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Biochemical Engineering Journal 32 (2006) 7983

Dynamic microwave-assisted extraction of flavonoids from


Saussurea medusa Maxim cultured cells
Min Gao a,b , Bao-Zhen Song a , Chun-Zhao Liu a,b,
a

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering,


Chinese Academy of Sciences, Beijing 100080, PR China
b Graduate School of the Chinese Academy of Sciences, Beijing 100049, PR China

Received 27 June 2006; received in revised form 7 September 2006; accepted 8 September 2006

Abstract
An approach for automated, continuous and rapid extraction of flavonoids from Saussurea medusa Maxim dried cell cultures has been developed
in a new-designed dynamic microwave-assisted extraction system. The main factors affecting the extraction process namely power of microwave
irradiation, liquid/solid ratio, flow rate of solvent and irradiation time were optimized. The yield of flavonoids reached 4.97% in 60 min under the
optimum microwave-assisted extraction conditions: 1200 W of radiation power, 50:1 (v/w) of the liquid/solid ratio, and 50 ml s1 of solvent flow
rate. The dynamic microwave-assisted extraction showed obvious advantages in short duration and high efficiency to extract flavonoids without
causing degradation of target components from the S. medusa dried cell cultures in comparison with the dynamic solvent extraction without
microwave assistance.
2006 Elsevier B.V. All rights reserved.
Keywords: Dynamic microwave-assisted extraction; Flavonoids; Dynamic solvent extraction; Saussurea medusa Maxim; Plant cell cultures

1. Introduction
Saussurea medusa Maxim is one of the most important traditional medicinal plants in China and officially listed in the
Chinese pharmacopoeia. The most important bioactive compounds in this elite medicinal species are flavonoids including
rutin, jaceosidin, hispdulin and so on. These flavonoids have
shown significant scavenging of free oxygen radicals and antidecrepitude activity [1]. Owing to over-exploitation of the wild
plants for commercial purpose and the difficulty of cultivation,
S. medusa is now almost extinct and has been listed as the second
grade national protected wild plant in China [2]. In view of these
problems, production of bioactive flavonoids by S. medusa cell
cultures has considerable importance not only in the protection
of natural plant resources, but also in its potential commercial
interest [35].
Extraction is the first step for the recovery and purification
of bioactive phytochemicals from plant materials. A number of
Corresponding author at: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing
100080, PR China. Tel.: +86 10 82622280; fax: +86 10 82622280.
E-mail address: czliu@home.ipe.ac.cn (C.-Z. Liu).

1369-703X/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2006.09.004

traditional extraction methods have been employed in the past


few years, including solvent extraction, heat reflux extraction,
Soxhlet extraction, and so on. These traditional extraction processes are time-consuming and laborious, and involve lengthy
operation techniques, bulk amount of solvents and ultimately
thermal decomposition of the target molecules at continuous
high temperature. Microwave-assisted extraction of biologically
active compounds has many advantages over these traditional
extraction methods such as shortened extraction time and lower
consumption of solvents. Numerous biologically active compounds have been extracted with application of microwaveassisted extraction, such as extraction of glycerrhizic acid from
Glycyrrhizia glaubra root [6], extraction of notoginseng from
cultured cells of Panax notoginseng [7], and extraction of camptothecin from Nothapodytes foetida [8].
Room temperature extraction and Soxhlet extraction have
been employed to recover flavonoids from wild plant materials of S. medusa. However, microwave-assisted extraction of
flavonoids from S. medusa cultured cells has not been reported.
The objective of the current study was aim at checking the
performance of a new constructed dynamic microwave-assisted
device for the extraction of flavonoids from S. medusa cultured
cells.

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M. Gao et al. / Biochemical Engineering Journal 32 (2006) 7983

2. Materials and methods


2.1. Plant material
Calli of S. medusa were cultivated on Murashige and
Skoog medium [9] supplemented with 0.5 mg l1 6-benzylaminopurine (6-BA), 2 mg l1 naphthalene acetic acid (NAA),
30 g l1 sucrose and 5 g l1 agar for production of flavonoids.
The medium pH was adjusted to 5.8 with 1 M NaOH before
autoclaving. The cultivation was carried out in 250 ml flasks
containing 50 ml of the above medium at 25 C under 16 h
light per day. Fresh calli were collected after 25 days, and
then dried at 60 C in an oven until a constant weight was
obtained. The dried cells were grounded to 0.45 mm before
extraction.
2.2. Apparatus and operation
The dynamic microwave-assisted extraction system was constructed of three stainless steel tanks (35 mm i.d. 350 mm H)
with 1.0 l working volume, and each tank was equipped with a
microwave irradiation system (2450 MHz) with a maximum irradiation power of 2000 W (Fig. 1). The inner side of microwave
cavity was constructed by quartz and microwave penetrated the
quartz and then was absorbed by solvent and material. The
stainless steel outside of the microwave cavity was used to let
microwave reflect back in order to keep safety. A refrigerant
system was put outside each microwave cavity to take away
excess heat. The microwave-assisted extraction system was connected with 2.5 l container through a liquid pump (H = 10 m)
with the addition of temperature measurement and time
controlling.
A given amount of S. medusa dried cell cultures and 2.0 l of
80% ethanol were put into the container, and then were mixed
homogenously by a magnetic stirrer. The mixed extraction sam-

ple was recycled between the container and three extraction


tanks by the liquid bump. The suspension was irradiated with
60 s of power ON to reach the desired temperature of about
80 C, and then was irradiated periodically with microwaves in
a pre-setting procedure (15 s of power ON for heating followed
by 15 s of power OFF for cooling without allowing the suspension to super-boil).
For dynamic solvent extraction, the same amount of S.
medusa dried cell cultures and 2.0 l of 80% ethanol were put
into the container, and then were mixed homogenously by a
magnetic stirrer. The mixed extraction sample in container with
a desired temperature at 80 C heated by water bath was recycled
between the container and three extraction tanks at flow rate of
50 ml s1 without microwave assistance.
2.3. Analysis
The content of total flavonoids was determined by a spectrophotometric method [10,11]. Briefly, 1 ml extraction solution was pipetted into 5 ml test tube and diluted to 2 ml to
which 0.15 ml 5% (w/w) NaNO2 , 0.15 ml 10% (w/w) AlCl3
and 2 ml 4% (w/w) NaOH were added in the order stated. The
absorbency value of the final solution was measured at 510 nm.
Flavonoids content was determined using rutin as a standard.
The yield of total flavonoids was defined as following: yield of
flavonoids (w/w) = mass of total flavonoids extracted/mass of
material (dried cell cultures of S. medusa) 100%.
Qualitative and quantitative analysis of flavonoids in cell
cultures of S. medusa was carried out by the HPLC method
described by Liu et al. [12] with minor modification. Agilent
1100 HPLC system is composed of a quaternary pump with a
degasser, a variable wavelength detector, an auto-sampler and
1100 ChemStation software. Sample analyses were performed
on an Alltech C18 column (250 mm 4.6 mm i.d., 5 m) with
a gradient elution of 0.1% phosphoric acid (A) and methanol
(B) as follows: AB (90:10) to AB (10:90) in 50 min. The
flow-rate was 0.8 ml min1 and the effluent was monitored
at 365 nm by UV detector. The reference standard of rutin
was supplied by National Institute for the Control of Pharmaceutical and Biological Product with the purity no less than
98%.
All the experiments were repeated twice, and all values
were the means of replicates S.D. One-way analysis of
variance (ANOVA) was conducted to determine the statistically significant. Trends were considered significant when
result of compared parameters differed at P < 0.05 significance
level.
3. Results and discussion

Fig. 1. Scheme of the dynamic microwave-assisted extraction system.

The current research focused on the establishment of an efficient extraction process for flavonoids recovery from S. medusa
dried cell cultures in a new constructed microwave-assisted
extraction system, and the effects of microwave power, flow
rate of extraction solvent and amount of plant material on yield
of flavonoids were investigated as follows.

M. Gao et al. / Biochemical Engineering Journal 32 (2006) 7983

Fig. 2. Effect of microwave power on yield of flavonoids from S. medusa dried


cell cultures in the dynamic microwave-assisted extraction system. Values are
means of triplicate standard deviation.

3.1. Effect of microwave power


A 40 g dried cell cultures of S. medusa and 2.0 l ethanol
(80%, v/v) were used to investigate the influence of microwave
power (400, 800 and 1200 W) on flavonoids extraction in the
dynamic microwave-assisted extraction system with a solvent
flow rate of 50 ml s1 . As shown in Fig. 2, a maximum yield of
flavonoids was 4.90%, 4.94% and 4.97% at microwave power of
400, 800 and 1200 W, respectively. This result suggested that the
microwave powers investigated here had no significant effect on
the yield of flavonoids. However, it needed 105, 90 and 60 min
to reach the maximum yield of flavonoids at microwave power
of 400, 800 and 1200 W. Similar result was also reported previously in microwave-assisted extraction of notoginseng saponins
from cultured cells of Panax notoginseng [7]. The accelerated
extraction of flavonoids from the cultured cells by increasing
microwave power is related to the direct effects of microwave
energy on bio-molecules by ionic conduction and dipole rotation which result in power dissipated inside the solvent and plant
material and then generate molecular movement and heating
[13,14]. More electromagnetic energy was transferred to extraction system quickly and shortened the extraction time when the
microwave power increased from 400 to 1200 W. An even larger
microwave power was also attempted, but the extraction temperature was found difficult to control.
3.2. Effect of liquid/solid ratio
A given amount of S. medusa dried cell cultures and
2.0 l of 80% ethanol were put into the container, and were
mixed homogenously by a magnetic stirrer before the dynamic
microwave-assisted extraction. Fig. 3 shows that the yield of
flavonoids from S. medusa dried cell cultures increased with
increasing liquid/solid (volume of extraction solvent/amount
of dried cells) ratios from 25:1 (ml:g) to 100:1 (ml:g). The
amount of dried cell cultures interacted with the electromagnetic

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Fig. 3. Effect of liquid/solid ratio on yield of flavonoids from S. medusa dried


cell cultures in the dynamic microwave-assisted extraction system. Values are
means of triplicate standard deviation.

field, and directly influenced the microwave energy transfer process. Microwave energy was absorbed and dispersed by a larger
amount of the dried cell cultures, which disadvantaged to the
extraction process [15,16]. If the extraction was carried out under
high liquid/solid ratio, the concentration of flavonoids in extraction solution was low. It meant that more energy and time were
needed to condense the extraction solution in later separation
and purification process. Therefore, the liquid/solid ratio of 50:1
(ml:g) was suitable to reach the high yield of flavonoids from the
dried cell cultures. The amount of plant materials and the volume
of extraction solvent used in the microwave-assisted extraction
reported before were usually limited to a laboratory scale of
milligram and milliliter [17,18]. In the current experiment, the
amount of plant materials and the volume of extraction solvent
treated in one extraction cycle reached 40 g and 2 l, respectively.
The dynamic microwave assisted extraction presented here may
be useful for large-scale flavonoids recovery from S. medusa
dried cell cultures.
3.3. Effect of solvent ow rate
The extraction process was performed under the dynamic
state in the new constructed microwave-assisted extraction system where the extraction solvent and plant materials were mixed
homogenously. It was very important to test the influence of solvent flow rate on yield of flavonoids because the solvent flow rate
affected irradiation time of microwave on plant materials and
mass/heat transfer in the extraction system. As shown in Fig. 4,
the maximum yield of flavonoids reached 4.97% in 60 min at a
solvent flow rate of 50 ml s1 . At a higher solvent flow rate, the
dried cells were mixed well with extraction solvent, and then
mass/heat transfer during the extraction process was improved.
At a lower solvent flow rate, the mass transfer became worse
because the dried cells were deposited easily at the bottom of
each extraction tank and pipeline. Over-heating of plant materials leading to phytochemicals degradation and over-boiling of
the extraction solvent were observed.

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M. Gao et al. / Biochemical Engineering Journal 32 (2006) 7983

Fig. 4. Effect of solvent flow rate on yield of flavonoids from S. medusa dried
cell cultures in the dynamic microwave-assisted extraction system. Values are
means of triplicate standard deviation.

3.4. Comparison of dynamic microwave-assisted extraction


and dynamic solvent extraction
The yield of flavonoids from S. medusa dried cell cultures
in dynamic solvent extraction without microwave assistance
and dynamic microwave-assisted extraction under the optimized
conditions were compared, and the results were summarized in
Fig. 5. Dynamic solvent extraction without microwave assistance is time-consuming process based on heat and mixing to
increase the mass transfer rate in the extraction system. In contrast, dynamic microwave-assisted extraction is fast extraction
process enhanced by physical field under fluidization state where
microwave energy is delivered efficiently to materials through
molecular interaction with the electromagnetic field and offers
a rapid transfer of energy to the extraction solvent and raw plant
materials [19]. Furthermore, the direct interaction of microwave

Fig. 5. Comparison of flavonoids recovery from S. medusa dried cell cultures


between dynamic microwave-assisted extraction and dynamic solvent extraction
without microwave assistance.

Fig. 6. HPLC chromatograms of the extracts from S. medusa dried cell cultures
by dynamic solvent extraction without microwave assistance (A) and dynamic
microwave-assisted extraction (B).

with solvent also resulted in the rupture of the plant cells and
release of intracellular products into the solvent quickly [20].
As shown in Fig. 6, the phytochemical profile of the extract
from S. medusa dried cell cultures with dynamic microwave-

Fig. 7. Comparison of peak area of qualitative compound in the S. medusa


dried cell cultures between dynamic solvent extraction without microwave assistance and dynamic microwave-assisted extraction. Values are means of triplicate
standard deviation.

M. Gao et al. / Biochemical Engineering Journal 32 (2006) 7983

assisted extraction was similar to that without microwave assistance. Of the 10 compounds extracted by microwave-assisted
extraction, only one compound (peak 8) was not detected in the
extract by dynamic solvent extraction without microwave assistance. The yield (indicated by peak area) of individual qualitative
compound in the extract by microwave-assisted extraction was
higher that in the dynamic solvent extraction without microwave
assistance (Fig. 7), such as rutin as a marked compound in S.
medusa dried cell cultures. The result showed that dynamic
microwave-assisted extraction has the capability to effectively
extract target components without causing degradation of target
components in the S. medusa dried cell cultures.
4. Conclusions
An optimized process for acceleration of flavonoids extraction from S. medusa dried cell cultures has been developed in a
newly designed dynamic microwave-assisted extraction system.
By comparing the dynamic microwave-assisted extraction with
dynamic solvent extraction without microwave assistance, the
dynamic microwave-assisted extraction showed obvious advantages in short extraction duration and high efficiency. It was
identified as the best extraction approach for flavonoids from
dried cell cultures of S. medusa, and also showed great potential
for large-scale industrial application to extraction of other plant
metabolites from cultured cells.
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