Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 146–151

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Short communication

Subcritical water extraction of alkaloids in Sophora flavescens Ait. and

determination by capillary electrophoresis with field-amplified sample stacking
Haiyan Wang a , Yuchao Lu a , Jie Chen a , Junchao Li b , Shuhui Liu a,c,∗
a
College of Science, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi 712100, China
b
College of Life Sciences, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi 712100, China
c
Shaanxi Key Laboratory of Molecular Biology for Agriculture, No. 3 Taicheng Road, Yangling, Shaanxi 712100, China

a r t i c l e i n f o a b s t r a c t

Article history: The extraction and determination of cytisine, sophocarpine, matrine, sophoridine and oxymatrine in
Received 24 July 2011 Sophora flavescens Ait. were performed using subcritical water extraction and capillary electrophoresis
Received in revised form with field-amplified sample stacking. The effects of extraction temperature, pressure, time and cycle
16 September 2011
number on the extraction yields were investigated systematically for accelerated solvent extraction with
Accepted 18 September 2011
ethanol (ASE) and accelerated solvent extraction with water (subcritical water extraction, SWE). The
Available online 22 September 2011
extraction yields obtained using SWE, ASE, water ultrasonic extraction and chloroform soaking extrac-
tion methods were compared. The electrophoresis separation buffer was monosodium phosphate (pH
Keywords:
Subcritical water extraction
3.0; 110 mM)-isopropanol (85:15, v/v). The effect of phosphoric acid added to the sample matrix on
Accelerated solvent extraction the reproducibility of the peak heights of the analytes was also examined. Cytisine, sophoridine and
Capillary electrophoresis oxymatrine showed good linearity (R2 > 0.999) within 0.125–4.0 ␮g/mL, and sophocarpine and matrine
Sophora flavescens Ait. exhibited good linearity (R2 > 0.998) within 0.0625–2.0 ␮g/mL, with the detection limits in the range of
Alkaloids 0.004–0.0013 ␮g/mL. The five alkaloid contents in medicinal plants from different sources and Sophora
instant granule were determined and compared.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction samples. ASE becomes more preferred because of its apparent

Sophora flavescens Ait. (Sophora), in the family Fabaceae, is a tra-
ditional Chinese medicine. Researchers at home and abroad have
studied its chemical components, which are mainly quinolizidine
alkaloids and flavonoids. Thus far, over twenties alkaloids have
been isolated from the root, leaves or flowers of Sophora which
are reported to exhibit sedative, analgesic and other central ner-
vous system inhibition effects [1] as well as antipyretic, anti-tumor,
anti-myocardial activities [2].
The extraction methods of the alkaloids in Sophora are mainly
chloroform soaking extraction [3–8] and solvent reflux extrac-
tion [9,10] in which shortcomings include long extraction time,
high cost of non-environmental friendly organic solvents, and
organic solvent residuals in the products extracted. Only a couple of
research groups used ultrasonic extraction [11,12] and supercritical
fluid extraction [13].
Accelerated solvent extraction (ASE) is a new highly efficient
sample preparation technique for extraction of solid or semi-solid
∗ Corresponding author at: College of Science, Northwest A&F University,
No. 3 Taicheng Road, Yangling, Shaanxi 712100, China. Tel.: +86 029 87092226;
fax: +86 029 87092226.
E-mail address: shliu815@126.com (S. Liu).
advantages such as less solvent consumption, automatic proce-
dure for simultaneous extraction of multiple samples, short sample
preparation time, and higher extraction recoveries. However, ASE
using organic solvents is not a promising extraction procedure in
term of environment issue. Subcritical water extraction (SWE) uses
pure water as the solvent and is an innovative green sample prepa-
ration technique. Subcritical water is also called superheated water,
pressurized hot water, whose polarity, surface tension and viscosity
can be changed by controlling temperature and pressure. When the
temperature is increased, the polarity decreases, and the solubility
of organic compounds in the subcritical water increases dramati-
cally. The device used for ASE is also suitable for SWE [14]. Several
reviews on SWE have been published recently [15,16].
So far, capillary electrophoresis (CE) has been widely used in
pharmaceutical analysis due to its high separation efficiency, low
consumption of organic solvent and in-line sample enrichment.
Several papers described the determination of alkaloids in Sophora
using CE [5–8]. Due to its self-limiting nature, the reproducibility
of CE is poor, especially when using in-line field-amplified sample
stacking (FASS), in which an internal standard has to be added dur-
ing the quantitative analysis [6–8]. However, the selection of the
internal standard substances is rather difficult. They often inter-
fere with other peaks from real samples, and some are quite toxic
and harmful.

0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jpba.2011.09.014
 H. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 146–151 147

O O 2.2. Chemicals and samples

H H
N Cytisine and matrine were purchased from Aladdin reagent
NH N
H company (Shanghai, China). Sophoridine was supplied by the
H
N cal Products of China (NICPBP, Beijing, China). Sophocarpine and
H H H H
O N N
Cytisine Sophocarpine Matrine
O (Tianjing, China).
O H
N sources, including a local hospital, a local drug store and the campus
H
H of Northwest A&F University (Yangling, Shannxi Province, China),
N
H
H
N
H ground into a fine powder (60 mesh), the samples were stored at
H N H
O
Sophoridine Oxymatrine
Scheme 1. The structures of the five alkaloids.
In this paper, the extraction of five active compounds (Scheme 1)
from Sophora using SWE was investigated and compared with ASE,
water ultrasonic extraction and chloroform soaking extraction. The
contents of the analytes were analyzed using CE with FASS, for
which the effort was made to improve the reproducibility. By inte-
grating SWE and CE, we attempted to provide a new, efficient and
environmentally friendly analysis approach for the medicinal plant
quality control.
2. Materials and methods
2.1. Instruments and apparatus
All electrophoresis experiments were performed on a Beck-
man P/ACETM MDQ Capillary Electrophoresis System equipped
with a photodiode array detector (PDA) and controlled by Sys-
tem Gold software (Beckman Coulter Inc., Fullerton, CA, USA).
Uncoated fused-silica capillaries with a total length of 50 cm (40 cm
to the detector) and 50 ␮m I.D., from Ruipu Chromatogram Equip-
ment Company (Yongnian County, Hebei Province, China) were
maintained at 25 ◦ C in a cartridge with a 100 ␮m × 800 ␮m detec-
tion window. The PDA was set at an acquisition range from 190
to 300 nm (bandwidth of 6 nm), at a spectral acquisition rate
of 4 Hz. Electropherograms were recorded at 214 nm. Peak iden-
tity was confirmed by post-run diode-array detection analysis
of the absorption maximum of individual peaks in a mixture.
Separation was performed at a constant voltage of 25 kV (anode
at injection end). The pH of all solutions was measured by pH
213 equipment (Hanna instruments, Italy). Water was purified
using Millipore Direct-Q 3 system (Millipore Corporation, Bedford,
MA, USA).
A new capillary was rinsed with 0.1 M sodium hydroxide for
30 min, deionized water for 30 min, and finally with the background
electrolyte (BGE) solution for 30 min before use. Between each
run, the capillary was rinsed with water for 1 min, 0.1 M sodium
hydroxide for 3 min, water for 1 min, and then the BGE for 5 min,
successively. The electrolytes were filtered through 0.22 ␮m mixed
cellulose ester filters (Shanghai Minimo Separation Technology Co.
Ltd.) prior to use.
National Institute for the Control of Pharmaceutical and Biologi-
oxymatrine were purchased from Sigma–Aldrich (St. Louis, MO,
USA). Sodium hydroxide and sodium dihydrogen phosphate (all
of analytical grade) were from Tianjin BODI Chemical Reagent Co.
Ltd. (Tianjing, China). Ethanol, methanol and 2-propanol (of HPLC
grade) were purchased from Luomiou Chemical Reagent Co. Ltd.
The crude drug Sophora root was obtained from different
which were all authenticated by Prof. Junchao Li (College of Life Sci-
ences, Northwest A&F University). After being dried naturally and
dry place under room temperature. Sophora instant granule, which
was a new commercial product made from the crude drug Sophora
root, was purchased from a local hospital.
Stock solution of 200 mM sodium dihydrogen phosphate was
prepared by dissolving the corresponding substance in water. The
pH of the run buffer was adjusted with 0.2 M hydrochloric acid
or 0.2 M sodium hydroxide. Stock standard solutions with cyti-
sine (200 ␮g/mL), sophocarpine (100 ␮g/mL), matrine (100 ␮g/mL),
sophoridine (200 ␮g/mL) and oxymatrine (200 ␮g/mL) were pre-
pared in water and stored at 4 ◦ C in a refrigerator, when in use,
diluted to the final concentration with water.
2.3. Sample extraction
2.3.1. Accelerated solvent extraction method
A Dionex ASE 200 Accelerated Solvent Extractor was performed.
First, 0.5 g Sophora sample was accurately weighed, mixed thor-
oughly with 3.0 g diatomite and then transferred to an 11-mL
stainless steel extraction cell. A filter paper was placed on the
bottom of the extraction cell. Ethanol was used as the extraction
solvent. Extraction pressure, temperature, static time and cycles are
specified in Section 3.2 (preheating period, 5 min). After extraction,
the thimble was rinsed with fresh extraction solvent (60% of the
extraction cell volume) and purged with a flow of nitrogen (150 psi
during 90 s). Solvent was collected in a 40 ml vial and then trans-
ferred to a 25 ml flask, which was rotary evaporated at 50 ◦ C. The
residue was redissolved in 25 mL of water. Then, 1 mL of the sample
solution was diluted to an appropriate concentration with water,
filtered through a 0.22-␮m filter and then analyzed.
2.3.2. Subcritical water extraction
The same ASE 200 (Dionex, USA) instrument was used. First,
0.5 g Sophora sample was accurately weighed, mixed thoroughly
with 3.0 g diatomite and then transferred to an 11-mL stainless steel
extraction cell. Water was deoxygenated by degassing for 20 min
prior to its use as extraction solvent. The same conditions were used
as in Section 2.3.1. After finishing extraction, water was added to
the extraction solution to reach a final volume of 25 mL. Then, 1 mL
of the sample solution was diluted to an appropriate concentration
with water, filtered through a 0.22-␮m filter and then analyzed.
2.3.3. Ultrasonic extraction
First, 0.5 g Sophora sample was weighed and placed in an Erlen-
meyer flask with a plug. Then, 25 mL of water was added as the
extraction solvent. The mixture was weighed and shaken until it
was mixed well. The sample was allowed to sit at room temperature
for 3 h, followed by ultrasonic extraction for 30 min. The flask was
weighed again, and the lost water was made up. The mixture was
148 H. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 146–151

Fig. 1. The influence of phosphoric acid added to a sample solution on the repro-
ducibility of the analyte peak heights. Each analyte, 1 ␮g/mL; buffer: monosodium
phosphate (pH 3.0; 110 mM)-isopropanol (85:15, v/v); water plug: 0.5 psi × 3 s;
injection: 8 kV × 8 s.

shaken, centrifuged and filtered. Then, 1 mL of the sample solution

was diluted to an appropriate concentration with water, filtered
through a 0.22-␮m filter and analyzed.

2.4. Soaking extraction

According to the Sophora extraction method described in the

1995 edition of Chinese Pharmacopoeia [17], 0.5 g Sophora sample
was weighed and placed in an Erlenmeyer flask with a plug. After
25 mL of chloroform was added as the extraction solvent, 0.3 mL of
concentrated ammonia was added to the mixture, which was then
shaken until it was mixed well. The flask was incubated at room
temperature overnight (16 h), then centrifuged and filtered. The
extraction solution was rotary evaporated at 40 ◦ C, and the residue
was redissolved in 50 mL of water. Then, 1 mL of the sample solution Fig. 2. Electropherogram of a standard mixture solution (a) and the extract of
Sophora using SWE (b). Both the standard and the actual sample solution were sup-
was diluted to an appropriate concentration with water, filtered
plemented with 0.8 mM phosphoric acid. Other conditions as described in Fig. 1.
through a 0.22-␮m filter and analyzed. 1 = cytisine, 2 = sophocarpine, 3 = matrine, 4 = sophoridine, 5 = oxymatrine.

3. Results and discussion

3.1. Development of CE analysis method
Based on the pilot experiments and our previous paper [8],
monosodium phosphate (pH 3.0; 110 mM)-isopropanol (85:15,
v/v) was used as the electrophoresis buffer. The FASS was con-
ducted by preparing the samples in water and electro-injecting.
Although a water plug was injected at 0.5 psi for 3 s to improve
the reproducibility [8], it could not meet the requirements without
the addition of an internal standard. Phosphoric acid was chosen
to add into a sample solution to overcome this drawback [18]. As
can be seen in Fig. 1, the addition of phosphoric acid improved the
reproducibility significantly. Nevertheless, the amount of phospho-
ric acid added had certain effects on the stacking efficiency. With
the addition of a small amount of phosphoric acid, the protonation
of the analytes was increased, which helped the electrostacking and
increased the analyte peak heights. The addition of more phospho-
ric acid to the sample caused an increase in solution conductivity,
which decreased the field strength for sample electro-injection,
thus the heights of analytes peaks declined. Based on these consid-
erations, 0.8 mM phosphoric acid was appropriate concentration.
The electropherogram of a five-standard mixture solution is shown
in Fig. 2(a).
Under the optimal electrophoresis conditions, the correlation
between the concentrations of the analytes and the peak heights
was analyzed. Cytisine, sophoridine and oxymatrine showed
good linearity (R2 > 0.999) within 0.125–4.0 ␮g/mL, and sopho-
carpine and matrine exhibited good linearity (R2 > 0.998) within
0.0625–2.0 ␮g/mL, respectively. The RSD values of the migration
times and peak heights that were determined by five replicate
injections of a standard mixture solution under the optimum con-
ditions were below 1.5% and 4.8%, respectively. The detection limits
calculated using a signal-to-noise criterion of 3 were in the range
of 0.004–0.0013 ␮g/mL.
3.2. Optimization of SWE and ASE
In this study, after Sophora samples (from the campus of North-
west A&F University) were extracted, the contents of the five active
compounds was determined by CE under optimized conditions
developed to indicate the influence of different extraction methods
and extraction conditions on extraction efficiency.
3.2.1. Effect of temperature and pressure
Pressure and temperature are the two most important physi-
cal parameters in SWE and ASE, and they have both theoretical and
 H. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 146–151 149

Table 1
Extraction efficiency of the alkaloids from Sophora flavescens Ait. via SWE and ASE expressed as the alkaloids contents of three replicates ±%RSD.

Content of the alkaloids (mg/g, ±%RSD, n = 3)

Sophocarpine Matrine Sophoridine Oxymatrine Total alkaloids

SWE
Temperature ( ◦ C)
70 0.20 ± 4.2 0.76 ± 4.1 6.25 ± 4.5 11.85 ± 3.3 19.06
100 0.65 ± 3.2 1.77 ± 4.4 7.01 ± 3.9 13.50 ± 2.1 22.93
130 1.53 ± 2.8 3.75 ± 3.8 7.40 ± 4.0 8.03 ± 5.0 20.71
160 3.08 ± 3.1 6.03 ± 4.9 7.64 ± 4.3 5.88 ± 3.6 22.63
190 3.29 ± 2.1 6.85 ± 2.8 7.85 ± 4.8 4.81 ± 4.1 22.80
Pressure (psi)
600 0.58 ± 2.6 1.50 ± 3.0 6.48 ± 4.3 12.68 ± 4.1 21.24
1000 0.60 ± 3.3 1.72 ± 2.2 6.95 ± 2.7 13.25 ± 3.8 22.52
1500 0.73 ± 1.6 1.75 ± 0.4 7.09 ± 2.5 13.38 ± 2.9 22.95
2000 0.60 ± 3.9 1.49 ± 3.5 6.45 ± 2.8 12.41 ± 4.8 20.95
Extraction time (min)
5 0.64 ± 4.1 1.73 ± 3.0 6.91 ± 3.2 12.92 ± 2.6 22.20
8 0.63 ± 1.3 2.10 ± 2.6 7.05 ± 0.6 14.39 ± 0.4 24.16
11 0.82 ± 3.0 1.76 ± 3.3 7.87 ± 2.5 13.50 ± 1.8 23.95
14 0.69 ± 5.2 1.75 ± 4.4 7.17 ± 3.4 12.74 ± 3.0 22.35
Cycles
1 0.57 ± 4.0 0.95 ± 4.3 6.76 ± 2.7 14.77 ± 1.9 23.05
2 0.63 ± 1.3 2.10 ± 2.6 7.05 ± 0.6 14.39 ± 0.4 24.16
3 0.67 ± 5.1 1.72 ± 3.7 6.66 ± 4.5 13.47 ± 2.5 22.52

ASE
Temperature (◦ C)
70 0.10 ± 5.1 0.46 ± 4.5 3.87 ± 1.6 11.42 ± 5.1 15.85
100 0.15 ± 3.6 1.25 ± 3.4 6.63 ± 4.8 10.83 ± 5.7 18.86
130 0.58 ± 3.3 2.28 ± 4.8 5.67 ± 2.9 7.38 ± 5.2 15.91
160 0.93 ± 2.9 3.05 ± 2.1 5.15 ± 4.0 5.25 ± 4.9 14.38
Pressure (psi)
600 0.14 ± 4.2 1.20 ± 5.5 5.83 ± 3.1 9.77 ± 3.7 16.94
1000 0.18 ± 4.8 1.24 ± 5.4 6.55 ± 5.6 10.43 ± 2.6 18.40
1500 0.14 ± 4.7 0.96 ± 4.2 5.35 ± 2.8 9.73 ± 4.3 16.18
2000 0.19 ± 5.2 1.35 ± 3.9 6.42 ± 1.6 10.97 ± 4.5 18.93
Extraction time (min)
5 0.13 ± 2.3 1.26 ± 0.7 6.22 ± 1.9 11.11 ± 1.1 18.72
8 0.24 ± 3.2 1.45 ± 3.6 6.27 ± 1.7 9.70 ± 1.9 17.66
11 0.25 ± 1.9 1.46 ± 3.3 6.48 ± 3.2 9.98 ± 1.6 18.17
14 0.16 ± 3.2 1.28 ± 0.9 6.13 ± 3.8 10.12 ± 5.3 17.69
Cycles
1 0.09 ± 5.1 0.60 ± 4.9 5.68 ± 4.6 11.71 ± 4.2 18.08
2 0.11 ± 2.7 1.27 ± 2.5 6.32 ± 3.7 10.89 ± 3.7 18.60
3 0.17 ± 5.1 1.33 ± 5.5 6.00 ± 5.2 9.50 ± 1.9 17.00

practical implications for the extraction process. In this experiment,
the effect of temperature on the yields of extracted compounds was
investigated with an extraction pressure of 1000 psi, a static extrac-
tion time of 5 min and a cycle number of 2 (Table 1). When SWE
was used, the yields of sophocarpine, matrine and sophoridine had
upward tendencies with increasing temperature from 70 to 190 ◦ C.
The yield of oxymatrine increased firstly with temperature ranging
from 70 to 100 ◦ C, then dropping significantly from 100 to 190 ◦ C
(cytisine could not be detected in all the samples). When ASE was
used, increasing the temperature from 70 to 160 ◦ C increased the
yields of sophocarpine and matrine. The yield of sophoridine first
increased and then decreased, with the highest yield at 100 ◦ C. The
yield of oxymatrine decreased with increasing temperature. With
an extraction solvent of either water or ethanol, the total alkaloid
yield reached its maximum at 100 ◦ C.
At an extraction temperature of 100 ◦ C, a static extraction time
of 5 min and a cycle number of 2, four levels of pressure (600,
1000, 1500, 2000 psi) were applied to the extraction, and the
corresponding extracted yields are reported in Table 1. For SWE
with increasing extraction pressure, the yields of sophocarpine,
matrine, sophoridine and oxymatrine first increased slightly and
then decreased, and the yield of each active compound reached its
maximal level at 1500 psi, which was then selected as the optimal
value for SWE. For ASE with increasing extraction pressure, the
yields of sophocarpine, matrine, sophoridine and oxymatrine first
increased, then decreased and increased again. The maximum
levels reached at both 1000 psi and 2000 psi. Since the latter was
excluded because of a safety issue with the equipment, 1000 psi
was selected in the subsequent experiments. It is also noticed that
the extraction pressure did not influence the yields as obviously
as the extraction temperature.
3.2.2. Effect of extraction time and cycle number
When SWE was used, the effect of extraction time on the yield
of the active compound was examined with an extraction tempera-
ture of 100 ◦ C, an extraction pressure of 1500 psi and a cycle number
of 2 (Table 1). With increasing extraction time, the yield of each
analyte generally showed an initial increasing and then decreasing
trend, and the total alkaloid yield reached its maximum at 8 min.
The effect of the extraction time on the yield by ASE was tested
with an extraction temperature of 100 ◦ C, an extraction pressure of
1000 psi and a cycle number of 2 (Table 1). As the extraction time
increased, the yields of sophocarpine and matrine and sophoridine
showed the same variance trend as in SWE, while the yield of oxy-
matrine decreased firstly and then kept stable. An extraction time
of 5 min was chosen due to the highest total yield obtained.
The effect of the cycle number on the yield was examined
for SWE with an extraction temperature of 100 ◦ C, an extraction
150 H. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 146–151

Table 2
Comparison of the alkaloid contents in medicinal plant from different sources and Sophora instant granule (mg/g, n = 3).

Samples Sophocarpine Matrine Sophoridine Oxymatrine Total alkaloids content

Content RSD% Content RSD% Content RSD% Content RSD%

Crude No. 1 2.21 4.0 5.50 2.0 2.33 3.3 28.34 1.2 38.37
Crude No. 2 1.21 3.8 3.72 4.4 6.09 6.2 21.04 5.5 32.06
Crude No. 3 0.63 1.3 2.10 2.6 7.05 0.6 14.39 0.4 24.17
Granule 14.56 2.7 51.81 2.1 0.00 – 13.73 7.5 84.11

pressure of 1500 psi and an extraction time of 8 min (Table 1).

The yield of sophocarpine showed a slight upward trend with
increasing cycle number. The yields of matrine and sophoridine
first increased and then decreased, showing the maximal levels
at 2 cycles. However, the yield of oxymatrine decreased with an
increase in cycle number. The effect of the cycle number on the
yields was investigated for ASE with an extraction temperature of
100 ◦ C, an extraction pressure of 1000 psi and an extraction time
of 5 min (Table 1). The yields of sophocarpine and matrine showed
upward trends with increasing cycle number. Sophoridine mani-
fested an initial upward and then downward yields. The yield of
oxymatrine dropped obviously with increasing cycle number. The
total alkaloid yields reached their maximal levels at a cycle number
of 2 whichever extraction method was used.
In summary, the optimal SWE conditions were as follows:
an extraction temperature of 100 ◦ C, an extraction pressure of
1500 psi, a static extraction time of 8 min and a cycle number of 2.
The optimal ASE conditions were as follows: an extraction tempera-
ture of 100 ◦ C, an extraction pressure of 1000 psi, a static extraction
Fig. 3. Comparison of the yield of each analyte using different extraction methods.
time of 5 min and a cycle number of 2.

results, SWE is preferred to the other three extraction methods for

3.2.3. Recovery yields
Six replicates of 0.5 g Sophora sample were accurately weighed,
in which appropriate amounts of cytisine, sophocarpine, matrine,
sophoridine and oxymatrine were spiked then. After extracting and
analyzing under the optimal SWE/CE and ASE/CE conditions, the
recoveries of the analytes were determined. For SWE, the aver-
age recoveries of six replicates of spiked samples ranged from 95%
to 119%, and for ASE, the average recoveries ranged from 62% to
106%. The relatively standard deviations of the recoveries (n = 6)
were < 8.7% indicating the reported method achieved satisfactory
repeatability.
3.3. Comparison of different extraction methods
After establishing the optimal conditions for SWE and ASE, the
extraction efficiencies of the different extraction methods were
compared based on the extraction yields (Fig. 3). Compared with
other methods, SWE showed a relatively high yield for each analyte.
When ASE was performed, the yield of oxymatrine was the lowest
among the four methods, but this method seemed to be slightly
better than chloroform soaking extraction and water ultrasonic
extraction for matrine and sophoridine. Chloroform soaking extrac-
tion and water ultrasonic extraction were particularly effective in
terms of extraction of oxymatrine, but they worked less efficiently
for other three alkaloilds. For the total alkaloild yields, SWE mani-
fest to be the best, closely followed by water ultrasonic extraction,
which gave a higher value than chloroform soaking extraction and
ASE.
Additionally, as SWE and water ultrasonic extraction used water
as solvent, which is suitable for CE analysis without solvent evapo-
ration, which is a serious advantage in terms of sample preparation
time as well as the accuracy of the method. As can be seen from the
the extraction of alkaloids in Sophora.
3.4. Comparison of analytes contents in different medicines
The SWE-CE method developed was used to extract and analyze
the alkaloids in Sophora samples from different sources and Sophora
instant granule, and the electropherogram of a real sample is illus-
trated in Fig. 2(b), the contents in Table 2. The amounts of matrine
and oxymatrine in the three crude samples were close to those
extracted by supercritical fluid extraction [13]. It is also noted that
the amounts of matrine and sophocarpine in the instant granule
manifested ca. 10–20 times higher than those in the crude sam-
ples, while the amount of oxymatrine in the former fell within the
same range in the latter, and sophoridine could not be detected in
the former. A conclusion can be drawn that the production process
of Sophora instant granule from crude Sophora plants influenced
the content ratio of the active compounds to a great degree, which
should be noticed.
4. Conclusions
This study describes an efficient and environmentally friendly
extraction and analysis approach for the determination of alkaloids
in Sophora. SWE exhibited the highest extraction efficiency for the
total alkaloid yield. And its particular advantages over traditional
extraction methods lies on a short extraction time, no need for
organic solvent consumption and high extraction efficiency. Adding
a certain amount of acid to the sample matrix was a simple way
to improve the electro-injection reproducibility in CE with field
amplified stacking. Due to the advantages of both SWE and CE, this
method may have a promising potential in application to the quality
control of natural medicine.
 H. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 146–151
Acknowledgements
The authors acknowledge with gratitude and appreciation
financial support from Northwest A&F University.
References
[1] Y.F. Miao, B. Wu, W.Q. Zhang, Y.M. Qiu, B. Li, X.J. Lu, Neuroprotective effect of
sophocarpine against transient focal cerebral ischemia via down-regulation of
the acid-sensing ion channel 1 in rats, Brain Res. 1382 (2011) 245–251.
[2] A.D. Kinghorn, M.F. Balandrin, Quinolizidine alkaloids of the leguminosae:
structural types, analysis, chemotaxonomy and biological activities, in: S.W.
Pelletier (Ed.), Alkaloids: Chemical and Biological Perspectives, vol. 2, Wiley,
New York, 1984, pp. 105–148.
[3] B.Q. Wang, Z.G. She, X.H. Wang, X.L. Hu, Determination of matrine by thin
layer chromatography-fluorescence quenching, Chin. J. Anal. Chem. 25 (1997)
693–695.
[4] F.Q. Yang, J.Q.T.Y. Zhang, Y. Ito, Preparative separation of alkaloids from the root
of Sophora flavescens Ait. by pH-zone-refining counter-current chromatogra-
phy, J. Chromatogr. A 822 (1998) 316–320.
[5] J.Y. Yin, Y.H. Xu, J. Li, E.K. Wang, Analysis of quinolizidine alkaloids in Sophora
flavescens Ait. by capillary electrophoresis with tris(2,2 -bipyridyl) ruthenium
(II)-based electrochemiluminescence detection, Talanta 75 (2008) 38–42.
[6] Y.Q. Yu, P.L. Ding, D.F. Chen, Determination of quinolizidine alkaloids in Sophora
medicinal plants by capillary electrophoresis, Anal. Chim. Acta 523 (2004)
15–20.
[7] Y.J. Wu, Q. Shao, Z.Q. Zhen, Y.Y. Cheng, Determination of quinolizidine alka-
loids in Sophora flavescens and its preparation using capillary electrophoresis,
Biomed. Chromatogr. 20 (2006) 446–450.
[8] S.H. Liu, Q.F. Li, X.G. Chen, Z.D. Hu, Field-amplified sample stacking in capillary
electrophoresis for on-column concentration of alkaloids in Sophora flavescens
Ait., Electrophoresis 23 (2002) 3392–3397.
151
[9] J.P. Lai, X.W. He, Y. Jiang, F. Chen, Preparative separation and determina-
tion of matrine from the Chinese medicinal plant Sophora flavescens Ait. by
molecularly imprinted solid-phase extraction, Anal. Bioanal. Chem. 375 (2003)
264–269.
[10] X.Y. Su, L. Kong, X. Lia, X.G. Chen, M. Guo, H.F. Zou, Screening and analysis of
bioactive compounds with biofingerprinting chromatogram analysis of tradi-
tional Chinese medicines targeting DNA by microdialysis/HPLC, J. Chromatogr.
A 1076 (2005) 118–126.
[11] L. Zhang, L. Xu, S.S. Xiao, Q.F. Liao, Q. Li, J. Liang, X.H. Chen, K.Sh. Bi,
Characterization of flavonoids in the extract of Sophora flavescens Ait. by high-
performance liquid chromatography coupled with diode-array detector and
electrospray ionization mass spectrometry, J. Pharm. Biomed. Anal. 44 (2007)
1019–1028.
[12] J.Z. Song, H.X. Xu, S.J. Tian, P.P. But, Determination of quinolizidine alkaloids
in traditional Chinese herbal drugs by nonaqueous capillary electrophoresis, J.
Chromatogr. A 857 (1999) 303–311.
[13] J.Y. Ling, G.Y. Zhang, Z.J. Cui, C.K. Zhang, Supercritical fluid extraction
of quinolizidine alkaloids from Sophora flavescens Ait. and purification by
high-speed counter-current chromatography, J. Chromatogr. A 1145 (2007)
123–127.
[14] Y. Cheng, S.M. Li, Analytical method development of long-chain ketones in PM2.
5 aerosols using accelerated solvent extraction and Gc/FID/MSD, Int. J. Environ.
Anal. Chem. 84 (2004) 367–378.
[15] J. Kronholm, K. Hartonen, M.L. Riekkola, Analytical extractions with water at
elevated temperatures and pressures, Trends Anal. Chem. 26 (2007) 396–412.
[16] C.C. Teo, S.N. Tan, J.W.H. Yong, C.S. Hew, E.S. Ong, Pressurized hot water extrac-
tion (PHWE), J. Chromatogr. A 1217 (2010) 2484–2494.
[17] Pharmacopoeia Commission of the Ministry of Public Health, The People’s
Republic of China, Pharmacopoeia of the People’s Republic of China: Part 1,
Guangdong Academic Press, 1995, pp. 176–177.
[18] C.X. Zhang, W. Thormann, Head-column field-amplified sample stacking in
binary system capillary electrophoresis. 2. Optimization with a preinjection
plug and application to micellar electrokinetic chromatography, Anal. Chem.
70 (1998) 540–548.