ORIGINAL ARTICLE

J. Chem. Metrl. 3:1 (2009) 13-23

Optimization of microwave assisted extraction of bioactive

flavonolignan - silybinin
Mahaveer Dhobi1, Vivekananda Mandal2 and Siva Hemalatha1*
1

Pharmacognosy Research Laboratory, Department of Pharmaceutics, Institute of Technology,

Banaras Hindu University, Varanasi, 221005, India

Division of Pharmacognosy, Department of Pharmaceutical Technology, Jadavpur University,

Kolkata 700032, India.
(Received April 17, 2009; Revised January 5, 2010; Accepted January 6, 2010)

Abstract: In this paper, a novel method of microwave assisted extraction (MAE) used for the extraction of a
potent hepatoprotective bioactive silybinin, as a main flavonolignan from Silybum marianum is presented. The
extracts were quantified for silybinin by HPLC. To prove the efficiency of the proposed MAE technique, it was
compared with traditional methods like Soxhlet, maceration and stirring extraction. If the extraction yield
obtained from MAE was to be considered as 100% performance level then, 12 h of Soxhlet extraction and 24 h
of maceration and stirring extraction could attain 79.6%, 26.3% and 35% performance (in terms of extraction
yield of silybinin) efficiency with high degree of reproducibility. The optimum extraction conditions were 600
W microwave power, 12 min extraction time (spread over two extraction cycles of 6 min each), 80% v/v ethanol
as the extraction solvent, 20 min preleaching time and 25:1 (mL/gm) as the solvent to material loading ratio. A
synergistic mechanism of heat and mass transfer was proposed to account for the accelerated extraction due to
microwave.
Keywords: Microwave assisted extraction; silybinin; soxhlet; HPLC

1. Introduction
Silymarin is a standardized extract from the milk thistle Silybum marianum (L.) Gaertn that
has been used as a medical remedy for almost 200 years [1]. In particular, it is used as a therapeutic
agent in many types of acute and chronic liver diseases; in United States, 10-15% of patients attending
liver disease clinics reported having taken milk thistle derivatives [2]. In addition, silymarin protects
experimental animals from various hepatotoxicants such as CCl4, acetaminophen and phalloidin, and
has been shown to have anticholestatic properties as well [3]. Silymarin is composed of a mixture of
several flavonolignans, with the most important being silybinin, isosilybinin, dehydroisosilybinin,
silidianin and silichristin. The extract contains also a few flavonoids, mainly taxifolin and quercetin
*

Corresponding author: E Mail: [email protected] Tel.: +91 9415256481 (O); +91 0542 2575810
(R); Fax: +91 0542 2368428
The article was published by Academy of Chemistry of Globe Publications
www.acgpubs.org/JCM Published 07/ 01/2010
Online ISSN:1307-6183

Microwave assisted extraction of silybinin

14

[1]. All these compounds account for 65% - 80% of the whole extract content, with the remaining
fraction being a chemically not well defined fraction, composed mostly of polymeric and oxidized
polyphenolic compounds [4,5]. Silybinin (C25H21O10), which forms the bulk amount of the silymarin
complex, is actually a mixture of two diastereoisomers in approximately 1:1 proportion. It is the main
flavonolignan of silymarin, and has been proposed to be its major active component [4,5,6]. Silymarin
and silybinin prevent lipid peroxide formation in liver cells, mainly due to their free radical
scavenging properties [7]. Silymarin also has antifibrinogenic properties, and is able to increase the
synthesis rate of rRNA by activating RNA polymerase I; this enhances the biosynthetic apparatus, thus
increasing the synthesis rate of both structural and functional proteins [8].
Phytochemicals extracted from plant materials are of great importance to the pharmaceutical
and the dietary supplement industries. Even though extraction is the starting step in qualitative and
quantitative analysis of medicinal plant constituents, but until date very substantial amount of work
has been done to improve the efficiency of this crucial step. An incomplete extraction process
producing a poorly prepared extract is sufficient to produce the most erroneous results even with the
best chromatographic system [9]. Ideally, an extraction procedure should be exhaustive with respect to
the constituents to be analyzed, rapid, simple, inexpensive and with high degree of automation.
Usually, the traditional techniques like soxhlet, maceration, reflux and hydrodistillation, which have
been used over decades forms the first choice for extraction of phytochemicals. However, these
techniques also suffer from severe drawbacks such as long extraction time and low efficiency
particularly when trace amount of compounds are present. Moreover, many natural products are
thermally unstable and could be degraded with increasing temperature during the extraction. The use
of large volumes of organic solvent associated with conventional methods are detrimental to
environment and their subsequent disposal also becomes an issue of concern. In this respect, a
procedure that could obtain most of the effective constituents in a shortest processing time with low
production cost and using minimum organic solvent will be an ideal technology. Modern techniques,
such as microwave assisted extraction (MAE), in recent times has proved to be a promising ideal
extraction tool for the extraction of phytochemicals from botanicals [9, 10]. Many reports on the
beneficial effects of MAE with respect to medicinal plants have been published, with significant
improvements over conventional extraction methods offering much lowered extraction time and
enhanced efficiency [9,10]. Compared with the traditional methods, MAE has many advantages, such
as shorter extraction time, lesser solvent consumption, higher extraction rate and better products with
lower cost. Direct interaction of microwaves with the free water molecules presents in the glands and
vascular systems, causes a tremendous increase in internal pressure inside the plant cell due to
evaporation of the internal moisture content which result in the subsequent rupture of the plant tissue
and the release of the active compounds into the organic solvent [10]. Therefore, MAE is an
interesting alternative to conventional extraction methods, especially in the case of botanical
extractions.
In this study, an effective time saving extraction model using microwave energy for the
improved yield of bioactive compound silybinin from Silybum marianum is presented. The proposed
extraction model has been compared with several conventional extraction methods and the effects of
various experimental conditions on the extraction yield are studied (microwave power, extraction time,
ethanol concentration, preleaching time solvent volume and extraction cycle) in a systematic fashion.
Until now, the extraction of silybinin from Silybum marianum with MAE method has not been
reported. The purpose of this study is to develop a novel, ecofriendly, rapid MAE method for the
efficient large scale extraction of silybinin as a potential biomarker, and to evaluate the efficiency of
the proposed extraction technique against conventional extraction methods for the extraction of
silybinin

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Dhobi et al., J. Chem.Metrl. 3:1 (2009) 13-23

2. Materials and Methods

2.1 Reagents
All solvents were from S.d Fine Chemicals (Mumbai, India) and those used for HPLC were of HPLC
grade. For sample and solvent filtration, 0.45 m membrane filters (Millipore, Germany) were used,
and solvents were degassed prior to use. Silybinin was purchased from Sigma (St. Louis, MO).
2.2 Apparatus
The extraction system comprised of a modified domestic microwave oven extractor equipped with
a magnetron of 2450 MHz with a nominal maximum power of 700 W, a reflux unit, 10 power levels,
time controller, exhaust system, beam reflector and a stirring device.
2.3 Conventional extraction techniques
Dried seeds of Silybum marianum were provided as gift sample from Indian drugs and
Botanicals (Delhi, India) and were used as received without any pretreatment. A voucher specimen
(BHU/Pcog/04/2008) was deposited in the herbarium of Department of Pharmaceutics, Institute of
Technology, Banaras Hindu University. Seeds were milled to homogenous 40 mesh powder (selected
by sieve), immediately before the experiment.
Three conventional extraction techniques namely, Soxhlet, maceration and stirring extraction were
used for comparison with MAE technique. Percentage extraction yield (w/w) for silybinin was
obtained by using the formula

2.4 Soxhlet Extraction

Exhaustive Soxhlet extraction was performed using a classical Soxhlet apparatus with accurately
weighed 1 g of the powdered seeds (screened through sieve 40) for 12 h. Extraction was performed
with 80% v/v ethanol as the extracting solvent. The dried residue was washed twice with 10 mL
petroleum ether and then finally dissolved in 10 mL methanol in a volumetric flask. The solution of
the samples were transferred in the HPLC vials by filtering through 0.45 m membrane filter.
2.5 Maceration and stirring extraction
Maceration was carried out in a closed conical flask for 24 h. Stirring extraction was carried
out by continuous stirring for 24 h with the help of a magnetic stirrer in a closed conical flask. In both
the cases 1 g powdered drug sample (screened through sieve 40) and 100 ml 80% v/v ethanol was used
as the extracting solvent. Heat was not applied in either of the cases. The suspension after
maceration/stirring was centrifuged and the supernatant evaporated under reduced pressure. The
residue was then washed twice with 10 mL petroleum ether, and finally dissolved in 10 mL methanol
for HPLC analysis as described earlier.
2.6 Microwave assisted extraction (MAE)
For MAE accurately weighed 1 g of the homogenous powder was mixed with 25 mL ethanol.
After allowing a preleaching time of 5 min the suspension was irradiated with microwave at different
experimental conditions for the optimization of the extraction parameters. The sample was treated

Microwave assisted extraction of silybinin

16

under microwave irradiation in an intermittent way, i.e. irradiation:cooling:irradiation. The microwave

irradiation time was 1 min and cooling time of 1 min was used to cool the sample solution between
two irradiations. After extraction, the samples were centrifuged at 4000 rpm (3520g) and the
supernatant evaporated under reduced pressure. The dried residue was washed twice with 10 mL
petroleum ether and then finally dissolved in 10 mL methanol in a volumetric flask. The solution of
the samples were transferred in the HPLC vials by filtering through 0.45 m HPLC filter.
2.7 HPLC analysis
The content and composition of the main flavonolignan, silybinin was analysed by using the
HPLC method of Kvasnicka et al. [12]. Briefly, the extracts were chromatographed on a HPLC system
(Waters 746, Millipore Corporation, Milford, MA,USA), equipped with a reversed phase column. A
mixture of 85% phosphoric acid-methanol-water (0.5:46:64, v/v) served as mobile phase. The elution
was made in an isocratic mode at a flow rate of 1ml/min, at room temperature. The eluted peaks were
monitored with an UV detector (wavelength 288 nm). Pure silybinin was used as external standard for
quantitative analysis. A calibration curve in the concentration range of 1 mg/mL and 0.065 mg/mL
was constructed for the quantification of silybinin.
2.8 Statistical analysis
The one way ANOVA test was used to calculate the significance of the differences of the yield
of silybinin [13]. The results of HPLC analysis were expressed as means of yield S.D.
3. Results and Discussion
3.1 Optimization of extraction parameters
In this study, the effects of several influential extraction parameters (microwave power,
irradiation time, type of solvent, solvent composition, preleaching time, loading ratio and extraction
cycle) were systematically studied for set up of the optimal extraction conditions to obtain the
maximum yield of silybinin. Ethanol was used as the extraction solvent.
3.2 Effect of microwave power

extraction yield (% w/w)

0.8
0.7
0.6

2 min

0.5

4 min

0.4

8 min

0.3
0.2
200

400

600

800

microwave power (W)

Figure 1. Effect of microwave power on the yield of silybinin

*Extraction conditions: Extraction conditions: 25 mL ethanol as extraction solvent and 5 min of preleaching time

Figure 1 highlights the typical yield power plots for the extraction of silybinin. In general, the
extraction efficiency was improved by raising the microwave power from 200 to 800 W. During short
irradiation time, (2 and 4 min) yield of silybinin was enhanced with microwave power increasing.
When the extraction solutions were heated long enough (8 min), the yields under different powers

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Dhobi et al., J. Chem.Metrl. 3:1 (2009) 13-23

were similar. The difference of the silybinin yield between 200 to 600 W appears more significant
with short irradiation times compared to long irradiation times. Since significant increase in extraction
yield was noticed at 600 W microwave power for all extraction time, hence it was considered
optimum. The accelerated extraction of silybinin by increasing microwave power can be correlated to
the direct effects of microwave energy on phytomolecules by ionic conduction and dipole rotation
which result in power dissipated in a volumetric fashion inside the solvent and plant material and then
generate molecular movement and heating. More electromagnetic energy was transferred to the
extraction system quickly and improved the extraction efficiency when the microwave power
increased from 200 to 800 W. Similar explanation was also given to support the effect of microwave
power on the MAE of flavonoids from Radix astragali [13].

extraction yield (% w/w)

3.3 Effect of irradiation time

0.8
0.7
0.6
0.5
0.4
0.3
2 min

4 min

6 min

8 min

10 min

extraction time

Figure 2. Effect of extraction time (irradiation time) on the yield of silybinin

*Extraction conditions: microwave power: 600 W, extraction solvent: 25 ml ethanol 5 min of preleaching time.

Figure 2 shows the duration of microwave radiation of 2, 4, 6, 8, and 10 min at 600 W

microwave power on the extraction yield of silybinin. Three phases were observed in the process of
microwave extraction. The first phase (1) is represented by the rise in extraction between 2 min and 4
min which characterizes the first quantities extracted, located at the surface of vegetable particles. This
is followed by the second phase (2) characterized by the rise in extraction yield between 4 min to 6
min representing the intern diffusion of the target analyte from the midst of the particles towards the
external medium involved by the intern warming of the natural moisture located in the plant cells. The
third phase (3) which begins after 6 min marks the end of the extraction process. MAE reached the
highest extraction yield of 0.72% w/w when irradiation time was 6 min. However, further increase in
irradiation time resulted in no improvement in the extraction performance. Similar observations were
also reported for MAE of artemisnin [14] and slavia miltiorrhiza [11]. Since no significant difference
in extraction yield was obtained between 6 min and 8 min of the extraction time, the former was
considered optimum for maximum extraction.

Microwave assisted extraction of silybinin

18

3.4 Effect of solvent composition

Figure 3. Effect of ethanol concentration on the yield of silybinin

*Extraction conditions: microwave power: 600 W, 6 min extraction time, 25 mL extraction solvent and 5 min of
preleaching time.

Figure 3 shows that the yield of silybinin was greatly influenced by the aqueous ethanol
concentration. Highest yield was obtained with 80% v/v ethanol concentration. Further increase in
water content resulted in fall in extraction yield. Presence of some amount of water can increase the
mass transfer process by increasing the relative polarity of the solvent thus improving its solubilizing
capacity and through effective swelling of the plant material, thus increasing the surface area for solute
solvent interaction. However, presence of excess amount of water can cause excess thermal stress due
to rapid heating of the solution on account of effective absorption of microwaves by water.

extraction yield (% w/w)

3.5 Effect of preleaching time

1.2
1.1
1
0.9
0.8
5 min

10 min 15 min 20 min 25 min

preleaching time

Figure 4. Effect of preleaching time on the yield of silybinin

*Extraction conditions: microwave power: 600 W, extraction time: 6 min, ethanol concentration: 80% v/v,
solvent volume: 25 mL.

Preleaching time can be defined as the contact time between sample matrix and extracting
solvent before microwave irradiation. Fig 4 shows that extraction performance kept improving until
preleaching time reached 20 min which was considered most favorable for enhancing the extraction
yield. Further increase in preleaching time did not show any promising effect on the extraction
performance. Preleaching time of 20 min allows sufficient swelling of the plant matrix. This increased
hydrated status helps is bursting of the cell wall due to internal thermal stress and enlargement of the

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Dhobi et al., J. Chem.Metrl. 3:1 (2009) 13-23

cellular pores thus facilitating leaching of the target analyte. Similar observations were also made in
the MAE of tanshinones from Salvia miltiorrhiza [11].

extraction yield (% w/w)

3.6 Effect of solvent to material ratio

1.2
1.1
1
0.9
0.8
25

30

35

40

loading ratio (ml/gm)

Figure 5. Effect of preleaching time on the yield of silybinin

*Extraction conditions: microwave power: 600 W, extraction time: 6 min, ethanol concentration: 80% v/v,
solvent volume: 25 mL.

The solvent volume must be sufficient to ensure that the entire sample is immersed, especially
when having a matrix that will swell during the extraction process. Generally in conventional
extraction techniques a higher volume of solvent will increase the extraction performance, but in MAE
a higher solvent volume may give lower yield [14,15]. To investigate the influence of solvent to
material ratio on the yield of silybinin, several loading ratios (25:1, 30:1, 35:1, 40:1, mL/g) were
examined. Fig. 5 shows that the yield of silybinin decreased with the increasing solvent volume
beyond 30:1 ml/gm. Since no significant difference in the yield of silybinin was observed between
25:1 mL/gm and 30:1 mL/gm loading ratio hence the former was selected as the optimum. This was
probably due to an inadequate stirring of the solvent when the microwaves are applied at larger solvent
volumes. Moreover, larger volume of solvent (80% v/v ethanol) will cause more absorption of
microwave energy and thus sufficient microwave energy may not be available for facilitating the cell
breakage for effective leaching out of the target analyte [16]. Similar effects were also recorded during
the MAE of artemisnin [14] and tea polyphenols and tea caffeine [17].
3.7 Effect of extraction cycle
The effect of repeated and successive extractions of the residue (extraction cycle) was
investigated in this experiment. The extraction conditions were set at the optimum parameters obtained
so far in the study. A second successive extraction of the residue yielded further 0.26% w/w silybinin
taking the final extraction yield to 1.37% w/w. The above data reflects that 81% of the extraction was
over in the first extraction cycle itself. A successive third extraction did not show any presence of
silybinin.
Hence the final optimum extraction conditions as obtained from the study is as follows: 600 W
microwave power, 12 min extraction time (spread over two extraction cycles of 6 min each), 80% v/v
ethanol as the extraction solvent, 20 min preleaching time and 25:1 (mL/gm) as the solvent to material
loading ratio.

20

Microwave assisted extraction of silybinin

3.8 Proposed extraction mechanism

Microwave treatment affects the structure of the cell due to the sudden temperature rise and
the internal pressure increase. The higher temperature attained by the cell wall, during MAE, causes
dehydration of cellulose and reduces its mechanical strength, which allows the solvent to gain an easy
entry inside the cellular channels [15,18]. During the cell wall rupture process, a rapid exudation of
the chemical substance within the cell into the surrounding solvents takes place. This mechanism of
MAE based on exposing the analytes to the solvent through cell rupture is different from that of heat
reflux extraction that depends on a series of permeation and solubilization processes to bring the
analytes out of matrix.
3.9 Stability studies
Stability at the optimum conditions derived were performed by subjecting standard silybinin
(at two concentration level; 0.75 mg/mL and 1 mg/mL in 80% v/v ethanol) acid to MAE for 6 min at
600 W microwave power. The recovery of silybinin was taken as the indicative marker for the stability
of silybinin at the derived operating extraction conditions. Data is indicated in Table 1. Results
showed that average complete recovery at the operating extraction conditions varied from 96% to
97.3% with no change in retention time of silybinin, thus abolishing any fear of thermal degradation at
the selected conditions.
3.10 Repeatability
Table 1. Stability studies of standard silybinin under optimum MAE conditions
Compound

Initial concentration
(mg/mL)

Recovered
concentration after
MAE, mg/mL

Relative
Standard
Deviation
(RSD%)
n=3

Average
recovery
(%)

silybinin

0.075

0.073

0.94

97.33

1.000

0.96

1.25

96.00

*Extraction conditions: 600 W microwave power, 6 min extraction time, 25 ml extraction solution volume
prepared with 80% v/v ethanol.

To determine the reproducibility of the novel extraction method five samples of 1 g each were
processed under the optimum extraction conditions as obtained from the systematic study of different
extraction parameters. The mean percentage extraction of silybinin obtained was found to be 1.37%
w/w, which was 25.7% more efficient than 12 hrs of conventional Soxhlet extraction. The calculated
R.S.D. value was 3.8%, which shows that the proposed microwave extraction method has an
acceptable precision. The repeatability of the chromatographic process was also considered. An
amount of 1 g sample was processed under the optimal MAE conditions. The sample was analyzed
repeatedly for five times under the same chromatographic conditions. The R.S.D. of the
chromatographic analysis was 0.68%.

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Dhobi et al., J. Chem.Metrl. 3:1 (2009) 13-23

3.11 Comparison of MAE with other conventional extraction techniques

Table 2. Comparison profile of MAE with traditional extraction techniques
Extraction method

Extraction time

Solvent volume

R.S.D (%)

MAE

6 min

25 ml

3.8 (n=5)

Response
(% w/w)
1.37

Maceration

24 h

100 ml

12.4 (n=5)

0.36

Soxhlet extraction

12 h

100 ml

7.6 (n=5)

1.09

Stirring extraction

24 h

100 ml

11.2 (n=5)

0.48

Response= % extraction of silybinin, w/w. Weight of the powdered leaf sample = 1 g. MAE was performed at
the optimum set up as obtained in the study

The selection of an extraction method would mainly depend on the advantages and
disadvantages of the processes, such as extraction yield, complexity, production cost, environmental
friendliness and safety. In general, maceration and heat reflux extraction are the most frequently used
extraction procedures. The drawbacks of maceration and heat reflux extraction are the large amount of
solvent and long extraction time needed. Considering the expensive solvent consumption and the long
extraction period, these extraction methods are not favorable from a commercial perspective. The
principle of heating during MAE is based on the direct effect of microwaves on molecules by ionic
conduction and dipole rotation. Ionic conduction is the electrophoretic migration of ions when an
electromagnetic field is applied. The resistance of the solution to this flow of ions will result in friction
and therefore heat the solution. Dipole rotation means realignment with the applied field. At 2.45
GHz, which is the frequency used in commercial systems, the dipoles align, randomize and jostle 4.9
109 times per second and this results in heating [19]. Based on that mechanism, either polar samples or
polar extraction solvents are required for efficient heating. However, compared with the conventional
extraction methods, MAE method showed prominent advantages with strong penetration force, high
extraction efficiency, reduced extraction time, less exposure to organic solvents which can lead to
better products with lower cost. In the current study, MAE was compared with the other conventional
extraction techniques for the extraction of silybinin from Silybum marianum. The conditions of
different techniques and their results are summarized in Table 2. It must be noted that all the extraction
techniques were used under their optimized conditions. Table 2 showed that in terms of yield of target
analyte, the best results were obtained by MAE, which gave significantly higher values. On extraction
time, MAE was also the fastest extraction method with only 12 min of extraction time (spread over
two extraction cycles of 6 min each)with preleaching time of 20 min. Stirring and heat reflux
extractions are time consuming processes based on heat or mixing to increase the mass transfer rate. If
the extraction yield obtained from MAE was to be considered as 100% performance level then, 12 h of
Soxhlet extraction and 24 h of maceration and stirring extraction could attain 79.6%, 26.3% and 35%
performance (in terms of extraction yield of silybinin) efficiency. These features would position MAE
as a valuable and cost effective technology suitable for todays highly competitive industries with
growing demand for increased productivity, improved efficiency and reduced cycle time.
4. Conclusion
The effectiveness and efficient applicability of MAE technique for the extraction of bioactive
compounds has been demonstrated. The technique can also be useful for the extraction of marker
compounds which are present in trace amount and often get degraded when extraction is attempted
through conventional techniques. Hence the proposed method can be very useful in case of chemical
standardization of botanicals to meet global standards. Comparison with conventional extraction

Microwave assisted extraction of silybinin

22

methods revealed that MAE could save a lot of time and electrical energy. Besides, the quantity of
solvent consumed in MAE was the least which proves its environment friendly or ecofriendly feature.
That means, it would save the production cost greatly. Henceforth the proposed extraction method can
be called as green extraction technique with an ecofriendly edge. In addition, the green aspect of the
total procedure becomes a key feature since research concerning new alternatives and new solvents in
chemistry are at the moment, for earth and environment protection, a key challenge that we cannot
disregard.
Acknowledgements
Financial supports from University Grant Commission (UGC), India for providing Junior
Research Fellowship (JRF) is acknowledged. The authors also wish to thank Ranbaxy Research
Laboratories (Gurgaon, India) for providing HPLC equipment for chromatographic analysis.

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