Separation and Purication Technology 74 (2010) 155159

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Separation and Purication Technology
j our nal homepage: www. el sevi er . com/ l ocat e/ seppur
Preparation of bergenin from Ardisia crenata sims and Rodgersia sambucifolia
hemsl based on microwave-assisted extraction/high-speed counter-current
chromatography
Jianchao Deng, Xiaohua Xiao, Xing Tong, Gongke Li

School of Chemistry and Chemical Engineering, Sun Yat-sen University, Xingang Xi Road 135, Guangzhou 510275, China
a r t i c l e i n f o
Article history:
Received 22 February 2010
Received in revised form 15 May 2010
Accepted 22 May 2010
Keywords:
Bergenin
Microwave-assisted extraction (MAE)
High-speed counter-current
chromatography (HSCCC)
Ardisia crenata sims
Rodgersia sambucifolia hemsl
a b s t r a c t
In this paper, a simple method for the rapid extraction, separation and purication of bergenin from
Ardisia crenata sims and Rodgersia sambucifolia hemsl by microwave-assisted extraction (MAE) coupled
with high-speed counter-current chromatography (HSCCC) was developed. The MAE conditions were
optimized and 2.0g sample was extracted using 60% (v/v) aqueous methanol as extraction solvent with
liquid/solid ratio of 10/1(mL/g) at 60

C for 15min. The crude extract of MAE was separated and puried
directly by HSCCC using ethyl acetaten-butanolwater (3:2:5, v/v/v) solvent system. In less than 3.5h,
18.6 or 25.0mg of bergeninwas obtainedfrom160mg crude extract of A. creanta or R. sambucifolia inone-
step separation, respectively. The purity of bergenin was over 99% determined by HPLC and its chemical
structure was further identied by ESI-MS,
1
HNMR and UV. The results indicate that microwave-assisted
extraction coupled with high-speed counter-current chromatography is very suitable for the extraction,
separation and purication of bergenin from A. creanta and R. sambucifolia.
2010 Elsevier B.V. All rights reserved.
1. Introduction
Bergenin is the major component of Ardisia creanta sims (A. cre-
anta) and Rodgersia sambucifolia hemsl (R. sambucifolia) which are
popular medicinal plants listed in the 2005 Edition of the Chinese
Pharmacopoeia. Bergenin (Fig. 1) possesses a wide range of biolog-
ical activities such as antiulcer [1], hepatoprotective [2,3], anti-HIV
[4], antidiabetic [5,6], antiarrhythmic [7], anti-inammatory [8],
anti-arthritic and antitussive activities [9,10]. In view of its good
pharmacological activities, the study on the separation and puri-
cation of bergenin from Chinese medicinal plant is necessary.
The conventional methods for the separation and purication of
bergenin are maceration [3,9], heat reux [11] and soxhlet extrac-
tion(SE) [12], followedbysilicagel column[2,8]. Thesemethods are
time-consuming and require relatively large quantities of organic
solvents. Microwave-assisted extraction (MAE) was widely used in
food [13], agriculture [14] and natural products [15,16] due to its
highextractionefciency. High-speedcounter-current chromatog-
raphy (HSCCC) invented by Ito [17] is a support-free liquidliquid
partition chromatographic technique. It eliminates the irreversible
adsorptive loss of samples onto the solid support matrix used in
conventional chromatographic columns, and offers excellent sam-

Corresponding author. Tel.: +86 20 84110922; fax: +86 20 84112245.

E-mail addresses: xiaoxhua@mail.sysu.edu.cn (X. Xiao),
cesgkl@mail.sysu.edu.cn (G. Li).
ple recovery compared with silica gel column method. So it is very
suitable for the separation and purication of components from
Chinese herbal medicines [18]. Microwave-assistedextractioncou-
pled with high-speed counter-current chromatography had been
successfully used for preparative separation of active components
from Radix angelicae sinensis [19], Anoectochilu roxburghii (wall)
Lindl [20], Sarcandra glabra [21] and Corydalis saxicola bunting [22].
In this paper, a simple method for the rapid extraction,
separation and purication of bergenin fromA. creanta and R. sam-
bucifolia by microwave-assisted extraction (MAE) coupled with
high-speed counter-current chromatography (HSCCC) was devel-
oped. Bergenin obtained was determined by HPLC and its chemical
structure was further identied by ESI-MS,
1
H NMR and UV.
2. Experimental
2.1. Instrumentation
The MAS-II microwave oven (2450MHz, Sineo Microwave
Chemistry Technology Company, Shanghai, China) was used. The
preparative HSCCC instrument was a GS10A HSCCC (Beijing UE
Biotechnology, Beijing, China). The apparatus was equipped with
a PTFE multilayer coil of 110m1.6mm i.d. with a total capacity
of 240mL and a 12mL sample loop. The revolution radius or the
distance between the holder axis and central axis of the centrifuge
(R) was 5cm, and the value varied from 0.5 at the internal termi-
nal to 0.8 at the external terminal (=r/R, where r is the distance
1383-5866/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.seppur.2010.05.018
156 J. Deng et al. / Separation and Purication Technology 74 (2010) 155159
Fig. 1. Chemical structure of bergenin.
from the coil to the holder shaft, and R, is the revolution radius or
the distance between the holder axis and central axis of the cen-
trifuge). The revolution speed of the apparatus can be regulated
witha speedcontroller inthe range 01000rpm. The HSCCCsystem
was equipped with a model NS-1007 constant-ow pump (Beijing
UE Biotech., Beijing, China), a model 8823A UV detector (Beijing
Institute of New Technology Application, Beijing, China) operat-
ing at 254nm and a model HW2000 chromatography workstation
software (Qianpu Co., Ltd., Shanghai, China).
A HPLC system (Shimadzu, Kyoto, Japan) consisted of two LC-
20AT pump, a SPD-M20A photodiode array detector, an SIL-20
A auto injector, a DGU-20 A
3
degasser and a CBM-20A system
controller. Identication of bergenin was carried out by ESI-MS
(ThermoFisher, Waltham, USA) and Nuclear Magnetic Resonance
(NMR) spectrometer (Mercury-plus 300, Varian, USA).
2.2. Reagents and materials
HPLC grade acetonitrile was used (Merck, Darmstadt, Ger-
many). Methanol, ethyl acetate and n-butanol were of analytical
grade (Guangzhou Chemical Factory, Guangdong, China). Standard
bergenin was purchased from National Institute for the Control of
Pharmaceutical and Biological Products (Beijing, China).
The dried A. creanta and R. sambucifolia were collected from
Chengdu, Sichuan, China and was ground into powder (4060
mesh) before use.
2.3. Optimization of MAE conditions
An orthogonal test design L
9
(3)
4
was employed for the opti-
mization of MAE conditions. The experiment was carried out with
four factors and three levels, namely extraction temperature (40,
50 and 60

C), liquid/solid ratio (10/1, 15/1 and 20/1mL/g), extrac-

tion time (5, 10 and 15min) and concentration of methanol (60%,
80%and100%, v/v). Moreover, microwave power andconcentration
of methanol were also optimized with single factor experiment.
For each test, 2.0g sample was added to a 50-mL glass vessel with
appropriatesolvent volumeandstirredat 500rpmusingamagnetic
stirring bar for MAE. At the same time, 2.0g sample was reuxed
with100mL methanol for 12hbysoxhlet extraction(SE). After MAE
or SE, the extract was ltered, and then the ltrate was diluted
and used for HPLC analysis. All the experiments were performed in
triplicate.
The extraction solution was concentrated to dryness by rotary
evaporator under reducedpressure. Driedextract was usedfor sub-
sequent HSCCC separation.
2.4. Measurement of partition coefcient (K)
Kvalues weredeterminedbyHPLCmethod. Approximately2mg
of the dried extract was dissolved with 10mL solution composed of
the upper and lower phases (1:1, v/v) of the HSCCC solvent system
in a 10mL test tube. The test tube was capped and shaken violently
for several minutes. Then bergenin in the upper layer and lower
layer were analyzed by HPLC. The K value is dened as the peak
areas in the upper phase divided by that in the lower phase.
2.5. HSCCC procedure
In this study, two-phase solvent system of ethyl acetaten-
butanolwater (3:2:5 v/v/v) was used for HSCCC. Solvent mixture
was thoroughly equilibrated in a separated funnel at roomtemper-
ature and the two phases were separated before use. The sample
solution was prepared by dissolving dried extract in 5mL lower
phase of solvent system for the separation and purication of
bergenin.
HSCCC separation was performed as follows: the coiled column
was rst entirely lled with the upper phase of the solvent system.
The lower phase was then pumped into the head end of the col-
umn at a ow rate of 2.0mL/min, while the apparatus was run at a
revolution speed of 900rpm. After hydrodynamic equilibrium was
reached, as indicated by a clear mobile phase eluting at the tail out-
let, 5mL sample solution was introduced into the column through
the injection valve. The efuent of the column was continuously
monitored with UVvis detector at 254nm. The retention of the
stationary phase was computed from the volume of the stationary
phase collected from the column after the separation.
2.6. HPLC analysis and identication of HSCCC fractions
The extracts and the fractions obtained from HSCCC were
analyzed by HPLC. The column was a Diamonsil C
18
column
(200mm4.6mm I.D., 5m, Dikma, China). The mobile phase
was acetonitrile and water (10/90, v/v). The ow rate was set at
1.0mL/min, and the temperature was 30

C. The detection wave-

length was 270nm. The identication of HSCCC peak fractions was
further carried out by ESI-MS and
1
H NMR spectra.
The external standard method was used for quantitative analy-
sis of bergenin. The linear range of the method was 0.8160mg/L
with correlation coefcient (r) 0.9999, the relative standard devi-
ation (RSD) for six replicates was 0.5%, the detection limit was
0.24mg/L which was evaluated on the basis of a signal-to-noise
ratio of 3.0. The recoveries were 98.4% and 99.3% with RSD 0.5%
and 1.3% for A. creanta and R. sambucifolia, respectively. The typical
HPLC chromatograms of bergenin standard and the MAE extracts
of A. creanta and R. sambucifolia are shown in Fig. 2.
Briey, the extraction yield of bergenin was dened as follow-
ing:
Extraction yield (mg/g)
=
quantity of bergenin in MAE extraction (mg)
quantity of original sample (g)
3. Results and discussion
3.1. Optimization of MAE conditions
The primary step in MAE is to optimize the operating condi-
tions to obtain an efcient extraction of target compounds and
avoid the co-extraction of undesired compounds such as starch,
saponin, etc. Extraction temperature (A), ratio of liquid to solid (B),
J. Deng et al. / Separation and Purication Technology 74 (2010) 155159 157
Fig. 2. (A) HPLC chromatograms of standard solution of 80mg/L bergenin (A),
the MAE extract of A. crenata (B) and R. sambucifolia (C). Experiment conditions:
Diamonsil C
18
column (200mm4.6mm I.D., 5m); column temperature: 30

C;
mobile phase: acetonitrilewater (10:90, v/v); ow rate: 1.0mL/min; detection
wavelength: 270nm; injection volume: 5L; peak 1: bergenin.
extraction time (C) and concentration of methanol (D) were impor-
tant factors and optimized using an orthogonal L
9
(3)
4
test design,
and the results are shown in Table 1. The inuence to the mean
extractionyieldof bergenindecreasedwiththe order of D>A>C>B
according to the R values. The concentration of methanol was
found to be the key factor for MAE. Since the choice of solvent
is fundamental for obtaining an optimal extraction process, some
consideration should be give to the microwave absorbing proper-
ties of the solvent, the interaction of the solvent with the matrix
and the analyte solubility in the solvent [23]. Further systemati-
cal investigation on the effect of concentration of methanol from
0% to 100% (v/v) on the extraction of bergenin was performed.
20.43, 23.60, 24.39, 24.16 and 20.19mg/g bergenin was obtained
from 0, 30%, 60%, 80% and 100% (v/v) aqueous methanol, respec-
tively. The results showed that the extraction yield improved with
the concentration of methanol was 60% (v/v), while the yield of
bergenin was decreased as 100% methanol or pure water was used.
Extraction temperature is also an important factor contributing to
improve sample wetting, matrix penetration and extraction ef-
ciency. The extraction yield of bergenin was increased with the
temperature in the range of 4060

C. Higher temperature could

accelerate the solvent transfer between the sample and solvent,
and better extraction yield was obtained. The ratio of liquid to solid
and extraction time only showed slightly effect on the extraction
yield of bergenin in comparison with the other two factors. A liq-
uid/solid ratio of 10/1(mL/g) and extraction time of 15min were
adopted in the present work. Moreover, the effect of microwave
power on the extraction yield of bergenin was also optimized
by single factor experiment and the extraction yield was almost
steady and 23.740.34mg/g bergenin was obtained from 300 to
900W. Therefore, the optimized MAE conditions for extraction
of bergenin from Ardisia crenata was performed using 60% (v/v)
aqueous methanol as extraction solvent with liquid/solid ratio of
10/1(mL/g) at 60

C for 15min under 500W microwave power.

Since the amount of starch in R. sambucifolia was higher than
that in A. crenata [24], lower extraction temperature coupled with
higher liquid/solid ratio in a fast procedure would be benet from
avoiding the effect of starch on the extraction of bergenin from
R. sambucifolia. And then, the optimized extraction conditions for
R. sambucifolia were extraction temperature of 50

C, liquid/solid
ratio of 15/1(mL/g) and 5min of extraction time, the main MAE
condition, 60% (v/v) aqueous methanol as extraction solvent, was
the same as that for A. crenata.
Under the optimum MAE conditions, the extraction yields of
bergenin in A. crenata and R. sambucifolia were 26.50.3 and
32.30.1mg/g, respectively. Moreover, to evaluate the extraction
efciency of MAE, bergenin was also extracted with the typical tra-
ditional SE method. The extraction yields of bergenin by SE from
A. crenata and R. sambucifolia were 27.70.4 and 32.60.1mg/g,
respectively. Compared with SE method, similar extraction yield
of bergenin by MAE was obtained with the greatly reducing time
(15min instead of 12h). Therefore, MAE was validated as a rapid,
efcient and reliable method for extraction of bergenin from A.
creanta and R. sambucifolia.
3.2. Selection of two-phase solvent system in HSCCC
In order to determine the optimal two-phase solvent system
for the HSCCC separation, a series of optimization experiments
were performed in the present study. According to the gold rules in
selecting optimum conditions introduced by Ito [25], solvent sys-
tem of ethyl acetaten-butanolwater would be suitable for polar
compound such as bergenin. And then, several kinds of solvent
systems composed of ethyl acetaten-butanolwater at different
volume ratios were selected and assessed for K values. The K val-
ues of bergenin were 1.53, 1.38, 1.20, 0.68 and 0.19 for the ethyl
acetaten-butanolwater solvent system under the volume ratios
Table 1
The results of orthogonal design L
9
(3)
4
(n=3) for MAE of bergenin from A. crenata.
No. Factors Yield of bergenin in
A. crenata (mg/g)
A (extraction temperature,

C) B (liquid/solid ratio, mL/g) C (extraction time, min) D (concentration of methanol, %)

1 40 10 5 60 24.2 0.6
2 40 15 10 80 23.4 0.2
3 40 20 15 100 20.3 0.4
4 50 10 10 100 22.1 0.5
5 50 15 15 60 26.3 0.6
6 50 20 5 80 24.5 0.2
7 60 10 15 80 26.9 0.6
8 60 15 5 100 21.3 0.1
9 60 20 10 60 26.3 0.3
k
1
22.633 24.400 23.333 25.600
k
2
24.300 23.667 23.933 24.933
k
3
24.833 23.700 24.500 21.233
R 2.200 0.733 1.167 4.367
Optimal level A
3
B
1
C
3
D
1
k
A
i
=

(yield of bergenin at A
i
)
3
, R
A
i
= max{k
A
i
} min{k
A
i
}.
158 J. Deng et al. / Separation and Purication Technology 74 (2010) 155159
Table 2
Effect of sample loading on the retention of the stationary phase of HSCCC and the purity of bergenin.
Chinese medicinal plant Original sample (g) Dry extract (mg) Bergenin in extract (mg) Bergenin in obtained
from HSCCC (mg)
Retention of the
stationary phase (%)
Purity (%)
A. crenata
0.2 40 4.7 4.2 41.7 >99
0.4 80 9.5 9.4 35.0 >99
0.6 120 14.2 13.9 31.7 >99
0.8 160 18.9 18.6 28.3 >99
R. sambucifolia
0.2 40 6.5 6.4 39.2 >99
0.4 80 12.9 12.7 35.8 >99
0.6 120 19.4 18.9 30.0 >99
0.8 160 25.9 25.0 27.5 >99
of 1:4:5, 2:3:5, 3:2:5, 4:1:5 and 5:0:5 (v/v/v), respectively. It can
be seen that the solvent system of ethyl acetaten-butanolwater
at the volume ratio of 5:0:5 and 4:1:5 had minor K value, and the
retention of the stationary phase was less than 30%. The other sol-
vent systems of ethyl acetaten-butanolwater at volume ratio of
1:4:5, 2:3:5and3:2:5hadappropriateKvalues. Further experiment
was performed and the results showed that two-phase solvent
system composed of ethyl acetaten-butanolwater (3:2:5, v/v/v)
could obtain relative better the retention of stationary phase of 40%
for preparative HSCCC, along with its good separation and accept-
able separationtime, it was selectedas the solvent systemof HSCCC
in the following studies.
3.3. Optimization of sample concentration of HSCCC
Successful separation in HSCCC greatly depends on the reten-
tion of the stationary phase, in general, the higher the retention of
the stationary phase, the better the peak resolution [23]. Although
high sample loading can enlarge the yield of obtained analytes
in a single separation and purication procedure, it is commonly
unfavorable to the retention of the stationary phase, resulting in
reducing the separation and purication efciency in HSCCC. The
sample loading was optimized under the sample concentration
of 8, 16, 24 and 32mg/mL, i.e. 40, 80, 120 and 160mg of dried
extract dissolved in 5mL of the HSCCC lower phase, respectively.
The effect of sample loading on the retention of the stationary
phase and purity of obtained bergenin is shown in Table 2. It
was found that, with the increase of the sample loading from 40
to 160mg, the corresponding peaks were similar and the purity
of obtained bergenin was over 99%, but the peak resolution of
bergenin decreased slightly. Meanwhile, the retention of the sta-
tionary phase greatly decreased from 41.7% to 28.3% and 39.2% to
27.5% for A. creanta and R. sambucifolia, respectively. Larger sample
loading (>160mg) could lead to excessive lost of the retention of
the stationary phase, and it was not suitable for the separation and
purication. Thus, the maximumsample loading was 160mg dried
extract.
3.4. Purity determination of the separated peak
Under the optimum MAE and HSCCC conditions, bergenin was
separated and puried from A. creanta and R. sambucifolia. The
HSCCC chromatograms of A. creanta and R. sambucifolia are shown
in Figs. 3A and 4A, respectively. 18.6 or 25.0mg of bergenin was
obtained from 160mg dried extract of A. creanta or R. sambucifolia,
respectively, within 3.5h.
Peak fractions from HSCCC were analyzed by HPLC, the HPLC
chromatograms and UV spectra of the collected fractions from A.
creanta and R. sambucifolia are shown Figs. 3B and 4B, respec-
tively. The obtained bergenin was determined according to the
peak area with the standard. The purity of bergenin was dened
as the amount determined with HPLC by the peak area divided
by the measured mass of bergenin in the collected efuent. The
results of HPLCshowedthat peak1whichcorrespondedtobergenin
possessed the purity of more than 99%.
The structural identication of peak fractions was performed
with ESI-MS and
1
H NMR spectra follows. ESI-MS (m/z): 328 [M
+
];
Fig. 3. (A) HSCCCchromatogramof thecrudeextract fromA. creanta; (B) HPLCanaly-
sis andUVspectrumof bergeninobtainedfromA. crenata; HSCCCconditions: solvent
system: ethyl acetaten-butanolwater (3:2:5, v/v/v); column volume: 240mL;
mobile phase: lower phase; ow rate: 2mL/min; rotation speed: 900rpm; sample
loading: 160mg, sample volume: 5mL; temperature, 25

C; detection wavelength,
254nm. HPLC conditions were the same as shown in Fig. 2.
Fig. 4. (A) HSCCC chromatogram of the crude extract from R. sambucifolia; (B)
HPLC analysis and UV spectrum of bergenin obtained from R. sambucifolia. HSCCC
conditions: solvent system: ethyl acetaten-butanolwater (3:2:5, v/v/v); column
volume: 240mL; mobile phase: lower phase; ow rate: 2mL/min; rotation speed:
900rpm; sample loading: 160mg, sample volume: 5mL; temperature, 25

C; detec-
tion wavelength, 254nm. HPLC conditions were the same as shown in Fig. 2.
J. Deng et al. / Separation and Purication Technology 74 (2010) 155159 159
1
H NMR (DMSO-d
6
, 300MHz): 6.98 (1H, s, H-7), 5.62 (1H, d,
H-10b), 4.98 (1H, d, H-4a), 3.98 (1H, d, H-4), 3.86(2H, d, H-11),
3.77 (3H, s, H-12), 3.65 (1H, m, H-2), 3.56 (1H, d, H-3), which was
accorded with the literature [26], indicating the structural identi-
cation of bergenin.
4. Conclusion
A new MAE coupled with HSCCC method was developed for
the preparation of bergenin from the traditional Chinese medicine
of A. creanta and R. sambucifolia. The crude extract of MAE was
separated and puried directly by HSCCC using ethyl acetaten-
butanolwater (3:2:5, v/v/v) solvent system. Under optimum
conditions, 18.6 or 25.0mg of bergenin was obtained from 160mg
dried extract of A. creanta or R. sambucifolia within 3.5h with purity
over 99%. Theresults indicatedthat thepresent methodof MAEcou-
pled with HSCCC was suitable for the separation and purication
of bergenin from A. creanta and R. sambucifolia.
Acknowledgements
This work supported by the project of the National Key
Technologies R&D Programme of the 11th-ve-year Plan (No.
2006BAK03A08), by the National Natural Science Foundation of
China (No. 20905080), and by Science and Technology Planning
Project of Guangdong Province of China (No. 2009B010900021).
Special thanks to Mr. Zhou Xiaonan for his useful advices on the
design of the LTV-MAE device.
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