneable absorbance UV leading to reduce the solvent power of supercritical CO2 . The
detector and equipped with an Eclipes XDR-C18 reversed- effect of temperature on solute solubility is different at pres-
phase column (25 cm × 4.6 mm × 5 ␮m, Supelco, USA). The sures in the critical range. Near the system critical pressure,
volume of the injection loop was 20 ␮l. Classic Millenium 2010 the fluid density is very sensitive to temperature. This might
software was used for the manipulation of data processing. be the reason that flavonoid yield was changed significantly
The mobile phase used for analysis was solvent A: TFA (tri- when temperature was changed over the range of 40–60 ◦ C. A
fluoroacetic acid) 2.5 pH in deionized water and solvent B moderate increase in temperature can lead to a large decrease
consists of methanol 100% (HPLC grade). MeOH 70% was also in fluid density, with a consequent reduction in solute solubil-
required for washing the system. The flavonoids were detected ity (Roop et al., 1989). However, the increase in temperature
at 280 nm. The temperature was set to room temperature and will also accelerate mass transfer and improve the extrac-
flow rate was set at 1.0 ml/min. All of the main flavonoid
compounds were identified by matching their retention time
Table 1 – Experimental levels of the factors used in
against those of standard compounds. Quantity calculations
complete randomized design (CRD) full factorial.
were made according to the linear calibration curves of stan-
Factors Levels
dards.
1 2 3
2.6. Statistical analysis
Pressure (bar) 100 200 300
Temperature (◦ C) 40 50 60
The optimization of the method can be carried out step by step Dynamic time (min) 30 60 90
or by using an experimental design. In the present study the
70 food and bioproducts processing 8 9 ( 2 0 1 1 ) 67–72

Table 2 – Results obtained at the experimental condition using complete randomized design (CRD) full factorial.
Parameter Yielda (mg/g) Yielda (mg/g) Yielda (mg/g) Rb
L1c L2c L3c

Pressure 40.69 ± 4.43 51.07 ± 3.21 45.13 ± 4.72 10.38

Temperature 40.48 ± 5.32 45.36 ± 5.61 51.06 ± 5.43 10.58
Time 40.28 ± 3.45 48.21 ± 4.43 48.40 ± 6.3 8.12

a
Values are mean ± SD of duplicate runs.
b
R-value means range between three average responses of each level about extraction yield.
c
Average responses of each level about extraction yield.

Fig. 2 – Effect of temperature on the extraction yield of

crude extract at the constant pressure.
Fig. 4 – Effect of dynamic extraction time on the extraction
yield of crude extract at the constant pressure.
tion yield (Wang et al., 2008). The increase of temperature
can increase the vapour pressure of the extractable com-
results, the extraction yield increased with pressure from 100
pounds. Thus, the tendency of the compounds to be extracted
to 200 bar, which was due to increase of SC-CO2 density at
is increased to pass in the supercritical fluid phase (Reverchon
higher pressures. However, an increase in the pressure level
and De Marco, 2006). For a volatile solute, there is competi-
above 200 bar led to an unexpected reduction in the extrac-
tion between its solubility in supercritical carbon dioxide and
tion yield. This unexpected result can probably be related to
its volatility (Pourmortazavi and Hajimirsadeghi, 2007). There-
the reduced diffusion rates of the extracted compounds from
fore, it is difficult to predict the effect of temperature. In the
the plant matrix to the supercritical fluid medium (Rezaei and
present study, the extraction yield increased with temperature
Temelli, 2000).
and the highest extraction yield (60.57 mg/g) was obtained at
An increase of pressure can result in an increase in the
60 ◦ C. In this manner, the solute vapour pressure played a key
fluid density, which alters solute solubility. Gomes et al. (2007)
role leading to increase in the extraction yield.
have indicated that a higher recovery of volatile fractions and
a lower recovery of non-volatile fractions are obtained at high
3.1.2. Effect of pressure
pressure. Therefore, it is interesting to control the composi-
Fig. 3 presents the effect of pressure on the extraction yield
tion of the extract using pressure. In this study, the flavonoid
of spearmint (M. spicata L.) leaves in SC-CO2 using three pres-
yield increased with increasing pressure to a certain value.
sure levels of 100, 200 and 300 bar. According to the obtained
Over this range of pressure, increasing fluid density is presum-
ably the main mechanism leading to a higher flavonoid yield.
Above this range of pressure, a decreasing flavonoid yield with
increasing pressure was observed. The volatility and polar-
ity of extracted analytes might be responsible for the result
(Gomes et al., 2007; Wang et al., 2008).

3.1.3. Effect of dynamic extraction time

Fig. 4 shows the effect of dynamic time on the extraction
yield of spearmint (M. spicata L.) leaves in SC-CO2 by applying
three different levels of dynamic time including: 30, 60 and
90 min. At 100 bar pressure the extraction yield was increased
with dynamic time until 90 min. However, at higher pressures
(200 and 300 bar) the extraction yield increased with dynamic
extraction time until 60 min. It can be concluded that the sol-
vent power of supercritical CO2 density is reduced at 100 bar
pressure due to the lower CO2 density and maximum yield
Fig. 3 – Effect of pressure on the extraction yield of crude was obtained at 90 min. However, at higher pressures (200 and
extract at constant temperature. 300 bar) the extraction rate is higher and as a consequence the
 food and bioproducts processing 8 9 ( 2 0 1 1 ) 67–72 71

Table 3 – Identification and quantification of bioactive flavonoid compounds extracted by conventional soxhlet extraction
(CSE) and supercritical carbon dioxide (SC-CO2 ) extraction.
Extraction mode Extraction yield Flavonoid content (mg/g)
(mg/g)a

Catechin Epicatechin Rutin Myricetin Luteolin Apigenin Naringenin

Conventional soxhlet extraction (CSE)

Methanol 267.33 ± 3.12e 0.144 0.163 0.161 0.041 0.093 0.392 0.054
Ethanol (99.5%) 218.0 ± 4.24c 0.081 0.114 0.109 0.090 0.154 0.246 –
Ethanol:water (70:30) 257.66 ± 3.47d 0.117 0.149 0.140 – 0.146 0.305 –
Petroleum ether 30.47 ± 2.34a – – – – – – –

SC-CO2 extraction 60.566 ± 3.14b 0.140 0.156 0.148 0.117 0.657 0.270 0.249
(60 ◦ C, 200 bar and 60 min)

Values in the yield column followed by letters are significantly different (p-value < 0.05).
a
Values are mean ± SD of duplicate runs.

extraction yield kept increasing but after 60 min of extraction its higher polarity than pure ethanol. For example, in ethanol
time, the extraction yield dropped (Reverchon and De Marco, soxhlet extraction apigenin with 0.246 mg/g had the highest
2006). Therefore, as shown in Fig. 4, the highest extraction concentration in contrast to ethanol 70% the concentration
yield was achieved at 60 min dynamic extraction time. of dominant detected flavonoid, apigenin, was reached to
0.305 mg/g. By adding water to the pure ethanol up to 30% for
3.2. Conventional soxhlet extraction (CSE) yields preparing ethanol 70% the polarity of solvent was increased.
Therefore, polar compounds, flavonoids, could be isolated bet-
Different solvents with different polarities were used to deter- ter from herb matrix.
mine which one gives the highest recoveries of bioactive
flavonoid compounds. Four solvents were used: (1) methanol, 3.3. Comparison of SC-CO2 extraction and CSE
(2) pure ethanol, (3) ethanol (70%) and (4) petroleum ether. methods
The extraction yield obtained by using each solvent is pre-
sented in Table 3. Based on the obtained results, the highest Different methods of natural matter extraction have differ-
extraction yield (267.3 mg/g) was found with methanol extrac- ent extraction yield and efficiencies. Higher concentration
tion and then with a little difference followed by ethanol of natural bioactive compounds in the extracts is an impor-
70% extraction (257.6 mg/g). The lowest extraction yield was tant factor in the production of natural products while a
obtained by using petroleumether (30.4 mg/g), suggesting that primary task in the industries is lower economic cost which
polar compounds in the plant matrix would be easier to can be achieved by better extraction yield (Grigonis et al.,
extract with a more polar solvent while lower polarity sol- 2005). Based on the results obtained with methanol conven-
vents enable to obtain the extracts with higher concentration tional soxhlet extraction the extraction yield and recovery
of bioactive compounds. The extracts which were obtained of flavonoid compounds were better but due to its toxic-
from methanol, pure ethanol, ethanol 70% and petroleum ity and less consumption in food industry comparison was
ether were then analyzed by HPLC to identify and quantify the performed between the obtained results of optimum SC-CO2
major bioactive flavonoid compounds profile. With different extraction condition and ethanol 70% conventional soxhlet
solvent, different flavonoid compounds were extracted. The extraction (Table 3). The composition of the extracts obtained
flavonoid compounds concentration in the extract obtained from SC-CO2 and conventional soxhlet extraction was strongly
with petroleum ether was undetectable. Using pure ethanol different. According to the extraction yield results, ethanol
five flavonoid compounds including catechin, epicatechin, 70% conventional soxhlet extraction (257.6 mg/g) had a higher
rutin, luteolin and apigenin were extracted from spearmint yield comparing to the SC-CO2 extraction (60.57 mg/g). But,
(M. spicata L.) leaves. Apigenin had the highest concentration supercritical carbon dioxide extract (optimum condition) was
(0.246 mg/g) among the other flavonoids, which are obtained found to have better quality and more main flavonoid com-
with pure ethanol conventional soxhlet extraction. pounds (seven flavonoids) comparing to the ethanol 70%
The highest extraction yield (267.33 mg/g) was obtained conventional soxhlet extraction (five flavonoids). Flavonoid
with methanol solvent, which extracted seven flavonoid compounds of plants have usually been extracted by the other
compounds including catechin, epicatechin, rutin, myricetin, conventional extraction methods such as solvent extraction,
luteolin, apigenin and naringenin. However, the concen- steam distillation. Some of the main disadvantages of all these
trations of myricetin and naringenin were low (0.041 and methods include: long extraction time, losses of volatile com-
0.054 mg/g, respectively) and apigenin had the highest con- pounds and degradation of unsaturated compounds, resulting
centration (0.392 mg/g). The obtained extraction yield from unfavourable off-flavour compounds due to heat (Grigonis et
ethanol 70% conventional soxhlet extraction was near to the al., 2005). Supercritical CO2 (SC-CO2 ) extraction has different
obtained extraction yield from methanol soxhlet extraction advantages over conventional soxhlet extraction (CSE) method
but extracted flavonoids were same as compounds which are such as low operating temperature, thus no thermal degra-
extracted with pure ethanol soxhlet extraction. Five flavonoid dation of most of the labile compounds, shorter extraction
compounds catechin, epicatechin, rutin, luteolin and apigenin duration and high selectivity in the extraction of target com-
were extracted from spearmint leaves with pure ethanol and pounds. SC-CO2 extraction also, seems to be a cost-effective
ethanol 70%. But higher concentrations of more bioactive process at laboratory scale, but a precise economic evaluation
flavonoid compounds were detected with ethanol 70% due to will need additional experiments for establishing large-scale
72 food and bioproducts processing 8 9 ( 2 0 1 1 ) 67–72

units (Qingyong Lang, 2001). Therefore, it can be recom- fluid extraction of antioxidant compounds from oregano
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flavonoid compounds from spearmint (M. spicata L.) leaves. vitro assays. J Supercrit Fluids, 38: 62–69.
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However, for the complete extraction of the other flavonoid
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compounds may be higher pressure and extraction times and its antioxidant behaviour. J Pharm Biomed Anal, 41:
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