Chinese Medicine, 2010, 1, 63-68
doi:10.4236/cm.2010.13013 Published Online December 2010 (http://www.SciRP.org/journal/cm)
Identification of Two Cold Water-Soluble Polysaccharides
from the Stems of Ephedra sinica Stapf
Yonggang Xia, Jun Liang, Bingyou Yang, Qiuhong Wang, Haixue Kuang*
Key Laboratory of Chinese Materia Medica, Heilongjiang University of Chinese Medicine,
Ministry of Education, Harbin, China
Received December 4, 2010; revised December 8, 2010; accepted December 10, 2010
Abstract
Two polysaccharides (ESP-A1 and ESP-A2) were isolated from the cold water extract of Ephedra sinica
Stapf and purified through ethanol precipitation, deproteinization and by ion exchange and gel-filtration
chromatography. Their molecular weight was determined using high performance size exclusion chromatog-
raphy and evaporative light scattering detector (HPSEC-ELSD) and their monosaccharide composition was
analyzed by high performance capillary electrophoresis (HPCE) based on pre-column derivatization with 1-
phenyl-3-methyl-5-pyrazolone (PMP). It was shown that ESP-A1 consisted of xylose, arabinose, glucose,
mannose and galactose and ESP-A2 consisted of xylose, arabinose, rhamnose and galactose, in a molar ratio
(%) of 3.2: 61.1: 11.1: 12.9: 11.6 and 20.6: 67.7: 5.0: 6.7, respectively. The molecular weights (Mw) of
ESP-A1 and ESP-A2 were 5.83 × 104 Da and more than 200 × 104 Da, respectively. To the best of our
knowledge, two neutral polysaccharides are now being reported for the first time in this study.
Keywords: Ephedra sinica Stapf, Polysaccharides, Isolation and Purification, Molecular Weight,
Monosaccharide Composition
1. Introduction
Ephedrae herba is the dried stem of Ephedra sinica Stapf,
E. intermedia Schrenk et C. A. Mey. and E. equisetina
Bge., which belongs to the Ephedraceae family [1]. It is a
crude drug widely used in Traditional Chinese Medicine
(TCM) for thousands of years for its medicinal qualities
as a diaphoretic, diuretic, anti-asthmatic to treat allergies,
asthma, pneumonia, bronchitis, hay fever and colds [2,3].
It is well-known that a series of ephedrine alkaloids have
for a long time been considered as the pharmacologically
active ingredients of E. sinica for treatment of various
diseases and symptoms [4], but they cannot account for
all the effects mentioned above and the water-soluble
polysaccharides are also demonstrated to be one of the
main bioactive constituents of E. sinica. Polysaccharides
from Ephedrae herba are a class of macromolecules that
have been shown obvious immunosuppressive effects by
carbon clearance test, delayed-type hypersensitivity reac-
tion and serum hemolysin analysis in vivo [2,3]. In the
present study, we extracted, isolated and purified the
polysaccharide components from the stems of E. sinica
and determined their molecular weight as well as their
monosaccharide composition so as to further investigate
its mechanism of action.
2. Experimental
2.1. Materials and Reagents
The dry stems of E. sinica were collected in March 2007
from Datong of Shanxi Province, China and identified by
Prof. Zhenyue Wang of Heilongjiang University of Chi-
nese Medicine. The voucher specimen (20070016) was
deposited at Herbarium of Heilongjiang University of
Chinese Medicine, Harbin, P. R. China.
D-mannose (Man), L-rhamnose (Rha), D-glucose (Glc),
D-galactose (Gal), L-arabinose (Ara), D-xylose (Xyl), D-
glucuronic acid (GlcUA), D-galacturonic acid (GalUA),
sulfuric acid (H2SO4), were purchased from Sigma (St.
Louis, USA). 1-Phenyl-3-methyl-5-pyrazolone (PMP),
purchased from Beijing Reagent Plant (Beijing, China),
was recrystallized twice from chromatographic grade
methanol before use. DEAE Sepharose Fast Flow,
Sephacryl S-400 HR, and Sephacryl S-200 HR were
from the Pharmacia Co. (Sweden). All other chemicals
were of the highest grade available.

64 Y. G. XIA ET AL.
2.2. Isolation of Polysaccharides from E. sinica
The dry stems of E. sinica were ground to powders, and
submitted to sequential extractions as follows: dry pow-
ders (1.0 kg) were extracted 3 times with 10 vol of 95%
EtOH under reflux for 3 h each time to remove lipids.
The residue was dried in air and then extracted 3 times
with 10 vol of distilled water for 24 h (each time) at 4°C.
The combined aqueous extracts were filtered, concen-
trated 10-fold, and 95% EtOH added to final concentra-
tion of 80%. The precipitate was dissolved in 600 mL of
water and deproteinated 15 times with 200 mL of 5:1
chloroform-n-butanol as described by Staub [5]. The
resulting aqueous fraction was extensively dialyzed
(cut-off Mw 3500 Da) against tap water for 48 h and dis-
tilled water for 48 h and precipitated again by adding a 5
fold volume of ethanol. After centrifugation, the precipi-
tate was washed with anhydrous ethanol and then dis-
solved in water and lyophilised to yield the crude poly-
saccharide A (8.5 g) was collected by centrifugation
(3000 rpm, 10 min, 20°C).
Crude polysaccharide A (3.0 g) was dissolved in dis-
tilled water and passed through two series connected
resin columns (Amberlite FPA90-Cl (Cl- form) and Am-
berlite IRC-84 (H+ form)) eluting with distilled water and
1.0 M NaCl to yield fractions Fr. A1 (1.2 g) and Fr. A2
(0.9 g), respectively. Fr. A1 (1.0 g) was chromatogra-
phed over DEAE-Sepharose F. F eluting with distilled
water and 0.2 M NaCl to yield subfractions Fr. A1-1
(400.0 mg) and Fr. A1-2 (380.0 mg), respectively. Fr.
A1-1 (400.0 mg) was further purified by gel-permeation
chromatography on a high resolution Sephacryl S-400
eluting with distilled water to afford Fr.-A1-1-1 (160.0
mg) and Fr.-A1-1-2 (180.0 mg). Fr.-A1-1-1 (160.0 mg)
was further purified by DEAE-Sepharose F. F eluting
with distilled water to afford ESP-A1 (130.0 mg). Fr.
-A1-1-2 was further purified by Sephacryl S-200 eluting
with distilled water to afford ESP-A2 (145.0 mg).
2.3. Identification on Purity of Polysaccharides
and Molecular Weight Determination
The molecular mass of the polysaccharide (5 mg/mL)
was determined by high performance liquid chromatog-
raphy (HPLC), using Waters 2695 HPLC and Alltech
ELSD 2000 detector. The separation was carried out on a
Shodex sugar KS-805 column (8.0 mm × 300 mm, 17
μm) coupled with a Shodex KS-G guard column (6 mm
× 50 mm, 7 μm). The Dextran standards (T-10, T-40,
T-70, T-500, T-2000) were used for the calibration curve.
The isocratic elution was employed using water with 0.5
mL/min at 30°C and the injection volume was 10 μL.
While the drift tube temperature for ELSD was set at 116
°C, the nitrogen flow rate was 3.3 L/min for the deter-
mination of polysaccharides. Their purities were over
98% by HPLC analysis. Total carbohydrate contents in
purified samples were determined by phenol-sulfuric
acid colorimetric method using glucose as the standard
(Dubois, Gilles, Hamilton, Rebers, & Smith 1956). Pro-
teins in the polysaccharides were detected by the method
of UV absorption on a TU-1800PC spectrophotometer
(Beijing Purkinje General Instrument Co., Ltd., China).
2.4. Analysis of Monosaccharide Composition
Monosaccharide composition was analyzed according to
the following procedure: Each polysaccharide sample
(20 mg) was dissolved in 2 ml of 2.0 M H2SO4 in an
ampoule (5 ml). The ampoule was sealed under a nitro-
gen atmosphere and kept in 110°C to hydrolyze the
polysaccharide into component monosaccharides for 6 h,
then cooled to room temperature and neutralized with 2
ml of 4.0 M sodium hydroxide. The reaction mixture was
diluted to 5 ml with deionized water and was centrifu-
galized at 1000 rpm for 5 min. Then the supernatant was
ready for the following experiments.
PMP derivatization of monosaccharides was carried
out as described previously [6]. 200 μl of individual
standard monosaccharide, or mix standard monosaccha-
ride solutions, or the hydrolyzed polysaccharide samples
were placed in the 2.0 mL centrifuge tubes, respectively,
then 0.5 M methanol solution (100 μl) of PMP and 0.3 M
aqueous sodium hydroxide (100 μl) were added to each.
Each mixture was allowed to react for 30 min at 70°C
water bath, then cooled to room temperature and neu-
tralized with 100 μl of 0.3 M HCl. The resulting solution
was performed on liquid-liquid extraction with same
volume of isoamyl acetate (two times) and chloroform
(one time), respectively. After being shaken vigorously
and centrifuged, the organic phase was carefully dis-
carded to remove the excess reagents. Then the aqueous
layer was filtered through a 0.45 μm membrane and di-
luted with water before HPCE analysis.
The analysis of PMP-labeled monosaccharides was
carried out on a P/ACE MDQ capillary electrophoresis
instrument (Beckman Coulter, Fullerton, CA, USA). An
integrated P/ACE 32 Karat Station (software version 4.0)
was used to perform the data collection and to control the
operational variables of the system. Separation was car-
ried out in an unmodified fused silica capillary (48.5 cm
× 50 μm i.d., effective length 40 cm) with direct UV
monitoring using a photodiode array detector at wave-
length 254 nm including 35 mM borate at pH 10.02, cap-
illary temperature 25°C and applied voltage 20 kV. The
molar ratio of the component monosaccharides is calcu-
lated as follows. The correction factor is shown in the

Y. G. XIA ET AL.
65

equation:

/// / ini in n
f
mA mA

n
, where A
i and A n
are the values of their peak areas in the standard mono-
saccharide, respectively. mi and mn are the values of their
weights of the standard monosaccharide, respectively.
The molar ratio value is shown in the equation: Ri/n = fi/n
×

/
i
A
A

, where A′i/A′n is the ratio value of peak area
for the component monosaccharide of tested samples and
fi/n is the correction factor.
3. Results and Discussion
3.1. Isolation of Polysaccharides from the Stems
of E. sinica
Though there are numerous literature reports on the ex-
traction of crude polysaccharides from TCMs, few re-
ports have studied the isolation, purification, molecular
weight and monosaccharide composition of neutral
polysaccharides from the stems of E. sinica [3,4]. In the
present work, the extraction and isolation of ephedrae
polysaccharides was performed by cold water and etha-
nol precipitation to yield crude polysaccharide, then the
Sevag method was used to remove protein components
after re-dissolution of the crude polysaccharides. The
solution was then dialyzed against against tap water for
48 h and distilled water for 48 h and precipitated by
adding a 5 fold volume of ethanol. The precipitate was
collected by centrifugation and washed successively with
absolute ethanol and acetone to give a light yellow pow-
der. For additional purification the ephedrae polysaccha-
rides were subjected to two series connected resin col-
umns (Amberlite FPA90-Cl (Cl- form) and Amberlite
IRC-84 (H+ form)) eluting with distilled water to yield
fractions Fr. A1. Fr. A1 continues DEAE-Sepharose F. F
column chromatography eluting with distilled water.
Each 5 mL eluted fraction was collected and the content
of sugar was monitored using the phenol-sulfuric acid
method. Fractions 5-60 and 140-160 were combined and
designated as the Fr. A1-1 and Fr. A1-2 fraction (Figure
1). ESP-A1 and ESP-A2 were acquired at last by EAE-
Sepharose F. F column and Sephacryl S-200 column,
respectively. Figures 2 and 3 showed that one main peak
was found in the elution trace by this method.
3.2. Molecular Weight Determination of
Polysaccharide
The two polysaccharides appeared as only a single and
symmetrical sharp peak in high performance gel permea-
tion chromatography by HPLC-ELSD with Shodex sugar
KS-805 column (Figure 4). The average molecular
weights were ca.5.83 × 104 Da and > 200 × 104 Da for
ESP-A1 and ESP-A2, respectively, by reference to the
Figure 1. Elution profile of Fr. A on DEAE-Sepharose F. F
column.
Figure 2. Elution profile of ESP-A1 from DEAE-Sepharose
F. F column.
Figure 3. Elution profile of ESP-A2 from Sephacryl S-200
column.
calibration curve (y = –2.2303, x + 31.68, r2 = 0.9936)
made from a Dextran T-series standard of known mo-
lecular weight (T10, T40 T70, T500, T2000). These
polysaccharides showed negative Fehling’s reagent and
iodine-potassium iodide reactions, indicating that they
didn’t contain reducing sugar and starch-type polysac-
charide. All two polysaccharides had negative responses
to the Bradford test and no absorption at 280 nm in the
V spectrum, indicating the absence of protein. U

Y. G. XIA ET AL.
66
(a)
(b)
Figure 4. (a) HPLC–ELSD chromatogram of ESP-A1 with sugar KS-805; (b) HPLC–ELSD chromatogram of
ESP-A2 with sugar KS-805.
3.3. Monosaccharide Composition of Polysac-
charide and its Molar Ratio
The lack of chromophores or fluorophores in the struc-
ture of monosaccharides limits their sensitive detection
in HPCE [7,8]. Therefore, carbohydrates are generally
tagged with a suitable chromophore or fluorophore to
obtain highly sensitive detection. The reagent 1-phenyl-3
-methyl-5-pyrazolone (PMP) is one of the popular labels
that react with reducing carbohydrate under mild condi-
tion, requiring no acid catalyst and causing no isomeriza-
ion [6-8]. This experiment was designed to develop a t

Y. G. XIA ET AL.
67
Figure 5. Electropherograms of PMP derivatives of monosaccharides in ESP-A1 (a), ESP-A2 (b) and standard
monosaccharides (c). Separation condition: 35 mM borate at pH 10.02, capillary temperature 25°C and applied
voltage 20 kV, 0.2 mM each. Peak identities: 1, Xyl; 2, Ara; 3 Glc; 4 Rha; 5, Man; 6, Gal; 7, GlcUA; 8, GalUA.
Detection, 254 nm direct mode; injection pressure, 0.5 psi for 5 s; capillary, fused-silica 48.5/58.5 cm (Ldet/Ltot);
separation temperature, 25°C.
rapid, repeatable and accurate CZE method with PMP
pre-column derivatization for the quantification of the
component carbohydrates in the cold water-soluble
polysaccharides from the stems of E. sinica. In the proc-
ess of structural analysis of polysaccharides, the molar
ratio of each monosaccharide in polysaccharides is one
of the most important parameters. It is unnecessary to
obtain the specific amount of each monosaccharide be-

68

Y. G. XIA ET AL.
cause researchers just want to know the molar ratios of
each one. Therefore, the molar ratio can be calculated by
the equation

// / inini n
RfAA


 
 
rather than specific
amount of each sugar obtained. We believe that the for-
mer is more favorable in the elimination of systematic
errors.
We have studied hydrolysis conditions of ephedra
polysaccharides and have found that 6 h at 110°C with
2M H2SO4 gave nearly quantitative release of neutral and
acidic sugars from several polysaccharides [6]. Therefore,
our previous method was used in this work. ESP-A1 and
ESP-A2 were hydrolyzed with 2M H2SO4, and PMP-
labeled as described in the experimental section and
finally, the released monosaccharide derivatives were
analyzed by the described CZE method under the opti-
mized conditions. Figures 5(a) and 5(b) showed typical
chromatograms of the cold water-soluble two polysac-
charide samples. As can be seen, the PMP derivatives of
the component monosaccharides released from the
ESP-A1 and ESP-A2 could be still baseline separated
and the component monosaccharides could be identified
by comparing with the chromatogram of the mixture of
standard monosaccharides (Figure 5(c)). Each mono-
saccharide peak in the order of increasing retention time
was identified as xylose, arabinose, glucose, rhamnose,
mannose, galactose, glucuronic acid and galacturonic
acid. It was shown that ESP-A1 consisted of xylose, ara-
binose, glucose, mannose and galactose and ESP-A2
consisted of xylose, arabinose, rhamnose and galactose,
in a molar ratio (mol%) of 3.2: 61.1: 11.1: 12.9: 11.6 and
20.6: 67.7: 5.0: 6.7, respectively. It was clear that the
predominantly composition monosaccharide in ESP-A1
and ESP-A2 was arabinose up to 61.1% and 67.7%
(mol%), respectively.
4. Conclusion
Two polysaccharides extracted from the stems of E.
sinica (ESP-A1 and ESP-A2) were successfully sepa-
rated and purified. The results reported in this paper pro-
vided an excellent example for the isolation, purification,
determination of molecular weights and molar ratios
among monosaccharides of complex hetero-lysaccharide
from E. sinica. The present finding provides a basis for
further structural analysis and evaluation of the bioac-
tivities of E. sinica polysaccharides for its application in
food and medicinal fields.
5. Acknowledgements
Our work was supported by the Major State Basic Re-
search Development Program of China (973 Program
2006CB504708), State Key Creative New Drug Project
of 12th Five-year Plan of China (2011ZX11102), Na-
tional Natural Science Foundation of China (No.
30973870) and Heilongjiang University of Chinese
Medicine Doctor Innovative Foundation.
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