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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-
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, 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,
(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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