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Biochemical Engineering Journal 12 (2002) 7177

Comparison of microwave-assisted extraction and conventional


extraction techniques for the extraction of tanshinones
from Salvia miltiorrhiza bunge
Xuejun Pan, Guoguang Niu, Huizhou Liu
Young Scientist Laboratory of Separation Science and Engineering, State Key Laboratory of Biochemical Engineering,
Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, China
Received 16 October 2001; accepted after revision 27 February 2002

Abstract
Extraction techniques using microwave-assisted extraction (MAE), extraction at room temperature (ERT), heat reflux extraction, ultrasonic extraction and Soxhlet extraction were evaluated for the extraction of tanshinones (Cryptotanshinone, Tanshinone I and Tanshinone
IIA) from Salvia miltiorrhiza bunge. The extracts were analyzed by high performance liquid chromatography (HPLC) without any treatment. The results showed that the percentage extraction of Cryptotanshinone, Tanshinone I and Tanshinone IIA from S. miltiorrhiza bunge
by MAE was equivalent with and in fact higher than that of conventional extraction methods. MAE only needs 2 min, whereas ERT, heat
reflux extraction, ultrasonic extraction and Soxhlet extraction need 24 h, 45, 75 and 90 min, respectively. Due to the considerable saving
of time and high extraction efficiency, MAE was more effective than the conventional methods.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Salvia miltiorrhiza bunge; Tanshinones; Microwave-assisted; Extraction method

1. Introduction
Dried root of Salvia miltiorrhiza bunge (Tan-Shen in
Chinese) is an ancient Chinese drug. It has been commonly used in traditional Chinese medicine for promoting
blood circulation to remove blood stasis, clearing away
heat, relieving vexation, nourishing blood, tranquillizing
the mind, cooling the blood to relieve carbuncles, treating
hemorrhages, menstrual disorders and miscarriages [1,2].
Tan-Shen is well known to contain abietane-type diterpenes (tanshinones), such as Cryptotanshinone, Tanshinone
I and Tanshinone IIA. They have shown some actions of
broad-spectrum bactericide, dilating coronary artery and
increasing coronary flow, cytotoxic activity and modulating the effect on mutagenic activity. They also protect the
myocardium against ischaemia [2,3].
Microwave-assisted extraction (MAE), which is known as
a more environmental-friendly process with economic advantages than the current extraction methods, has been used
for the extraction of biologically active compounds from
different matrices [4]. Recently, many research studies have

Corresponding author. Tel.: +86-10-6255-4264;


fax: +86-10-6256-1822.
E-mail address: hzliu@home.ipe.ac.cn (H. Liu).

been done, such as extraction of taxanes from Taxus biomass


[5], extraction of azadirachtin-related limonoids from neem
seed kernel [6], extraction of felodipine tablets [7], extraction of glycyrrhizic acid from licorice root [8] and extraction
of tea polyphenols and tea caffeine from green tea leaves [9].
Extraction of tanshinones from the root of S. miltiorrhiza bunge and subsequent analysis by high performance
liquid chromatography (HPLC) has been reported [1012].
The mixed-level orthogonal array design (OAD) procedure
was used to optimize the heat reflux extraction techniques
[13,14]. Until now, the extraction of tanshinones from root of
S. miltiorrhiza bunge with MAE has not been reported. The
purpose of this study was to develop a novel MAE method,
and to evaluate MAE and conventional extraction techniques
for the extraction of tanshinones from S. miltiorrhiza bunge.
2. Experimental
2.1. Plant materials
Dried root of S. miltiorrhiza bunge (Tan-Shen in Chinese)
was purchased from Tongrentang Medicine Cooperation
(China). Material size consisted of pieces, the diameter and
thickness of which was about 38 and 35 mm, respectively.

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X. Pan et al. / Biochemical Engineering Journal 12 (2002) 7177

2.2. Reagents
Ethanol (analytical reagent), methanol (reagent for
HPLC), tetrahydrofuran (reagent for HPLC), glacial acetic
acid (analytical reagent), Cryptotanshinone, Tanshinone
I and Tanshinone IIA standard were purchased from the
China Institute for Drugs and Biological Products Identification (Beijing, China).
2.3. Conventional extraction methods
Extraction at room temperature (ERT) was carried out in
the following manner. Root of S. miltiorrhiza bunge (10 g)
was mixed with an appropriate solvent (100 ml). The suspensions were left at the room temperature of about 25 C
and shaken periodically during the extraction time indicated
by the experimental design.
Heat reflux extraction using a water-bath was performed
with a 10 g root of S. miltiorrhiza bunge and 100 ml solvent in a flask (250 ml) and the suspensions were made to
boil. Mechanical stirrer was used during the extraction. At
the same time the temperature of the water-bath can be set
according to the experimental design for the purpose of the
experiments about the effect of water-bath temperature.
Ultrasonic extraction using an ultrasonic bath (Tianpeng
Electronic Technology Co. Ltd., Beijing) was performed.
Root of S. miltiorrhiza bunge (10 g) was mixed with an appropriate solvent (100 ml). The suspensions were sonicated
with continuous power for a pre-setting time.
Soxhlet extraction was carried out using a 10 g root of
S. miltiorrhiza bunge placed inside the Soxhlet apparatus
(250 ml). The solvent of about 100 ml remained in the
boiling state during the time indicated by the experimental
design.
2.4. Microwave-assisted extraction
A household microwave oven was modified in our laboratory [8]. Root of S. miltiorrhiza bunge was mixed with
an appropriate solvent (100 ml). The suspensions were irradiated with microwaves in pre-setting procedures (for 25 s
power was switched on at the desired temperature of about
80 C and then for heated for 2 s and the power was switched
off for cooling for 10 s) and not allowed to super-boil.
2.5. High performance liquid chromatographic analysis
In the present work, the suspensions following MAE
were centrifuged and filtered with a microporous (about
0.5 m) membrane and then analyzed. Separations were
achieved on a Zorbax-SB guard column and Zorbax-ODS
(5 m, 150 4.6 mm, Du Pont) column and was eluted
with methanoltetrahydrofuranglacial acetic acidwater
(16:37.5:1:45.5, v/v) at UV 254 nm at a flow rate of
1 ml min1 . Three tanshinones were completely separated
from other compounds. Retention time of Cryptotanshinone,

Fig. 1. Calibration curve of Cryptotanshinone, Tanshinone I and Tanshinone IIA.

Tanshinone I and Tanshinone IIA was about 4.8, 5.7 and


9.3 min, respectively. A good linear fit ranging from 0.16 to
1.4 g of Tryptotanshinone, Tanshinone I and Tanshinone
IIA was found (Fig. 1). The regression equations and correlation coefficients were respectively, Cryptotanshinone:
Y = 2360.7X 142.5, R = 0.9998 (N = 9); Tanshinone
I: Y = 3642.5X 195.2, R = 0.9999 (N = 9); Tanshinone IIA: Y = 2547.4 180.3, R = 0.9997 (N = 9).
This method is sensitive and accurate with good reproducibility. The analytical operation can be completed in
25 min.
In the present work, extraction efficiency was defined as
follows:
Percentage extraction(w/w)
Mass of tanshinone (in extracted solution)
=
100%
Mass of material (root of S. miltiorrhiza bunge)

3. Results and discussion


3.1. The effect of MAE time on percentage extraction
of tanshinones
Fig. 2 shows the effect of MAE time on percentage
extraction of tanshinones. The results indicate that the
percentage extraction of tanshinones increased with the
increase of MAE time. MAE can reach the highest percentage extraction in 2 min. If MAE time was more than 2 min,
the percentage extraction of tanshinones decreased with the
increase of MAE time because tanshinones easily decompose if they are kept at high temperature for a long period
of time [15] and readily undergo photooxidation [16]. So
MAE time of 2 min was optimal for reaching the highest
percentage extraction.

X. Pan et al. / Biochemical Engineering Journal 12 (2002) 7177

73

Fig. 2. The effect of MAE time on percentage extraction of tanshinones (solvent: 95% (v/v) of 100 ml ethanol, root of Salvia miltiorrhiza bunge: 10.0 g,
L/S = 10.0).

3.2. The effect of ERT time on percentage extraction


of tanshinones
Fig. 3 shows the effect of ERT time on the percentage
extraction of tanshinones. The results indicate that the percentage extraction of all tanshinones increased with the
increase of extraction time at room temperature. Similar results were also obtained with other studies in our laboratory
on extracting GA from G. Uralensis Fisch [17], and the ERT
method gave equal or slightly lower percentage extraction
of tanshinones compared with the MAE procedure without
considering the extraction time.
3.3. The effect of heat reflux extraction time on percentage
extraction of tanshinones
Fig. 4(a) and (b) show that when the heat reflux extraction method was used, the tanshinones reached the highest

percentage extraction after 45 and 60 min when 95 and 100%


(v/v) of ethanol was used, respectively. At higher extraction times, the percentage extraction was decreased, which
was similar to the result of the MAE procedure as shown
in Fig. 2. So heat reflux extraction time of 4560 min was
optimal to reach high percentage extraction of tanshinones.
3.4. The effect of water-bath temperature on percentage
extraction of tanshinones
The results of Fig. 5 show that the percentage extraction of
tanshinones increased with the increase of water-bath temperature till <65 C for 95 and 100% (v/v) ethanol. But at
70 C, the percentage extraction of tanshinones reached the
lowest, due partly to the decomposition [15]. If the temperature was increased from 70 to 80 C, the percentage extraction can reach the highest for 95 and 100% (v/v) ethanol,
as the extraction velocity simultaneously increased at the

Fig. 3. The effect of ERT time on percentage extraction of tanshinones (solvent: 95% (v/v) of 100 ml ethanol, root of Salvia miltiorrhiza bunge: 10.0 g,
L/S = 10.0, room temperature: about 25 C).

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X. Pan et al. / Biochemical Engineering Journal 12 (2002) 7177

Fig. 4. The effect of heat reflux extraction time on percentage extraction of tanshinones: (a) solvent: 95% (v/v) of 100 ml, root of Salvia miltiorrhiza
bunge: 10.0 g of ethanol, L/S = 10.0, water-bath temperature: 80 C; (b) solvent: 100% (v/v) of 100 ml ethanol, root of Salvia miltiorrhiza bunge: 10.0 g,
L/S = 10.0, water-bath temperature: 80 C.

higher temperature: And an interesting phenomena was also


seen while extracting TP and tea caffeine from green tea
[19]. The results show that the temperature for the extraction
of tanshinones from root of S. miltiorrhiza bunge should be
lower than 70 C or about 80 C.

traction time when the highest percentage extraction was


reached was 75 min after the initiation of the experiment.

3.5. The effect of ultrasonic extraction time on


percentage extraction of tanshinones

Fig. 7 shows the effect of Soxhlet extraction time on


percentage extraction of tanshinones. The results indicate
that the percentage extraction of all tanshinones increased
with the increase of Soxhlet extraction time from 30 to
90 min. On the contrary if Soxhlet extraction time was higher
than 90 min the percentage extraction decreased.

Fig. 6 shows the effect of ultrasonic extraction time on percentage extraction of tanshinones. The results indicate that
the percentage extraction of all tanshinones increased with
the increase of ultrasonic extraction time. In all experiments,
the extraction of tanshinones reached the highest percentage extraction in 75 min. The longer the extraction times,
the higher the temperature of the ultrasonic bath. At longer
extraction time, the percentage extraction was decreased,
due partly to decomposition. So the optimum ultrasonic ex-

3.6. The effect of Soxhlet extraction time on percentage


extraction of tanshinones

3.7. Comparison of MAE and conventional extraction


techniques
Fig. 8 shows that the same percentage extraction of tanshinones was reached after 2 min, 24 h, 75, 45 and 90 min

X. Pan et al. / Biochemical Engineering Journal 12 (2002) 7177

75

Fig. 5. The effect of water-bath temperature on percentage extraction of tanshinones: (a) solvent: 95% (v/v) of 100 ml ethanol, root of Salvia miltiorrhiza
bunge: 10.0 g, L/S = 10.0, heat reflux extraction for 45 min, (b) solvent: 100% (v/v) of 100 ml ethanol, root of Salvia miltiorrhiza bunge: 10.0 g;
L/S = 10.0, heat reflux extraction for 45 min.

Fig. 6. The effect of ultrasonic extraction time on percentage extraction of tanshinones (solvent: 95% (v/v) of 100 ml ethanol, root of Salvia miltiorrhiza
bunge: 10.0 g, L/S = 10.0).

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X. Pan et al. / Biochemical Engineering Journal 12 (2002) 7177

Fig. 7. The effect of Soxhlet extraction time on percentage extraction of tanshinones (solvent: 95% (v/v) of 100 ml ethanol, root of Salvia miltiorrhiza
bunge: 10.0 g, L/S = 10.0).

Fig. 8. Comparison of MAE and conventional extraction techniques (experimental conditions: solvent: 95% (v/v) of 100 ml ethanol, root of Salvia
miltiorrhiza bunge: 10.0 g, L/S = 10.0, MAE: 2 min, ERT: 24 h, heating reflux extraction: 45 min, ultrasonic extraction: 75 min, Soxhlet extraction: 90 min).

when MAE, ERT, ultrasonic extraction, heating reflux


extraction and Soxhlet extraction techniques were used,
respectively. MAE can obviously reduce the extraction
time. The results showed that the MAE technique gave
similar results to the more time-consuming classical extraction methods. Similar results have also been reported by
other researchers on comparing the conventional extraction
methods and MAE for the purpose of PAHs determination
[18], for extraction of organic contaminants from marine
sediments [19] and for the preparation of the systematic
analysis of additives in polyolefins [20].

4. Conclusions
Tanshinones decomposed easily when they were exposed
at high temperature for a long period of time. The percentage

extraction of tanshinones was very low at a water-bath temperature of 70 C. The efficiencies of tanshinones transfer
into various solvents from the roots of S. miltiorrhiza bunge
by five alternative extraction techniques were compared. The
effects of extraction mixture composition, temperature, time
and the recoveries were studied. Microwave energy was a
fast heating method achieving 80 C in a very short time.
So MAE was the most efficient method for the extraction
of tanshinones from the roots of S. miltiorrhiza bunge. This
was verified by the results presented in this study. When
compared with the conventional techniques such as ERT,
heat reflux extraction, ultrasonic extraction and Soxhlet extraction, the MAE procedure employed provides high extraction efficiency in short time, and is less labor intensive.
Therefore, MAE is an alternative extraction technique for
fast extraction of tanshinones from root of S. miltiorrhiza
bunge.

X. Pan et al. / Biochemical Engineering Journal 12 (2002) 7177

Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (No. 29836130).
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